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North American Journal
of
Medicine & Science
Vol. 7, Issue 2
April 2014
Folate Receptor Alpha Autoantibodies Modulate
Thyroid Function in Autism Spectrum Disorder
Richard E. Frye, MD, PhD; Jeffrey M Sequeira, MS;
Edward Quadros, PhD; Daniel A. Rossignol, MD
Labor Market Conditions and Food Security Status of
Emergency Food Assistance Program Clients in the
Recession
Qi Zhang, PhD; Rajan Lamichhane, PhD
Study of the Application of Clinical Pathways in
Varicella, Acute Bacillary Dysentery, Measles, Scarlet
Fever, and Rubella
Zhe Xu, MD, PhD; En-qiang Qin, MD; Min Zhao, MS;
Wei-ming Nie, MS; Zhi-ping Zhou, MS; Bo Tu, MS; Weiwei Chen, PhD; Dan Wu, BS; Fei Wang, MS; Jin Li, MS
False-Negative Interpretation of Breast Sentinel Lymph
Node Touch Preps: Analysis of the Causes with
Suggestions to Improve Diagnostic Accuracy
Frank Chen, MD, PhD, MBA; David Hicks, MD; Maria
Nava, MD; Richard Cheney, MD
Anti-HBV Activities of Xanthones From Swertia
Punicea Hemsl
Xiu-Qiao Zhang, PhD; Jia-Chun Chen, PhD; Feng-Jiao
Huang, MS; Luan-Yuan Tian, PhD; Yuan Tu, MS
Folate
Receptor
Alpha
Autoantibodies Modulate Thyroid
Function in Autism Spectrum
Disorder
The folate receptor alpha (FRα) is
essential for folate transportation across
the blood-brain barrier and is closely
associated with cerebral folate deficiency,
Validity of Fine Needle Aspiration Cytology in
Diagnosis of Prostatic Lesions and Correlation with
Trucut Biopsy
Judith J. Thangaiah, MD; Krishna Balachandran, MD;
Usha Poothiode, MD; Suresh Bhat, MD
Eosinophilic Esophagitis Presenting as Complete
Esophageal Desquamation: An Unusual Case of Chest
Pain
Kheng-Jim Lim, MD; Lanjing Zhang, MD, MS; Anish
Sheth, MD
Furosemide Induced Bullous Pemphigoid Associated
with Antihistone Antibodies
Matthew F. Helm, BS; Lin Lin, MD, PhD; Peter
Santalucia, MD; Brummitte Dale Wilson, MD; RW
Plunkett, PhD; Raminder Grover, MD
Sebaceous Tumors of the Skin and Muir Torre
Syndrome – A Mini-Review
Ayesha Arshad, MD; Christopher A. D’Angelis, MD, PhD
Acting Editors:
Published:
Distributed:
Frank Chen, MD, PhD
C. Cameron Yin, MD, PhD
Hans Iwenofu, MD
Boston, MA, USA
Worldwide
ISSN:
1946-9357
NORTH AMERICAN JOURNAL
OF
MEDICINE & SCIENCE
April 2014 (Volume 7, Issue 2)
Original Research
53
Folate Receptor Alpha Autoantibodies Modulate Thyroid Function in Autism Spectrum Disorder
Richard E. Frye, MD, PhD; Jeffrey M Sequeira, MS; Edward Quadros, PhD; Daniel A. Rossignol, MD
57
Labor Market Conditions and Food Security Status of Emergency Food Assistance Program Clients in the
Recession
Qi Zhang, PhD; Rajan Lamichhane, PhD
63
Study of the Application of Clinical Pathways in Varicella, Acute Bacillary Dysentery, Measles, Scarlet Fever, and
Rubella
Zhe Xu, MD, PhD; En-qiang Qin, MD; Min Zhao, MS; Wei-ming Nie, MS; Zhi-ping Zhou, MS; Bo Tu, MS; Wei-wei
Chen, PhD; Dan Wu, BS; Fei Wang, MS; Jin Li, MS
68
False-Negative Interpretation of Breast Sentinel Lymph Node Touch Preps: Analysis of the Causes with Suggestions
to Improve Diagnostic Accuracy
Frank Chen, MD, PhD, MBA; David Hicks, MD; Maria Nava, MD; Richard Cheney, MD
72
Anti-HBV Activities of Xanthones From Swertia Punicea Hemsl
Xiu-Qiao Zhang, PhD; Jia-Chun Chen, PhD; Feng-Jiao Huang, MS; Luan-Yuan Tian, PhD; Yuan Tu, MS
75
Validity of Fine Needle Aspiration Cytology in Diagnosis of Prostatic Lesions and Correlation with Trucut Biopsy
Judith J. Thangaiah, MD; Krishna Balachandran, MD; Usha Poothiode, MD; Suresh Bhat, MD
Case Report
81
Eosinophilic Esophagitis Presenting as Complete Esophageal Desquamation: An Unusual Case of Chest Pain
Kheng-Jim Lim, MD; Lanjing Zhang, MD, MS; Anish Sheth, MD
84
Furosemide Induced Bullous Pemphigoid Associated with Antihistone Antibodies
Matthew F. Helm, BS; Lin Lin, MD, PhD; Peter Santalucia, MD; Brummitte Dale Wilson, MD; RW Plunkett, PhD;
Raminder Grover, MD
Review
87
Sebaceous Tumors of the Skin and Muir Torre Syndrome – A Mini-Review
Ayesha Arshad, MD; Christopher A. D’Angelis, MD, PhD
www.najms.net
North American Journal of Medicine and Science
Apr 2014 Vol 7 No.2
53
Original Research
Folate Receptor Alpha Autoantibodies Modulate
Thyroid Function in Autism Spectrum Disorder
Richard E. Frye, MD, PhD;1* Jeffrey M Sequeira, MS;2
Edward Quadros, PhD;2 Daniel A. Rossignol, MD3
1
Arkansas Children’s Hospital Research Institute, Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AR
2
Department of Medicine, State University of New York – Downstate Medical Center, Brooklyn, NY
3
Rossignol Medical Center, 16251 Laguna Canyon Road Suite 175, Irvine, CA
The folate receptor alpha (FRα) is essential for folate transportation across the blood-brain barrier and is
closely associated with cerebral folate deficiency, a syndrome that commonly presents with autism
spectrum disorder (ASD) features. FRα autoantibodies (FRAAs) interrupt FRα function and have a high
prevalence in children with ASD. Since the FRα is also located on the thyroid, FRAAs could also interfere
with thyroid function. Interestingly, ASD has been inconsistently associated with hypothyroidism. The aim
of this study was to determine if thyroid dysfunction in ASD could be related to FRAAs. To this end we
investigated the relationship between serum FRAA titers (both blocking and binding) and thyroid
stimulating hormone (TSH) in 32 children with ASD. Blocking, but not binding, FRAAs were found to be
related to TSH levels. Higher FRAAs were significantly correlated with higher TSH concentrations (r =
0.36, p = 0.025), while ASD children who were positive for blocking FRAAs demonstrated a significantly
higher serum concentration of TSH than children who were negative for FRAAs (t(31) = 2.07, p = 0.02).
These results are consistent with the notion that blocking FRAAs are associated with reduced thyroid
function and suggest that thyroid function should be examined in children with ASD who are positive for
the blocking FRAAs.
[N A J Med Sci. 2014;7(2):53-56. DOI: 10.7156/najms.2014.0702053]
Key Words: Folate receptor autoantibody, autism spectrum disorders, thyroid function, hypothyroidism
INTRODUCTION
Folate is a water-soluble B vitamin that is essential for
numerous physiological systems and is critical for
neurodevelopment.1,2 Folate is transported across cellular
membranes using several mechanisms, including the folate
receptor alpha (FRα), the folate receptor beta,3 the reduced
folate carrier (RFC)4 and the proton-coupled folate
transporter.5 The FRα is located on the endothelium of the
choroid plexus,6 thyroid cells,7 the microvillus plasma
membrane of the placenta,8 as well as the epithelium of the
fallopian tubes, uterus, and epididymis, acinar cells of the
breast, submandibular salivary and bronchial glands and the
alveolar lining including pneumocytes.9 The folate receptor
beta appears to be important in the placental transport of
folate,3 while the proton-coupled folate transporter is critical
for gastrointestinal tract folate transport.5 The RFC is a
transmembrane protein that is expressed in a wide range of
tissues, including the placenta, kidney, intestine and both the
basolateral and apical surface of the choroid plexus.10 The
RFC is unique as it allows bidirectional transport of folate
Received: 03/07/2014; Revised: 03/26/2014; Accepted: 03/29/2014
*Corresponding Author: Arkansas Children's Hospital Research Institute,
Slot 512-41B, 13 Children's Way, Little Rock, AR 72202. Tel: 501-3644662, Fax: 501-364-1648. (Email: [email protected])
across the cellular membrane,11 is responsible for folate
transport across the blood-brain barrier when extracellular
folate concentrations are high6 and has a higher affinity for
reduced forms of folate as compared to the oxidized form
commonly known as folic acid.4
Autoantibodies that bind to the FRα and greatly impair its
function were described approximately a decade ago when
they were linked to cerebral folate deficiency (CFD).7 CFD is
a neurometabolic disorder characterized by severe
neurodevelopmental symptoms. CFD is defined by below
normal concentrations of 5-methyltetrahydrofolate (5MTHF)
in the cerebrospinal fluid despite normal systemic folate
levels. The FRα is located on both sides of the endothelial
surface of the choroid plexus and is believed to be the
primary transportation mechanism for folate across the
blood-brain barrier.6 As such CFD is believed to be due to
impaired FRα function, in large part due to FRα
autoantibodies (FRAAs). FRAAs have been linked to CFD in
cases with12 and without7 ASD. Recently, Frye et al.
measured FRAA titers on 93 children with ASD as part of a
medical workup.13 Overall, 60% and 44% were positive for
the blocking and binding FRAAs, respectively; 29% children
were positive for both FRAAs; 46% were positive for only
54
Apr 2014 Vol 7 No.2
one FRAA and 75% were positive for at least one FRAA.
The prevalence of blocking FRAA (60%) was much higher
than the prevalence reported in the general population in
Spain (7.2%),14,15 Ireland (12.6%)16 and the general U.S.
population (10-15%, unpublished data from Dr. Quadros’
laboratory). In addition, a recent study from Belgium has
verified the high prevalence of the blocking FRAA in ASD
children.17 In this study 47% of ASD children were found to
be positive for the blocking FRAA as compared to only 3.3%
of developmentally delayed non-ASD controls.17
Given the fact that the FRα is important for the transportation
of folate into other organs it is possible FRAAs could
interfere with the function of other organs that use the FRα.
FRα is essential for folate transportation into thyroid cells
and, interestingly, there have been inconsistent reports of
thyroid dysfunction in ASD. One study of 5 children with
ASD reported that 3 of them had congenital hypothyroidism
and two of the mothers in the study had probable
hypothyroidism during pregnancy.18 The basal and
thyrotropin-releasing hormone stimulated peak thyroid
stimulating hormone (TSH) levels were shown to be lower in
41 autistic boys as compared to children with mental
retardation, minimal brain dysfunction and typically
developing controls in a Japanese study.19 In another small
study, young adults with ASD were generally found to have
higher TSH levels but the magnitude of the difference
between the ASD and control participants was dependent on
the time of day due to the significant diurnal variation in TSH
levels.20 The largest study examined thyroxin levels at birth
in 784 children with ASD and 554 matched control children
born in California in two groups, a group born in 1994 and a
group born in 1995. The study found that very low thyroxin
levels at birth increased the risk of developing ASD but only
in the 1995 study group.21 Interestingly, one study of 308
children with ASD reported that autoimmune thyroid
disorders in family members was associated with ASD
regression in the child (OR=1.89; 95% CI=1.17-3.10).22 One
way in which thyroid dysfunction could adversely affect
neuronal migration is via the regulation of reelin as this is
dependent on adequate levels of triiodothyronine. 23 However,
several early studies have not found a relationship between
abnormal
concentrations
of
TSH,
thyroxin
or
triiodothyronine and ASD.24,25 One of the reasons that studies
are inconsistent is that there may be only a subgroup of
children with ASD who have abnormal thyroid function and
it may be difficult to detect this subgroup when looking at the
whole population of children with ASD. Thus, it is possible
that thyroid dysfunction may be related to the subgroup of
ASD children with FRAAs.
To determine if there is a relationship between FRAAs and
thyroid function, we examined the relationship between
blocking and binding FRAAs and thyroid stimulating
hormone (TSH) concentrations in a case-series of patients
diagnosed with ASD.
METHODS
Two of the authors (REF; DAR) offered FRAA testing as
North American Journal of Medicine and Science
part of the workup for medical conditions associated with
ASD. Approximately 1ml of serum was collected and sent to
the laboratory of Dr. Edward Quadros, PhD, at the State
University of New York, Downstate (Brooklyn, NY). The
assay for both the blocking and binding FRAAs has been
described previously.7,16 Blocking FRAAs were expressed as
pmoles of folic acid blocked from binding to FRα per ml of
serum and binding FRAAs were expressed as pmoles of IgG
antibody per ml of serum.
Thirty-two children with ASD who had FRAA and TSH
testing were included in this study. All children met the
Diagnostic and Statistical Manual of Mental Disorders –
Fourth Edition – Text Revision26 criteria for ASD and had
previously been diagnosed with ASD by a developmental
pediatrician, pediatric neurologist or clinical psychologist.
Review of each child’s medical record was obtained through
an Institutional Review Board approved protocol. Patient
laboratory values such as the TSH concentration were
abstracted from the medical record. For TSH interpretation,
the National Academy of Clinical Biochemistry standard for
children was used as a reference range, which is 0.4 to 5.0
mIU/L.
Statistical Analysis
The relationship between FRAAs and TSH was investigated
in two ways for both the blocking and binding FRAAs
separately. First, the Pearson correlation between the FRAA
titers and TSH was calculated. Second, patients were divided
into two groups, FRAA positive and FRAA negative, and the
two groups were compared. TSH was found to be log
distributed and was log transformed before analysis. An
alpha of 0.05 was used as a one-tailed test, since the
relationships between FRAAs and TSH were predicted to be
in one particular direction. Specifically, it is hypothesized
that higher FRAA titers would be related to higher TSH
concentrations and FRAA negative patients would have
lower TSH values than FRAA positive patients.
RESULTS
Subject Characteristics
The average age of the participants was 7y 2m (SD = 2y 8m)
and 91% were male.
Folate Receptor Alpha Autoantibody Titers
56% of the patients were positive for the blocking FRAA and
50% of the patients were positive for the binding FRAA.
28% were negative for both FRAAs, 44% were positive for
only one FRAA and 28% were positive for both FRAAs. The
average blocking FRAA titer was 0.44 (SD = 0.48) and
ranged from 0 to 1.44. The average binding FRAA titers was
0.48 (SD = 0.65) and ranged from 0 to 3.45.
Thyroid Stimulating Hormone Concentrations
The average TSH concentration was 2.76 (SD = 3.42) and
ranged from 0.81 to 20.8. Using the standard reference range,
0% of the ASD children demonstrated an abnormally low
TSH and 3% of the ASD children demonstrated an
abnormally elevated TSH.
North American Journal of Medicine and Science
Apr 2014 Vol 7 No.2
Relationship Between Folate Receptor α Autoantibodies
and Thyroid Stimulating Hormone
No significant relationship was found between the binding
FRAA and TSH. However, a higher blocking FRAA titer was
significantly related to a higher TSH concentration [r = 0.36,
55
p = 0.025; See Figure 1A]. Patients who were positive for
the blocking FRAA were found to have a significantly higher
TSH concentration as compared to patients who were
negative for the blocking FRAA [t(31) = 2.07, p = 0.02; see
Figure 1B].
Figure 1. The relationship between the blocking folate receptor alpha autoantibody and thyroid stimulating hormone. (A) The blocking folate
receptor alpha autoantibody titer is correlated with serum concentrations of thyroid stimulating hormone such that higher titers are related to
higher thyroid stimulating hormone levels (note that one individual with a very high thyroid stimulating hormone level is not included in this
graph). (B) Thyroid stimulating hormone levels are significantly higher for children with ASD who were positive for the blocking folate
receptor alpha autoantibody as compared to children with ASD who were negative for the blocking folate receptor alpha autoantibody.
DISCUSSION
This study has demonstrated a potential relationship between
the blocking FRAA and TSH in children with ASD,
suggesting that autoantibodies to the FRα could modulate
thyroid function in children with ASD. Several studies have
demonstrated that blocking FRAA titers have a relationship
to central 5MTHF concentrations, presumably by blocking
the transportation of folate through the FRα. This study
suggests that a disruption of folate transportation into thyroid
cells through the FRα may similarly disrupt folate levels in
thyroid cells leading to thyroid dysfunction. Since thyroid
dysfunction would presumably decrease the amount of
thyroxin or triiodothyronine produced, an increase in TSH,
that has been shown to be related to blocking FRAAs in this
study, would follow.
We have demonstrated that TSH is significantly elevated in
ASD children who are positive for the blocking FRAA as
compared to children who are negative for the blocking
FRAA. However, for the most part, all TSH levels were
within the range considered normal for children. There are
several possibilities why more children with abnormally
elevated TSH were not found. First, it is believed that the
current upper limit of the TSH reference range is too high to
detect subclinical hypothyroidism in adults27 and that high
normal TSH levels are associated with increased risk of
metabolic abnormalities.28 Thus, some investigators have
advocated reducing the reference range to detect these
subclinical cases of hypothyroidism in adults. 27 However,
this has not been studied in children to provide a basis to
lower the upper limit of the TSH reference range for children.
Second, iodine deficiency has been reported in some children
with ASD29 and some investigators think it may play a role in
ASD causation,30 especially in combination with pesticide
exposure.31 Iodine deficiency has been implicated as a cause
of thyroid dysfunction in children with ASD32,33 and had
been correlated with ASD severity in one study. 32 It is
possible that abnormalities in folate transport may
synergistically combine with other factors such as iodine
deficiency to result in thyroid dysfunction in children with
ASD. Third, since thyroid dysfunction in children with ASD
is highly correlated with thyroid dysfunction in their mothers,
abnormalities in thyroid function in children with ASD may
simply be an epiphenomenon of thyroid dysfunction during
gestation which can cause significant neurodevelopmental
consequences.32 Lastly, subtle abnormalities in thyroid
function during childhood could be a marker for more severe
thyroid dysfunction earlier in life when the sensitivity of
neurodevelopment to thyroid function is more critical.
Clearly there are several unanswered questions that require
further research. Unfortunately the current study did not
measure thyroxin or triiodothyronine levels to more
accurately determine thyroid function in this series of
children with ASD. Future studies to examine the FRAAs
should consider investigating thyroid function and studies
that investigate thyroid function should consider measuring
FRAA as a cause of thyroid dysfunction. As this is a limited
sample of children, it is difficult to make firm conclusions of
the exact relationship between FRAAs and thyroid function.
Indeed, larger cohorts are needed to confirm and extend the
preliminary findings of this study.
56
Apr 2014 Vol 7 No.2
ACKNOWLEDGEMENTS
This study was supported, in part, by funding from the Autism Research
Institute, the Jane Botsford Johnson Foundation and Autism Speaks.
CONFLICT OF INTEREST
Drs. Frye and Rossignol have no conflict of interest to declare. Two of the
authors (J.M.S. and E.V.Q.) are inventors on a US patent for the detection of
FR autoantibodies issued to the Research Foundation of the State University
of New York.
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North American Journal of Medicine and Science
Apr 2014 Vol 7 No.2
57
Original Research
Labor Market Conditions and Food Security
Status of Emergency Food Assistance
Program Clients in the Recession
Qi Zhang, PhD;1* Rajan Lamichhane, PhD2
2
1
School of Community and Environmental Health, Old Dominion University, Norfolk, VA
Department of Mathematics, Rhode Hall 231, MSC 172, Texas A &M University-Kingsville, Kingsville, TX
The latest economic recession severely threatens food security among low-income Americans. This study is
to examine how the severity of the economic recession is associated with food security among emergency
food assistance program (EFAP) clients in the U.S. We used Hunger in America 2010, a national survey of
61,085 clients of local EFAPs. We found that state unemployment had a positive association with risk of
low food security status measurements but negative association between county unemployment rates and
household food insecurity. Therefore, the economic recession had a significant impact on food insecurity
status among low-income American.
[N A J Med Sci. 2014;7(2):57-62. DOI: 10.7156/najms.2014.0702057]
Key Words: food insecurity, economic recession, hunger
INTRODUCTION
The scale, length and depth of the latest economic recession
exceeded all recessions since the Great Depression. 1 The U.S.
economy reached its worst level in 26 years, while the
national unemployment rate reached 9.3% in 2009, a 60.3%
jump from the previous year.2 This severe recession resulted
in a larger population in poverty: 6.3 million more people,
including 2.1 million children, slipped into poverty between
2007 and 2009.3 Prolonged unemployment and the length of
time living in poverty severely threatened the capabilities of
American households to provide adequate and quality food
for household members: 17.1 million households were food
insecure in 2008, a 4.1 million increase from 2007. 4 The
proportion of food-insecure households in 2008, 14.6
percent, was the highest ever since 1995, when federal
agencies monitored food insecurity.
Households with
children were disproportionally hit, with a 32.9% jump in the
food insecurity rate in 2008 compared with the previous year.
The severe recession pushed financially vulnerable
households to seek public and private sources for their food
supply. The number of participants in federally supported
food assistance programs, such as Supplemental Nutrition
Assistance Program (SNAP), increased significantly, from
12.7 million households in fiscal year 2008 to 15.2 million in
fiscal year 2009.5 In addition to public assistance programs,
more American relied on emergency food assistance, such as
food pantries. In the period of 2007-2009, the number of
households using food pantries increased by 44 percent, to
5.6 million, which topped the record established in 2001.
Received 01/08/2014; Revised 01/14/2014; Accepted 01/15/2014
*Corresponding Author: 3138 Health Sciences Building, School of
Community and Environmental Health, Old Dominion University, Norfolk,
VA 23529. Tel: 757-683-6870 Fax: 757-683-6333.
(Email: [email protected])
Emergency food assistance programs (EFAPs) face more
challenges in the economic recession than public food
assistance programs. For example, theoretically SNAP is not
an entitlement program. However, since requests for SNAP
funding have always been approved by the U.S. Congress, all
eligible households receive benefits regardless of the
economic cycle. EFAPs, on the other hand, have always
relied heavily on nongovernment sources of financing, such
as local donations and fund raising.6 In economic recessions,
EFAPs could face a spike in the demand and a dip in the
supply, simultaneously. Due to the volatile funding source,
EFAP clients could be more vulnerable to experience food
insecurity in the recession. Therefore, it is important to
examine food security status among EFAP clients in the
latest economic downturn. Moreover, due to the significant
regional disparity in the severity of the latest recession, it is
important to answer whether the food security status among
EFAPs was associated with state or local economic
conditions in the recession. If there was a significant
association, the governments at federal, state and local levels
should provide additional resources to the economically hardhit regions to ensure the basic human rights of having
sufficient food and quality food, even in times of recession.
Although EFAP clients’ food security status warrants
attention, few studies have been conducted at the national
level compared with research on food security status among
public food assistance program participants. 7-9 The primary
barrier is lack of quality national data on EFAP clients. One
of the most authoritative national studies on EFAP clients
was conducted almost a decade ago.6 An additional barrier is
the small sample size of very low food security households in
most nationally representative data, such as the Current
58
Apr 2014 Vol 7 No.2
Population Survey (CPS). These national datasets survey
households across income groups and were used in many
researchers to study the general food security, but not very
low food security. The prevalence of very low food security
(VLFS) has been low in all households (2009: 5.3%) and
extremely low in households with children (2009: 1.2%).10
Given the normal sample size of most national data sets, such
as 60,000 households in the CPS, the limited sample sizes of
VLFS households in these data sets makes it challenging to
conduct in-depth research on VLFS households or
households with children. The data limitations prevent policy
makers and researchers from understanding fully about the
factors contributing to VLFS in times of recession.
For these reasons, there is a significant knowledge gap in the
literature about the food security status of EFAP clients in the
latest recession. In this paper, we used data and a newly
completed national survey, Hunger in America, to assess the
relationship between unemployment rates and food security
status among EFAP clients and to compare the impact of
state vs. county labor market conditions on different levels of
food security status among low-income households.
METHODS
Data
Hunger in America 2010 (HIA 2010) is the largest and most
extensive study ever conducted in the U.S. on EFAPs and
their clients.11 Although the final report was released in 2010,
Feeding America completed the study in 2009 by
interviewing a sample of its 61,085 clients in face-to-face
interviews. Feeding America is the nation’s largest hungerrelief charity, representing 80% of EFAPs and serving 37
million Americans, including 14.3 million children. It
distributes 2.6 billion pounds of food products annually.
Among the 61,085 households surveyed in the HIA 2010,
there were 22,173 households with VLFS status; 7,158 of
these VLFS households had at least one child. Therefore,
HIA 2010 provides rich information and opportunities for
researchers to analyze large numbers of low-food-securitystatus households during this recent recession. HIA 2010
collected detailed information on households’ sociodemographics, household food security status as measured by
the standard USDA module and participation in federal food
assistance programs. Home zip codes were also collected.
More technical details about the survey methods are available
in the Final Report of HIA 2010.12
Measurement
Household and Childhood Food Security
HIA 2010 used a nine-item module to measure the food
security. The first six questions were adopted from the sixitem short form of the USDA module to measure the
household food security. 13 The module asks six questions
related to the household’s experience of food availability and
difficulty in obtaining sufficient food. It has been used since
1995 and is a reliable substitute for the standard 18-item U.S.
Household Food Security Survey Module and has a high
specificity and sensitivity to identify very low food
security.14 Based on the USDA’s recommendation, we
defined food security status as follows:
North American Journal of Medicine and Science
 Marginal food security: 1 positive answer
 Low food security: 2-4 positive answers
 Very low food security (VLFS): 5-6 positive answers
We also used the interval-level measures of food security
status based on the Rasch measurement model. 4 However,
the limitation of the six-item module is that it does not
directly measure children’s food security status. To remedy
the limitation, HIA 2010 also asked three questions related to
children’s food availability in addition to the six-item
household food security module. To be consistent with the
measurement of household food security, we defined the
childhood food security as follows:
 Marginal childhood food security: 1 positive answer
 Low childhood food security: 2 positive answers
 Very low childhood food security: 3 positive answers
To measure labor market conditions, we used annual state
and county unemployment rates reported by the Bureau of
Labor Statistics and linked them with HIA 2010 based on
clients’ home zip codes. We also controlled for the clients’
age, gender, race/ethnicity, marital status, employment status,
education, number of household members and SNAP
participation status.
Statistical Analyses
First we provided the descriptive statistics of the
sociodemographics of the EFAP clients; the prevalence of
marginal, low and very low food security status among all
households; households with children aged 18 years or
younger; and the prevalence of marginal, low and very low
childhood food security status. To analyze the association
between unemployment rates and food security status, we
adopted two approaches: linear models with interval
measures of food security scores and logistic models with
binary outcomes of food security status. Since these food
security statuses indicate ordinal levels of severity, we
created a series of binary indicators based on these cut-off
points: very low food security, low food security or worse,
and marginal food security or worse due to the collinearity
between state and county unemployment rates, we conducted
analyses with state and county unemployment separately.
Since multiple clients could be sampled from one EFAP, we
also tested the mixed effects model to accommodate the
hierarchical structure of the data. Likelihood ratio tests were
conducted to compare the mixed effects models with
traditional models. Commands of XTMELOGIT and
XTMIXED in Stata 11 were used.15 Coefficients, standard
errors of the estimates, P-values and 95% confidence
intervals were reported.
RESULTS
The analytical sample included 57,649 clients with complete
information on socio-demographics, food security status and
valid home zip codes. Average household size was 2.42
people, and over one third of the client households had
children. Less than half of the clients were male, and the
average age of the clients was 48.8 years. Approximately
28% of the clients had received some college education or a
bachelor’s degree. Most of the clients did not own their
North American Journal of Medicine and Science
Apr 2014 Vol 7 No.2
homes: 11.8% were living in a trailer, 29.4% were living in
an apartment, 4.8% were living motel or with friends and
14.3% were homeless. The employment situation among
clients was dire: only 19.9% were employed. Among the
unemployed, more than 50% of the clients had been jobless
59
for more than 2 years, and 4.5% of the clients had never
worked in their lives. Approximately 29.1% of the client
households had more than one unemployed adult. In
summary, the employment status of the EFAP clients was
miserable in the latest recession.
Table 1. Socio-demographics of Respondents in Hunger in America Surveys.
Mean or %
57649.0
2.4
34.9
40.3
48.8
Number of Households
Average number of people in a household
Proportion of households with children (%)
Male
Age (yrs)
SE
1.72
0.2
0.21
15.13
Race
Non-Hispanic white
Non-Hispanic black
Hispanic
Other
49.1
27.7
15.6
7.5
0.21
0.19
0.15
0.11
Married or living as married
Divorced or separated
Never been married
Widowed
31.5
30.7
26.6
11.3
0.2
0.2
0.19
0.13
Less than high school
High school graduate
Some college education
33.3
38.8
22.3
0.2
0.2
0.17
Bachelor’s degree or above
5.7
0.1
House or condo
Trailer
39.8
11.8
0.2
0.13
Apartment
Motel, living with friends
Homeless
29.4
4.8
14.3
19.9
0.19
0.09
0.15
0.17
<6 months
6~12 months
1~2 years
>2 years
Never worked
17.0
10.4
15.5
52.0
4.5
0.18
0.15
0.17
0.24
0.1
0
1
2
>=3
13.7
57.3
22.3
6.8
0.14
0.21
0.17
0.1
Marital status
Education
Shelter
Respondent is employed
Unemployment time if unemployed
Number of unemployed individuals
Apr 2014 Vol 7 No.2
60
North American Journal of Medicine and Science
sample had 85.3% food-secure households. In Figure 2,
EFAP households with children had 7.0% low-food-security
children, which was lower than the national sample of 10.6%.
But the prevalence of very low food security children was
significantly higher than the national sample (8.1% vs. 1.2%).
In summary, compared with the national population, EFAP
clients and their children faced a greater threat of food
insecurity in the recession.
Figures 1 and Figure 2 present the dramatic gap between the
food security status of EFAP households and other US
households. In Figure 1, 31.0% of the EFAP households had
low food security, compared to 9.0% in the national sample. 4
The prevalence of very low food security in EFAP client
households was 7.4 times greater than the national sample
(42.1% vs. 5.7%). Only 26.8% of EFAP households were
food secure or marginally food secure, but the national
Table 2. State and County Unemployment Rate Effects on Household Food Security.
No Mixed Effects
(logit model or OLS model)
Coeff.
SE
P-value
Mixed Effects
(logit model or OLS model)
95% CI
Coeff.
SE
P-value
95% CI
Outcome:
Very Low Food Security
0.262
0.073
<0.001
0.119
0.405
0.012
0.012
0.287
-0.010
0.035
-0.018
0.005
<0.001
-0.027
-0.009
-0.020
0.005
<0.001
-0.031
-0.009
0.323
0.085
<0.001
0.157
0.489
0.019
0.013
0.153
-0.007
0.046
-0.018
0.005
0.001
-0.028
-0.008
-0.018
0.006
0.004
-0.030
-0.006
0.379
0.106
<0.001
0.171
0.586
0.028
0.016
0.080
-0.003
0.059
-0.018
0.006
0.004
-0.030
-0.006
-0.017
0.007
0.024
-0.031
-0.002
0.109
0.077
0.007
0.058
0.360
0.015
0.011
0.153
-0.006
0.036
-0.016
0.005
0.001
-0.026
-0.006
-0.015
0.005
0.006
-0.025
-0.004
State Unemployment
County Unemployment
Outcome:
Low Food Security or
worse
State Unemployment
County Unemployment
Outcome:
Marginal Food Security
or worse
State Unemployment
County Unemployment
Outcome:
Food Security Scale
State Unemployment
County Unemployment
Table 3. State and County Unemployment Rate Effects on Childhood Food Security.
No Mixed Effects
(logit model or OLS model)
Coeff.
SE
P-value
Mixed Effects
(logit model or OLS model)
95% CI
Coeff.
SE
Outcome:
Very Low Food Security
State Unemployment
-0.152
0.234
0.515
-0.611
0.306
0.001
0.020
County Unemployment
Outcome:
Low Food Security
-0.013
0.014
0.340
-0.041
0.014
-0.013
0.013
State Unemployment
-0.100
0.182
0.581
-0.456
0.256
0.028
0.017
County Unemployment
Outcome:
Marginal Food Security
-0.013
0.011
0.217
-0.034
0.008
-0.003
0.010
State Unemployment
-0.093
0.141
0.510
-0.370
0.184
0.013
0.015
-0.005
0.008
0.567
-0.022
0.012
-0.004
0.008
County Unemployment
North American Journal of Medicine and Science
Apr 2014 Vol 7 No.2
61
Figure 1. Prevalence of Household Food Security Status among EFAP and
US Households. US statistics obtained from USDA (2010)4
Figure 2. Prevalence of Childhood Food Security Status among EFAP and
US Households. US statistics obtained from USDA (2010)4
Table 2 presents the results of models that examine the
relationship between food security status and unemployment
rates. For state unemployment rates, all the coefficients were
positive in all models, indicating higher likelihood of food
insecurity with worse state labor markets. In the models
without mixed effects, all coefficients were highly significant
(P-value < 0.01). However, after controlling the clustering
effect, the coefficients became insignificant. For the
likelihood of marginal food security or worse, the coefficient
was marginally significant (Coefficient = 0.03, P = 0.08). In
all the models with county unemployment rates, the
coefficients were all negative and statistically significant.
Except for the coefficient for marginal food security or worse
(P = 0.024), all the p-values were significant at a 1% level.
The coefficients varied little, with or without controlling for
the hierarchical models. Likelihood ratios tests indicated that
all mixed-effects models were better fitted with all Chisquare statistics < 0.001.
Our study complements the limited research that has been
done on the association between the economic environment
and food security. A majority of the studies on food
insecurity have focused on the impact of individual or
household characteristics on food security status.9,10,16,17
Limited research has been done at the state or county level
focusing on contextual characteristics. Bartfeld and Dunifon
found that state unemployment rates were significantly
related to food insecurity among low-income households
with children.18 Bernell et al. found that county-level
unemployment rates were positively related to food
insecurity, but the effect was not statistically significant, in
part due to the study being limited to Oregon only. 19 Using
the survey data of parents of elementary school children in
Wisconsin, Bartfeld and Wang found that county
unemployment rates were significantly related to household
food insecurity.20 However, both of these regional studies
were not sufficient to paint a more general picture of food
security and county unemployment rates across the country.
Our study targeted a nationally understudied population,
EFAP clients, and found that higher state unemployment
rates were associated with higher likelihood of household
food security, but the relationship was not statistically
significant. Moreover, higher county unemployment rates
actually were significantly associated with lower likelihood
of household food security among EFAP clients, which is
against what intuition would suggest. Our results also
indicated that childhood food security status was not
associated with any unemployment rates significantly.
Table 3 presents the coefficients of unemployment rates on
childhood food security. The coefficients of state
unemployment had mixed signs: For all non-mixed effects
models, the coefficients were all negative, but the
coefficients of mixed effects models were all positive. The
models using county unemployment rates were still all
negative, just as the household food security models.
However, all coefficients in the childhood food security
models were not statistically significant, which indicates that
childhood food security among EFAP clients was not directly
associated with the recession at the county or state levels.
DISCUSSION
This is the first study to examine the association between
EFAP clients’ food security status and the recession. We
used two different levels of unemployment rates as indicators
of the severity of the economic recession. Our results suggest
that the likelihood of EFAP clients’ food security status may
not be directly associated with state unemployment rates but
may actually be negatively related to county unemployment
rates. Our study contributes new information helping us to
understand the relationship between economic environment
and food security in the U.S.
To fully understand these counter-intuitive results, we need
to interpret the results carefully. Since this is a crosssectional study, the results cannot be compared with food
security levels before the recession. Therefore, our results did
not necessarily suggest that food security was not threatened
by the economic recession, only that there were no
significant disparities in food security across state economic
conditions. In 2010 the USDA reported a significantly higher
prevalence of food insecurity at the national level during the
recession than the prevalence before the recession. However,
only five states had significantly higher rates than the
national average. Therefore, another way to interpret our
62
Apr 2014 Vol 7 No.2
results is that the economic recession was so widespread
across the states that the disparity of food insecurity was no
longer statistically significant.
It seems counter-intuitive that county unemployment rates
were negatively related to likelihood of food insecurity.
However, our results echoed the latest findings by the USDA
that the prevalence of food insecurity and very low food
security among low-income Americans fell 2.2 and 2.0
percent respectively from 2008 to 2009, while the national
annual unemployment rates jumped from 5.8% to 9.3%
during the same period.21 Researchers at the USDA suggest
that the primary contributor to the decrease in food insecurity
could be the American Recovery and Reinvestment Act
(ARRA) of 2009.21
The ARRA of 2009 was a historical move to jump start the
U.S. economy and protect Americans during the severest
recession since World War II. It included specific clauses to
ensure the food security network for Americans and provided
over 20 billion dollars to supplement existing food assistance
programs, including the SNAP, Women, Infants and Children
(WIC), the National School Lunch Program, and the
Emergency Food Assistance Program (TEFAP). TEFAP is a
federal assistance program that provides food to the states,
which the states then distribute to local food banks, pantries
and soup kitchens. As the USDA states explicitly, “The
amount of food received by each state depends on its
population of unemployed persons and persons with incomes
below the poverty level”.22 Therefore, EFAPs in counties
with a higher number of unemployed persons may receive a
greater amount of food from TEFAP. The budget for TEFAP
in FY 2008 was $190 million for food and $50 million for
administrative costs. However, the ARRA provided an
additional $100 million for food and $50 million for
administrative cost, which was a 50% increase in funding for
food resources and a 100% increase in funding for
administrative costs.23 In 2009, over 800 million pounds of
food were distributed to EFAPs through TEFAP.4 Therefore,
the increases in food supply to EFAPs may have exceeded
the increase in food demand, which may have reduced food
insecurity in higher-unemployment regions.
Due to the lack of longitudinal data on EFAP clients’ food
security status, we are unable to conclude that the ARRP did
cause the negative relationship between county
unemployment rates and food insecurity. However, our
pioneering study highlights that a worse local economy may
not be associated with worse welfare among EFAP clients
and that strong support from the federal government may
achieve the original policy goal, namely, to ensure that
sufficient food is available for low-income Americans in a
tough economic period.
ACKNOWLEDGEMENTS
We thank Emily Engelhard from Feeding America for assistance with data
access.
North American Journal of Medicine and Science
REFERENCES
1.
National Bureau of Economic Research (NBER). US business cycle
expansions and contractions. http://www.nber.org/cycles.html.
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Bureau of Labor Statistics (BLS). Local area unemployment statistics.
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3.
Census Bureau. Income, poverty, and health insurance coverage in the
United States: 2009. http://www.census.gov/prod/2010pubs/p60238.pdf. Accessed on August 30, 2011.
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Andrews M. More Americans relied on food assistance during
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Nord M. Food stamp participation and food security. Food Rev. 2001;
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Yen ST, Andrews M, Chen Z, Eastwood DB. Food stamp program
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severity, and household characteristics. EIB-56. USDA/ERS. 2009.
11. Feeding America. Hunger in America 2010: Executive summary.2010.
http://feedingamerica.issuelab.org/research/listing/hunger_in_america_
2010_executive_summary. Accessed on September 4, 2011.
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report prepared for Feeding America. Mathematica Policy Research,
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form. 2008. http://www.ers.usda.gov/ briefing/foodsecurity/
surveytools/short2008.pdf. Accessed on September 4, 2011.
15. StataCorp. Stata 11. College Station, Texas. 20011.
16. Wilde PE, Nord M. The effect of food stamps on food security: A
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17. Gibson-Davis CM, Foster EM. A cautionary tale: Using propensity
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Serv Rev. 2006; 80(8):93-126.
18. Bartfeld J, Dunifon R. State-level predictors of food insecurity among
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1317-06. 2006.
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in SNAP Benefits. Economic Research Report, Number 116.
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ary.pdf. Accessed on September 15, 2011.
22. USDA. The Emergency Food Assistance Program (TEFAP).
http://www.fns.usda.gov/cga/FactSheets/TEFAP_Quick_Facts.htm.
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030209.pdf. Accessed on September 20, 2011.
North American Journal of Medicine and Science
Apr 2014 Vol 7 No.2
63
Original Research
Study of the Application of Clinical Pathways
in Varicella, Acute Bacillary Dysentery,
Measles, Scarlet Fever, and Rubella
Zhe Xu, MD, PhD;1 En-qiang Qin, MD;1 Min Zhao, MS;1* Wei-ming Nie, MS;1 Zhi-ping
Zhou, MS;1 Bo Tu, MS;1 Wei-wei Chen, PhD;1 Dan Wu, BS;1 Fei Wang, MS;2 Jin Li, MS2
1
Treatment and Research Center for Infectious Disease, 302 Hospital of PLA, Beijing, China
2
Department of medical affair, 302 Hospital of PLA, Beijing, China
In order to explore the clinical pathways that fit the actual situation of our country and department of
infectious diseases, an analysis was performed to evaluate the effectiveness of clinical pathways for
varicella, acute bacillary dysentery, measles, scarlet fever and rubella when compared with traditional
standard medical care. Using a retrospective comparative study design, varicella, acute bacillary
dysentery, measles, scarlet fever and rubella patients who were managed on a clinical pathway (clinical
pathway group) were compared with a retrospective group of patients who received traditional medical
care (control group) prior to the pathway's implementation. The following outcomes were measured:
length of hospital stay, hospitalization costs. There was a significant reduction in the median
hospitalization costs in the clinical pathway group patients in all five infectious diseases (P<0.05). The
clinical pathway group's length of hospital stay for varicella, measles, acute bacillary dysentery and
rubella were significantly shorter than the control group (P<0.05). The implementation of clinical
pathways in varicella, acute bacillary dysentery, measles, scarlet fever and rubella might contribute to
better quality of care and cost-effectiveness.
[N A J Med Sci. 2014;7(2):63-67. DOI: 10.7156/najms.2014.0702063]
Key Words: clinical pathway, varicella, acute bacillary dysentery, measles, scarlet fever, rubella
INTRODUCTION
In the face of a changing health care environment, health care
organizations must focus on improving outcomes while
considering both quality of care and cost containment.
Clinical pathways, also known as critical pathways, critical
paths, care maps, and care paths, are a popular initiative to
meet these challenges. They have gained multidisciplinary
acceptance as tools intended to reduce costs while
maintaining or improving quality of care. Such pathways
were first developed for use in the manufacturing industry to
identify and manage rate-limiting steps in production
processes. Subsequently, pathways have been developed and
used in many other areas, including medical care.1-3
Health outcomes research has previously focused on chronic
disease states and disease states that contribute significantly
to total healthcare costs such as asthma, coronary heart
disease and diabetes.4-5 In this study, we looked at the
application and utility of clinical pathways in the
management of five infectious diseases: varicella, acute
bacillary dysentery, measles, scarlet fever and rubella.
The details and results are reported as follows.
Received: 03/07/2014; Revised: 03/26/2014; Accepted: 03/29/2014
*Corresponding Author: Treatment and Research Center for Infectious
Disease, 302 Hospital of PLA, Beijing 100039, China. Tel: 86-01066933421. Fax: 86-010-66933402. (Email: [email protected])
METHODS
General Data
This study was conducted at the Treatment and Research
Center for Infectious Disease of 302 hospital of PLA. Using a
retrospective comparative study design, varicella, acute
bacillary dysentery, measles, scarlet fever and rubella
inpatients who were managed via clinical pathways from
April 2010 to January 2012 (clinical pathway group) were
compared with a group of patients who received traditional
medical care from January 2009 to March 2010 (control
group) prior to the pathway's implementation. This study was
conducted in accordance with the declaration of Helsinki and
approval from the Ethics Committee of 302 Hospital of PLA.
Written informed consent was obtained from all participants.
The diagnostic criteria for varicella, acute bacillary
dysentery, measles, scarlet fever and rubella were in
accordance with Practice of Infectious Diseases, 3rd edition
(People's Medical Publishing House) and are listed as
follows:

Acute bacillary dysentery: epidemiological data
(ate food or drank water contaminated with the bacteria);
clinical features (acute onset of fever and diarrhea,
abdominal pain, frequent passage of blood and mucus,
tenesmus, tenderness of left lower quadrant abdomen);
isolated shigellae from feces by bacterial culture.
64




Apr 2014 Vol 7 No.2
Rubella: acute onset of generalized maculopapular rash,
fever, arthralgia, arthritis, lymphadenopathy, or
conjunctivitis; epidemiological exposure to a laboratoryconfirmed case of rubella; positive serologic test for
rubella
immunoglobulin
M
(IgM)
antibody
determination.
Measles: history of fever for at least three days with at
least one of the three C's (cough, coryza, conjunctivitis);
typical measles rash or Koplik's spots; serologic
positivity for measles IgM antibodies.
Varicella: typical early "prodromal" symptoms;
characteristic rash;
positive
serologic
test
for
for varicella IgM antibodies.
Scarlet fever: (1) fever, sore throat, characteristic rash;
(2) contact
history with scarlet
fever or
pharyngitis/angina
patient;
(3)
marked
leukocytosis with neutrophilia and
conserved
or
increased eosinophils; (4) positive throat culture of
group A β-hemolytic streptococcus.
overnight cost, daily charges and relevant medicine prices
have not been adjusted in our Hospital. Monitoring and
specific treatment modalities were recorded as follows:


Methods
A clinical pathway management team was established to
create and strictly implement clinical pathway charts which
define the process of medical treatment and nursing
requirements for varicella, acute bacillary dysentery, measles,
scarlet fever and rubella. Hospitalization education was the
first part for patients of clinical pathway group when they
were hospitalized. The hospital education provides a variety
of oral verbal suggestions and written instructions that help
normalize hospital stays for patients. Informed consent is
obtained using a short form consent process. Medical history
taking, physical examination, higher authority physician's
ward round and explanation of clinical pathway contents
came next. Data with variation were recorded. The patients
were withdrawn from clinical pathway management when
negative variation happened.
The team consisted of a chief physician, a deputy chief
physician, two responsible physicians, a head nurse and a
responsible advanced nurse practitioner. Responsible
physicians were in charge of executing medical treatment
pathways and the nursing staff was in charge of
implementing clinical and nursing pathways. The chief
physician and the head nurse supervised the quality of
treatment protocols and nursing, including complication
observation, basic nursing and health education.
A clinical pathway chart included the following 10 aspects:
medical treatment measures, estimation of the severity of the
disease as well as patient’s sex and age, examinations and
assays, activities (requirement for inpatients: bed rest, avoid
exercise and strenuous exercise), treatment and nursing, diet,
education, monitoring, discharge planning, and medical care
results.
Compared to traditional medical care methods, the
management team removed vitamin preparations and
immunomodulators like thymosin and other medicines which
lacked EBM to prove beneficial to the treatment but were
applied in the past. From January 2009 until now, the
North American Journal of Medicine and Science


Varicella clinical pathway group: intravenous drip of
acyclovir at 5 mg/kg/time, q8h; liver protecting therapy
for hepatic dysfunction via intravenous administration of
150 mg diammonium glycyrrhizinate once daily for
adults and an appropriate dose reduction for children;
antipyretics like paracetamol were considered when the
temperature reached 38.5o C or above. Monitoring and
testing utilized routine examination of blood, urine and
stool samples, liver function tests and IgM anti-varicellazoster virus serology.
Measles clinical pathway group: radix isatidis granules
10g twice daily for adults and an appropriate dose
reduction in children; a half dose of antipyretic like
paracetamol was considered when the temperature
reached 38.5o C or above; multiple doses of compound
glycyrrhiza oral solution were given to patients who
suffered from severe cough, symptomatic aerosol
inhalation was provided: chymotrypsin 400U,
hydrocortisone 25 mg and normal saline 30ml were
divided into three doses; fluid infusions included ORS or
intravenous fluids and 3g smecta three times daily for
patients with diarrhea and dehydration (defined by no
less than five episodes of diarrhea accompanied by dry
mouth and hypourocrinia), and an appropriate dose
reduction in children; eyedrops of rifampicin
wereadministered several times for increased eye
secretions and congestion; liver protecting therapy for
hepatic dysfunction via intravenous administration of
150 mg diammonium glycyrrhizinate once daily for
adults and an appropriate dose reduction for children.
Monitoring and testing utilized routine examination of
blood, urine and stool samples, function tests of liver and
kidney, IgM anti-measles virus serology, and PA chest
x-ray.
Rubella clinical pathway group: radix isatidis granules
10g twice daily for adults and an appropriate dose
reduction in children; antipyretics like paracetamol
were considered when the temperature reached 38.5o C
or above. Monitoring and testing utilized routine
examination of blood, urine and stool samples, liver
function tests and IgM anti-rubella virus serology.
Scarlet fever clinical pathway group: intramuscular
injection of penicillin (2.4 million-4.8 million U daily for
adults or 20 thousand-40 thousand U/kg daily for
children) q12h or via intravenous drip at 50 thousand200 thousand U/kg q8h for 10 days, erythromycin was
used as an alternative therapy in patients allergic to
penicillin; antipyretics like paracetamol were considered
when the temperature reached 38.5o C or above.
Monitoring and testing utilized routine examination of
blood, urine and stool samples, and throat swab culture.
Discharge medication: a 10 day course of amoxicillin
dispersible tablets at 0.5-1g three to four times daily for
adults, or 50-100mg/kg three to four times daily for
children; erythromycin was used as an alternative
therapy in patients allergic to penicillin: a 10 day course
North American Journal of Medicine and Science

Apr 2014 Vol 7 No.2
at 1.6g daily divided into two to four oral doses for
adults, or 15-25mg/kg divided into two oral doses for
children.
Acute bacillary dysentery clinical pathway group:
intravenous drip of levofloxacin 0.2g twice daily for
adults, or oral calcium fosfomycin tablets 50-100mg/kg
divided into three to four daily doses for patients under
the age of 18; alternatives included ceftriaxone 5080mg/kg daily by intramuscular injection, or
levofloxacin hydrochloride and sodium chloride 0.2-0.3g
twice daily by intramuscular injection for 3 days.
Antipyretics like paracetamol were considered when the
temperature reached 38.5o C or above.
Discharge standards are listed as follows: varicella normothermia, scabbed over rash, no emerging rash; measles
- normothermia, deflorescence, quarantine period expiring;
rubella - normothermia, deflorescence, expired isolation;
scarlet fever - normothermia, deflorescence, 3 consecutively
negative throat swab cultures; acute bacillary dysentery normothermia, symptom resolution, 2 consecutively negative
stool cultures.
Evaluation criterion: (1) average length of hospital stay:
number of overnights; (2) average hospitalization costs:
diagnosis and treatment expenses during hospitalization.
65
Evaluation results are presented by comparing the length of
hospital stay and hospitalization costs between clinical
pathway groups and control groups.
Statistics Analysis
The statistical software SPSS 13.0 was used for data
processing. Hospitalization costs data managed by the
normality test did not fit a normal distribution, thus they were
expressed as a median (quartile range), Md (QR); those data
fitting a normal distribution were expressed as X  S
[mean±standard deviation]. The difference between both
groups was managed by a nonparametric rank-sum test, the
Mann-Whitney test. The comparison between clinical
pathway and control groups' data fitting a normal distribution
went through an independent-samples t-test with P < 0.05
defined as statistically significant.
RESULTS
Baseline Characteristic and Variances
There were 506 inpatients involved and the baseline
characteristics of each disease are presented in Table 1.
There was no significant difference in the findings for
gender, age, illness severity, admission day of illness course
and other general characteristics between clinical pathway
groups and control groups, so these data were comparable.
Table 1. The baseline characteristics of clinical pathway and control groups for each disease.
Disease
Clinical
pathway group
201
Control group
Varicella
Case load
Average age (years old, X  S )
Case load
20.30±7.36
19.23±6.38
Measles
75
104
17.44±14.26
15.76±13.90
Rubella
Average age (years old, X  S )
Case load
24
35
Average age (years old, X  S )
Case load
21.95±6.99
20.02±5.54
73
6.59±3.20
133
23.63±19.28
Scarlet
fever
Acute bacillary
dysentery
Average age (years old, X  S )
Case load
Average age (years old, X  S )
As shown in Table 1, the exclusion rates of each infectious
disease are all less than 20%. There were 21 records excluded
from 201 cases of the varicella clinical pathway group: 8
with basic diseases (1 patient with cerebral palsy, 1 with
medulloblastoma, 1 with severe anemia, 1 with favism, 3
with lymphoma and 1 with leukemia); 8 with complications
(2 patients with liver injury, 3 with bronchopneumonia, 1
with electrolyte disturbance, 1 with urinary tract infection
and 1 with EBV infection); 3 patients asked for early
discharge; and 2 patients with final diagnosis of nonvaricella.
There were 13 records excluded from 75 cases of the measles
clinical pathway group: 1 with basic disease (pre-
104
Exclusion number of
clinical pathway group
21
Exclusion rate
10.45%
13
17.33%
2
8.33%
39
6.38±3.84
4
5.48%
80
25.73±18.37
15
11.28%
hospitalization moderate skin scalding); 11 with
complications (6 pneumonia cases, 4 liver damage and 1
fungal infection); and 1 asked for early discharge.
There were 2 records excluded from 24 cases of the rubella
clinical pathway group: 1 with psoriasis and 1 with drug
eruption.
There were 4 records excluded from 73 cases of the scarlet
fever clinical pathway group: 1 with electrolyte disturbance
and bronchopneumonia, 1 with a basic disease (indirect
inguinal hernia) and insufficient course of treatment, 2 asked
for early discharge, and 1 with paronychia (staphylococcus
aureus infection).
Apr 2014 Vol 7 No.2
64
There were 15 records excluded from 133 cases of the acute
bacillary dysentery clinical pathway group: 5 with basic
diseases (1 with coronary heart disease, 1 with coronary heart
disease and uncontrollable diabetes mellitus, 1 with
hypertention and diabetes mellitus, 1 with severe anemia, and
1 with hepatic hemangioma); 4 with moderate to severe
electrolyte disturbances; 6 asked for early discharge with an
insufficient course of treatment but obviously improved
condition.
North American Journal of Medicine and Science
Hospitalization Costs
As shown in Table 2, the hospitalization costs of varicella,
acute bacillary dysentery, measles, scarlet fever and rubella
clinical pathway groups were significantly less than the
control group (P<0.05). Among the 5 diseases,
hospitalization costs of the varicella group had the largest
decrease by over 50%, from 2,072.77 yuan to 941.20 yuan.
Table 2. Comparison of the costs between the clinical pathway and control groups for each disease (yuan) M d (QR).
Disease
Clinical pathway group
Control group
Z
P
Varicella
941.20(467.53)
2072.77(1857.24)
-8.744
0.000
Measles
760.49(379.17)
1244.99(902.84)
-6.530
0.000
Average Length of Hospital Stay
As shown in Table 3, the average length of hospital stay of
varicella, measles, rubella, scarlet fever and acute bacillary
dysentery clinical pathway groups were less than the control
group. The clinical pathway management of varicella, for
example, resulted in a decrease in the average length of
Rubella
512.16(451.69)
882.83(617.33)
-3.082
0.002
Scarlet fever
853.91(330.55)
1206.60(780.98)
-5.370
0.000
Acute bacillary dysentery
677.49(443.82)
784.78(696.04)
-2.179
0.029
hospital stay from 7.28 days to 5.98 days. The comparison
between the 2 groups for varicella, measles, rubella, and
acute bacillary dysentery was statistically significant
(P<0.05). While there was a decrease in average length of
hospital stay from 5.15 days to 4.97 days for scarlet fever, it
did not reach statistical significance (P > 0.05).
Table 3. Comparison of the length of hospital stay between the clinical pathway and control groups for each disease (Χ±S, days).
Disease
Clinical pathway group
Control group
t
P
Varicella
5.98±1.82
7.28±2.17
5.233
0.000
Measles
3.87±1.54
5.38±2.24
4.509
0.001
DISCUSSION
For a long time, public hospitals in China have been harshly
criticized for their random prescriptions, high prices and
inadequate medical resources. Doctors were wrongly
encouraged to prescribe expensive or unnecessary drugs to
patients, from which hospitals usually sought profits. People
still bear a relatively heavy burden in covering their medical
fees for infectious diseases. Treating infectious diseases
efficiently and effectively is an essential criterion in gauging
a country's healthcare level. Thus, it is an important task to
find ways to improve management of medical treatment
quality and provide better medical service in infectious
diseases with lower prices and higher quality. 6-10
Clinical pathways are an important means in the modern
medical management, which gives consideration to both
quality and efficiency. However, there is little literature
available regarding the clinical pathway management in
infectious diseases.11-15
Through this study, the implementation of clinical pathways
in varicella, measles, rubella, scarlet fever and acute bacillary
dysentery strongly suggests their application is of great
practical significance as evidenced by the decrease in
hospital costs and average length of stay.
In our study, medical staff were motivated to offer quality
service and dispense prescriptions more reasonably, which
reduced patients' burden. Except for the scarlet fever group
Rubella
3.36±1.22
4.57±1.58
3.025
0.004
Scarlet fever
4.97±1.50
5.15±1.90
0.517
0.607
Acute bacillary dysentery
3.34±1.22
3.84±1.31
2.342
0.020
the median hospitalization costs and length of hospital stay
were reduced significantly with clinical pathway
management.
Healthcare costs decreased and the quality of infectious
disease treatment improved. The reduction in the median
hospitalization costs in this study was statistically significant.
The implementation of clinical pathways resulted in higher
quality, higher efficiencey and a lower budget by properly
allocatinge medical resources and controlling medical
expenses appropriately. Clinical pathway management
regulates medical behaviors and reduces unnecessary
examinations and treatment.16-19 Additionally, they help
avoid excessive medical spending, which may effectively
curb the practice of hospitals' relying on drug sales or
medical instrumentation for income. In doing so, they
establish a reasonable, effective and optimized medical
service system.
As soon as patients were admitted to hospital, they were told
about their treatment plan, including what to do for
examination and treatment, what to expect, how long the
treatment would be, and how much it would cost. This
explanation provided transparency of the medical process to
the patients. Patients also received a version of "clinical
pathways for patients" everyday, which lists anticipated
hospital stay, examination programs, responsibility of
medical personnel, costs and other relevant details. This
enhanced patients' participation and degree of satisfaction.
North American Journal of Medicine and Science
Apr 2014 Vol 7 No.2
The communication and cooperation between doctors, nurses
and medical technicians is of strategic importance for highquality patient care and for creating a positive work
environment for all health care professionals. In our study,
the medical processes in the clinical pathway management
groups were defined clearly, the communication and
cooperation between medical and other staff were
strengthened, and team spirit was improved. These factors
resulted in improved time management, work efficiency, and
helped alleviate bed shortages in our hospital via increasing
bed turnover. It also created a cohesive team approach which
helped defuse the difficulty of hospitalization.
Clinical pathway management may also be a way to improve
the departmental management in hospitals. In our study, we
noticed that clinical pathway management appeared to
decrease unnecessary differences of medical care, decrease
the incidence of technical accidents and play a positive role
in medical training. Similarly, implementation of clinical
pathway management, specifically a defined team approach,
may
decrease
medical negligence
or
even
medical malpractice resulting from differences in individual
medical staff ability.
The overall purpose of this study was to improve outcomes
of infectious disease entities by providing clinical pathway
management to coordinate care, reduce fragmentation and
ultimately costs. Our results demonstrate that it is possible to
achieve this goal. Compared with traditional standard
medical care, our study suggests the implementation of
clinical pathways will contribute to better quality of care and
cost-effectiveness. Although controversial elements still
exist, we think that clinical pathways can have a positive
impact on the quality in infectious disease care. 20-25
ACKNOWLEDGEMENT
The work in this paper was supported by grants from the National Grand
Program on Key Infectious Disease (2012ZX10004301 and
2013ZX10004203).
CONFLICT OF INTEREST
The authors have no conflict of interest to disclose.
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Apr 2014 Vol 7 No.2
68
North American Journal of Medicine and Science
Original Research
False-Negative Interpretation of Breast Sentinel
Lymph Node Touch Preps: Analysis of the Causes
with Suggestions to Improve Diagnostic Accuracy
Frank Chen, MD, PhD, MBA;1 David Hicks, MD;2 Maria Nava, MD;2 Richard Cheney, MD2
1
Department of Pathology, Buffalo General Medical Center, State University of New York, Buffalo, NY
2
Department of Pathology, Roswell Park Cancer Institute, Buffalo, NY
Sentinel lymph node (SLN) biopsy has become widely accepted as an important procedure in staging
breast cancer. False-negative results of touch prep (TP) examination at time of SLN biopsy requires
additional surgery, delaying treatment and increasing cost. Therefore, we have analyzed our experience
with false-negative interpretation on SLN TP’s. Eight-hundred and three consecutive SLN biopsies from
2003 to 2005 were obtained from the pathology archive of Roswell Park Cancer Institute. The
intraoperative consultation results were correlated with the final diagnoses.
Twenty-five SLN
intraoperative consultations had false-negative TP’s [false-negative rate = 3.1% (25/803), including 9
metastatic lobular carcinomas and 16 metastatic ductal carcinomas]. These cases were re-evaluated by 3
pathologists independently, and the metastases in the SLN sections were confirmed by positive cytokeratin
staining. Size of the metastatic focus, nuclear grade and the adequacy of TP’s were analyzed with regard
to the cause of false-negative results. On re-screening of TP’s, we found that rare tumor cells of low
nuclear grade were identified on 28% (7/25) of the TP’s (3 metastatic lobular carcinomas and 4 metastatic
ductal carcinomas). In the remaining 72% (18/25) of TP’s, re-screening revealed no evidence of tumor.
Evaluation of these TP’s demonstrated that 50% (9/18) were unsatisfactory for evaluation or limited by
scant cellularity. While cases that remained negative on re-screening tended to have smaller measured foci
of tumor in the SLN (Average 0.65 mm vs. 0.94 mm from cases that were positive on re-screening), there
was considerable overlap between these two groups. In conclusion, TP’s with scant cellularity,
unsatisfactory TP’s and failure to identify tumor cells with low nuclear grade were found to significantly
contribute to false-negative interpretations. We suggest that an additional TP or frozen section may be
necessary if the cellularity of the initial TP is limited. Correlation with the original core biopsy may be of
value to help in identifying cancer cells of low nuclear grade.
[N A J Med Sci. 2014;7(2):68-71. DOI: 10.7156/najms.2014.0702068]
Key Words: sentinel lymph node, breast cancer, false-negative interpretation
INTRODUCTION
A sentinel lymph node (SLN) is the first lymph node to
receive afferent lymphatic drainage from the primary tumor.
SLN biopsy examination is the current modality for
evaluating the axilla in breast cancer patients. 1-4 Numerous
studies have demonstrated that SLN biopsy can determine
axillary nodal status for breast cancer, predicting the risk of
additional nodal metastases.1,3-6 This procedure not only
allows the surgeon to make an individualized decision
regarding the need for completion axillary lymph node
dissection, but also permits it to be performed during the
same mastectomy procedure if metastatic tumor is found.2,7
However, intraoperative diagnostic techniques such as touch
prep examination, often carry the risk of false-negative
results.8 In this study, we have evaluated our experience with
Received: 03/07/2014; Revised: 03/26/2014; Accepted: 03/29/2014
*Corresponding Author: Department of Pathology, Buffalo General
Medical Center, State University of New York at Buffalo, Buffalo, NY
14203. (Email: [email protected])
false-negative interpretations on cytologic examination of
sentinel lymph nodes, analyzed the possible causes and
provided suggestions to improve the diagnostic accuracy.
METHODS
Pathology reports from 803 consecutive SLN biopsies from
2003 to 2005 were obtained from the pathology archive of
Roswell Park Cancer Institute. In all of these cases, during
intraoperative consultation, the SLN’s were serially sectioned
perpendicular to the long axis and touch preps were derived
from the exposed cut surfaces. Then, the SLN’s were
formalin-fixed for permanent sections. In this study, these
intraoperative consultation results were re-evaluated
retrospectively and correlated with the final diagnoses by
three pathologists independently. The metastases in the SLN
sections were confirmed by cytokeratin staining. Size of the
metastatic focus, nuclear grade and the adequacy of TP’s
were analyzed regarding the cause of false-negative results.
Apr 2014 Vol 7 No.2
North American Journal of Medicine and Science
RESULTS
We found that 25 out of 803 SLN intraoperative
consultations had false-negative interpretations, including 9
metastatic lobular carcinoma cases and 16 metastatic ductal
carcinoma cases (Table 1). The false-negative rate is 3.1%
(25/803). On re-screening, rare tumor cells of low nuclear
grade were identified on 28% (7/25) of TP’s, including 3
metastatic lobular carcinoma cases and 4 metastatic ductal
carcinoma cases. Examples of metastatic ductal carcinoma on
TP and in SLN are shown in Figure 1 and Figure 2,
69
respectively. In the remaining 72% (18/25) of TP’s, rescreening revealed no evidence of metastatic tumor.
Evaluation of these TP’s demonstrated that 50% (9/18) were
unsatisfactory for evaluation or limited by scant cellularity.
While cases that remained negative on re-screening tended to
have smaller measured foci of tumor in the SLN (Average
0.65 mm vs. 0.94 mm from cases that were positive on rescreening), there was considerable overlap between these two
groups.
Table 1. Axillary SLN with False Negative Interpretation between 01/2003-06/2005.
TP
TP Original
Dx
TP Rescreen
by A
TP Rescreen
by B
TP Rescreen
by C
1
Neg
Neg
Neg
Neg
2
Neg
Neg
Neg
Neg
3
Neg
Neg
Neg
Neg
4
Neg
Neg
Neg
Neg
5
Neg
Neg
Neg
Neg
6
Neg
Neg
Neg
Neg
7
Neg
Neg
Neg
Neg
8
Neg
Neg
Neg
Neg
9
Neg
Neg
Neg
Neg
10
Neg
Neg
Neg
Neg
11
Neg
Neg
Neg
Neg
12
Neg
Neg
Neg
Neg
13
Neg
Neg
Neg
Neg
14
Neg
Neg
Neg
Neg
15
Neg
Neg
Neg
Neg
16
Neg
Neg
Neg
Neg
17
Neg
Neg
Neg
Neg
18
Neg
Neg
Neg
Neg
19
Neg
Pos
Pos
20
Neg
Pos
Pos
Suspicious
Susp-prob
Pos
21
Neg
Pos
Pos
prob pos
22
Neg
Pos
Pos
Pos
23
Neg
Pos
Pos
Pos
24
Neg
Pos
Pos
Pos
25
Neg
Pos
Pos
Pos
Lymph Node
Dx
Micro mets
(1.0 mm)
Micro mets
(0.5 mm)
Micro mets
(1.1 mm)
Macro mets
(3.0 mm)
Micro mets
(2.0 mm)
Submicro
mets (0.1
mm)
Micro mets
(1.5 mm)
Micro mets
(0.7 mm)
Micro mets
(1.0 mm)
Micro mets
(0.3 mm)
Macro mets
(3 mm)
Submicro
mets (0.1
mm)
Micro mets
(0.3 mm)
Submicro
mets (0.1
mm)
Micro mets
(0.7 mm)
Micro mets
(2.0 mm)
Micro mets
(1.0 mm)
Micro mets
(0.2 mm)
Macro mets
(2.5 mm)
Micro mets
(1.5 mm)
Micro mets
(0.7 mm)
Submicro
mets (< 1
mm)
Micro mets
(0.9 mm)
Micro mets
(0.3 mm)
Micro mets
(0.5 mm)
Causes for
error
Limited by
SC* + DA**
Nuclear Grade
Primary Tumor Dx
II
Ductal CA
I
Ductal CA
I
Lobular CA
I
Ductal CA
I
Lobular CA
SAT#
Limited by
SC*
Limited by
SC* + DA**
Limited by
TS***
II
Lobular CA
Limited by
SC*
I
Ductal CA
II
Ductal CA
II
Ductal CA
II
Ductal CA
II
Ductal CA
SAT#
Limited by
TS***
II
Ductal CA
UNSAT##
II
Ductal CA
SAT#
II
Ductal CA
SAT#
I
Lobular CA
I
I
Lobular CA
Mixed
Ductal/Lobular CA
Mixed
Ductal/Lobular CA
UNSAT##
Limited by
SC*+ DA**
I
Tubulolobular CA
II
Ductal CA
II
Ductal CA
SAT#
Limited by
TS***
Limited by
TS***
I
Lobular CA
SAT#
I
Lobular CA
SAT#
II
Ductal CA
SAT#
I
Ductal CA
SAT#
I
*SC: Scant cellularity; **DA: Dry artifact; ***TS: Thick smear; #SAT: Satisfactory; ##UNSAT: Unsatisfactory.
SAT#
Limited by
SC*
Limited by
SC*
SAT#
SAT#
Apr 2014 Vol 7 No.2
70
Figure 1. Metastatic ductal carcinoma on touch prep.
DISCUSSION
SLN biopsy is commonly used in the evaluation of breast
cancer patients. Axillary lymph node status is considered the
most significant prognostic factor for breast cancer outcome,
and treatment decisions are based on the presence or absence
of nodal disease.1,2 According to the revised American Joint
Committee on Cancer (AJCC) staging: SLN metastases were
classified as follows;9 (1) immunohistochemistry positive if
only single keratin-positive cells or clusters were present and
were not observed with standard tissue stains; (2)
submicrometastatic if tumors were less than 0.2 mm
(excluding IHC positive); (3) micrometastatic if tumors were
larger than 0.2 mm but </=2 mm, or (4) macrometastatic if
tumors were larger than 2 mm. A previous study has found a
significantly poorer prognosis associated even with
metastases less than 2 mm in size (micro- and
submicrometastasis), suggesting that such small metastases
cannot be safely overlooked.10 In addition, Kamath et al
showed that sentinel lymph node micrometastases, regardless
of identification techniques, inferred a risk of 15.2% for non
sentinel lymph node (NSLN) involvement. As the volume of
tumor in the SLN increased, the risk of NSLN metastases
also increased.5
Touch prep is often used for intraoperative examination of
SLN’s in breast cancer. This allows axillary lymph node
dissection to be performed immediately for tumor-positive
nodes when mastectomy is the surgery of choice.2,7 However,
it has a high false-negative rate, particularly in patients with
micrometastases.5 In 2006, Puqliese et al reported that the
chances of false-negative intraoperative consultation
increased with decreasing size of the metastasis. 6 We
observed similar correlation between the size of metastatic
tumor and false-negativity. However, due to small sample
size, the correlation is not significant. We predict that future
studies with larger numbers of cases should verify the above
observation.
North American Journal of Medicine and Science
Figure 2. Metastatic ductal carcinoma in SLN.
Different methods have been tried to reduce the false
negativity rate of breast SLN biopsy. Cytokeratin
immunohistochemical staining of the breast SLN detects
micrometastatic disease, which is frequently missed on
routine H&E stain, providing more accurate staging of the
regional lymph nodes in patients with breast cancer. 11
However, the role of rapid immunohistochemistry for
cytokeratin
during
intraoperative
consultation
is
controversial. Johnston et al reported that rapid
immunohistochemistry for cytokeratin is a more sensitive
method for detecting breast cancer metastases in SLN’s than
TP’s and frozen sections.12 In contrast, Beach et al showed
that the method of rapid immunohistochemistry to detect
metastasis was the least sensitive when compared with TP’s,
frozen sections, and permanent sections. 13 Further,
Celebrioglu et al divided the metastases into micrometastases
and macrometastases, and found that the sensitivity for
detection of micrometastases was not substantially increased
by the use of intraoperative immunohistochemistry.14
Molecular techniques such as polymerase chain reaction
(PCR) offer even more sensitive methods for detecting occult
metastasis in SLN’s. However, it remains as a research tool
due to its high false positive rate.10
In this study, we have found that scant cellularity, technical
limitations (i.e. too thick, air drying) of TP’s and failure to
identify tumor cells with low nuclear grade significantly
contribute to false-negative interpretations. We suggest that
an additional TP or frozen section may be necessary if the
cellularity of the initial TP is unsatisfactory or if there are
correctable technical limitations on the initial TP.
Correlation with the original core biopsy may be of value to
help identify cancer cells of low nuclear grade.
CONFLICT OF INTEREST
The authors declare that there are no conflicts of interest.
North American Journal of Medicine and Science
Apr 2014 Vol 7 No.2
REFERENCES
1.
Bleiweiss IJ. Sentinel lymph nodes in breast cancer after 10 years:
rethinking basic principles. Lancet Oncol. 2006;7(8):686-692.
2.
Lyman GH, Temin S, Edge SB, et al. Sentinel Lymph Node Biopsy for
Patients With Early-Stage Breast Cancer: American Society of Clinical
Oncology Clinical Practice Guideline Update. J Clin Oncol.
2014;32(13):1365-1383.
3.
Guidroz JA, Johnson MT, Scott-Conner CE, De Young BR, Weigel
RJ. The use of touch preparation for the evaluation of sentinel lymph
nodes in breast cancer. Am J Surg. 2010;199(6):792-796.
4.
Vanderveen KA, Ramsamooj R, Bold RJ. A prospective, blinded trial
of touch prep analysis versus frozen section for intraoperative
evaluation of sentinel lymph nodes in breast cancer. Ann Surg Oncol.
2008;15(7):2006-2011.
5.
Kamath VJ, Giuliano R, Dauway EL, et al. Characteristics of the
sentinel lymph node in breast cancer predict further involvement of
higher-echelon nodes in the axilla: a study to evaluate the need for
complete axillary lymph node dissection. Arch Surg. 2001;136(6):688692.
6.
Pugliese MS, Kohr JR, Allison KH, Wang NP, Tickman RJ, Beatty JD.
Accuracy of intraoperative imprint cytology of sentinel lymph nodes in
breast cancer. Am J Surg. 2006;192(4):516-519.
7.
Kane JM III, Edge SB, Winston JS, Watroba N, Hurd TC.
Intraoperative pathologic evaluation of a breast cancer sentinel lymph
8.
9.
10.
11.
12.
13.
14.
71
node biopsy as a determinant for synchronous axillary lymph node
dissection. Ann Surg Oncol. 2001;8(4):361-367.
Forbes RC, Pitchford C, Simpson JF, Balch GC, Kelley MC. Selective
use of intraoperative touch prep analysis of sentinel nodes in breast
cancer. Am Surg. 2005;71(11):955-960; Discussion 961-962.
Thor A. A revised staging system for breast cancer. Breast J. 2004;10
(Suppl 1):S15-S18.
Treseler P. Pathologic examination of the sentinel lymph node: what is
the best method? Breast J. 2006;12 (5 Suppl 2):S143-S151.
Pendas S, Dauway E, Cox CE, et al. Sentinel node biopsy and
cytokeratin staining for the accurate staging of 478 breast cancer
patients. Am Surg. 1999;65(6):500-505; discussion 505-506.
Johnston EI, Beach RA, Waldrop SM, Lawson D, Cohen C. Rapid
intraoperative immunohistochemical evaluation of sentinel lymph
nodes for metastatic breast carcinoma. Appl Immunohistochem Mol
Morphol. 2006;14(1):57-62.
Beach RA, Lawson D, Waldrop SM, Cohen C. Rapid
immunohistochemistry for cytokeratin in the intraoperative evaluation
of sentinel lymph nodes for metastatic breast carcinoma. Appl
Immunohistochem Mol Morphol. 2003;11(1):45-50.
Celebioglu F, Sylvan M, Perbeck L, Bergkvist L, Frisell J.
Intraoperative sentinel lymph node examination by frozen section,
immunohistochemistry and imprint cytology during breast surgery--a
prospective study. Eur J Cancer. 2006;42(5):617-620.
Apr 2014 Vol 7 No.2
72
North American Journal of Medicine and Science
Original Research
Anti-HBV Activities of Xanthones
From Swertia Punicea Hemsl
Xiu-Qiao Zhang, PhD;1* Jia-Chun Chen, PhD;2
Feng-Jiao Huang, MS;1 Luan-Yuan Tian, PhD;1 Yuan Tu, MS1
2
1
School of Pharmaceutical Sciences, Hubei University of Chinese Medicine, Wuhan, Hubei, China
Tongji School of Pharmaceutical Sciences, Huazhong University of Science and Technology, Wuhan, Hubei, China
We studied the effects of two xanthones compounds isolated from Swertia punicea Hemsl (from
Geutianaceae), swertianolin (I) and bellidifolin (II), on Hepatitis B surface antigen (HBsAg) and e antigen
(HBeAg) in cultured human hepatocellular carcinoma cell line (HepG2). The HepG2 cells were first
cultured for 24h, various concentrations of these two xanthones were then added to the culture medium.
The culture medium containing the two xanthones was exchanged once every 4 days. After 8 days, the
cytotoxic activities of these two xanthones were assessed by cytopathic effect. The HepG2 cells were then
treated with the two compounds at a concentration of swertianolin (1.6, 3.1, 6.2, 12.5, 25g/ml) and
bellidifolin (2.0, 3.9, 7.8, 25.5, 31.2g/ml). Four or eight days later, the culture medium was collected and
the expression of HBsAg and HBeAg were determined by radioimmunoassay. Our results show that
swertianolin can suppress the expression of HBeAg with IC50 of 8.0g/ml, while bellidifolin can inhibit the
expression of HBsAg with IC50 of 13g/ml at the eighth days. The Therapeutic Index for swertianolin and
bellidifolin are 6.2 and 6.8, respectively. Our findings suggest that swertianolin and bellidifolin have antiHBV activities in vitro.
[N A J Med Sci. 2014;7(2):72-74. DOI: 10.7156/najms.2014.0702072]
Key Words: Swertia punicea Hemsl, swertianolin, bellidifolin, HepG2, HBsAg, HBeAg
INTRODUCTION
Hepatitis B is one of the most prevalent infectious diseases,
especially in Asia. It has been reported that more than 350
million people worldwide are persistent carriers of HBsAg. 1,2
Infection with hepatitis B virus (HBV) results in severe liver
diseases, including chronic hepatitis, cirrhosis and
hepatocelluer carcinoma.3 At present, interferon-α and
lamivudine are the main licensed drugs for the treatment of
chronic HBV infection. However, interferon-α is expensive
and is associated with severe side effects. Long-term
treatment with lamivudine may cause drug resistance. 4
Therefore, the development of more effective agents from
crude extracts with anti-HBV activity remains of great
importance. Swertia punicea Hemsl (from Geutianaceae) is a
traditional medicinal plant mainly used for the treatment of
hepatitis in some rural areas in China, and it has been
approved for pharmacological and clinical trials in Hubei and
Yunnan province in China. It has been reported that some of
its active components, such as oleanolic acid, mangiferin,
and swertiamarin, are useful for the treatment of liver
diseases.5-9 The HepG2 cells has been developed as a model
for screening novel agent with anti-HBV biological
activities.10,11 In this study, we reported that two active
Received 01/08/2014; Revised 04/11/2014; Accepted 04/15/2014
*
Corresponding Author: School of Pharmaceutical Sciences, Hubei
University of Chinese Medicine, Wuhan, China 430065. Tel: +86-2768890106. (Email: [email protected])
components isolated from the Chinese herb, Swertia punicea
Hemsl, suppressed HBsAg or HBeAg the expression of the
HepG2 cells. The structures of these two components were
identified as xanthones, namely, swertianolin (I) and
bellidifolin (II).
METHODS
Plant Collection and Identification
Swertia punicea Hemsl was collected at Hefeng county in
Hubei province of China and identified by Professor Jiachun
Chen, Tongji School of Pharmaceutical Sciences, Huazhong
University of Science and Technology. A voucher specimen
(No.040803) was stored in the herbarium of Hubei
University of Chinese Medicine.
Preparation of Tested Compounds
The plant materials were air-dried and ground to a fine
powder. Extraction was performed by soaking samples (500g
dry weight) in 95% ethanol (5000ml) for 24h at 25ºC. After
filtration through filter paper, the residue was washed twice
with 95% ethanol, followed by concentrating in vacuum at
40ºC. The extract was further extracted with petroleum ether
for 5 times to remove chlorophyll and subsequently
partitioned in ether, EtOAc and water. The aqueous and
EtOAc extractions were fractionated by chloroform and
methanol gradient of sequential gel column chromatograph,
respectively. The two compounds were obtained in
chloroform and methanol (90:10 and 75:25, v:v) and further
North American Journal of Medicine and Science
Apr 2014 Vol 7 No.2
purified by SephadexLH-20 column chromatography. The
structures of the two compounds were identified respectively
by comparing 1H-NMR,13C-NMR and MS data with
literature. For bioassay, the two compounds were first
dissolved in Dimethyl sulfoxide (DMSO), and then filtered
through 0.45μm filter.
Reagents and Chemicals
HBsAg and HBeAg radioimmunoassay kits were purchased
from the Chinese Isotope Co. (Beijing, China). Dulbecco’s
modified Eagle’s medium (DMEM) and L-glutamine were
obtained from Gibco Industries Inc. (Los Angeles, CA,
USA). Fetal Bovine serum (FBS) was obtained from Hyclone
(Logan, UT, USA). DMSO was obtained from Sigma
(Dorset, UK). All chemical reagents for chromatography
were of HPLC grade.
Cell Culture
HepG2 cells were obtained from the Mount Sinai School of
Medicine, USA, and were maintained in DMEM medium
supplemented with 10% FBS, 50U/ml streptomycin and 3%
L-glutamine. The cells were seeded into 96-well plates at a
density of 2.0×104 /well, and incubated in 5% CO2 at 37℃
for 24h. Various concentrations of the two xanthones were
then added to the culture medium. The medium was removed
every 4 days and fresh medium was added.
73
( )
Figure 1. The equation.
RESULTS
Screening of Active Substances
In the course of our search for natural plant products as antiHBV agents, the aqueous and EtOAc extracts of Swertia
punicea Hemsl were found to show significant anti-HBV
activity in vitro. Subsequent bioactivity fractionation resulted
in the isolation of two pure compounds as the active
compounds. The structures of these two active compounds
were identified as swertianolin (I) and bellidifolin (II),
respectively. 13-15
Cytotoxic Activity Assay
After 8 days, the viability of the cells was assesssed by
cytopathic effect. The median toxic concentration (TC50)
values were calculated according to the method of ReedMeuench.12
Determination of HBsAg and HBeAg
After the cytotoxic activity assay of these two xanthones, the
HepG2 cells were seeded into 24-well plates at a density of
1.0×105/well and allowed to attach overnight. The medium
was changed to DMEM without serum, HepG2 cells were
treated with the two compounds at a concentration of
swertianolin (1.6, 3.1, 6.2, 12.5, 25g/ml) and bellidifolin
(2.0, 3.9, 7.8, 25.5, 31.2g/ml). The medium was removed
every 4 days and fresh medium containing the two
compounds was added until the eighth day. The culture
medium of the fourth and eighth days was collected. The
HBsAg and HBeAg in culture medium, which was secreted
by HepG2 cells, was measured by a radioimmunoassay kit
according to the manufacture’s instructions (Chinese Isotope
Co.) and counted in a hemocytometer. The mean value (x) of
cycles per minute (cpm) and standard deviation (s) of both
experimental and control groups were calculated. The assays
were performed in triplicate and the results were averaged.
The antigen inhibition percentage (%) between the
experimental group and the control group, the half maximal
inhibitory concentration (IC50), and therapeutic index (TI)
were all calculated. The difference in cpm between the
experimental and control groups were calculated using the
Student’s test.
Figure 2. The structural formula of swertianolin and bellidifolin.
Cytotoxicity of the Two Compounds
We assessed the cytotoxicity of these two compounds by
cytopathic effect, and found that TC50 of swertianolin and
bellidifolin were 50μg/ml and 88μg/ml, respectively.
Suppression of HBsAg and HBeAg Production in HepG2
We assessed the effect of these two compounds on HBsAg
and HBeAg production in HepG2 cells at the fourth and
eighth days in culture. At the fourth day, IC50 of bellidifolin
and swertianolin suppressing HBsAg or HBeAg expression
of the HepG2 cells could not calculated according to the
experimental results. But the results from the eighth day
showed that bellidifolin effectively suppressed HBsAg
expression of the HepG2 cells with IC50 of 13μg/ml and TI of
6.8. Swertianolin inhibited HBeAg expression with IC50 of
8.0μg/ml and TI of 6.2. The suppression on HBsAg and
HBeAg expression of swertianolin and bellidifolin was not
due to any cytotoxic activity of these two compounds, since
the treated cells were still viable and continued to proliferate
slowly during the incubation period of 8 days (Table 1 and
Table 2).
Apr 2014 Vol 7 No.2
74
North American Journal of Medicine and Science
Table 1. Effect of swertianolin on HBsAg and HBeAg.
HBsAg
concentration
(g/ml)
1.6
3.1
6.2
12.5
25
control group
4d
cpm
(x  s)
24723541
233122552
208201486
20210989
206251196
232711710
HBeAg
8d
inhibition
ratio (%)
-6.24
-0.18
10.54
13.16
11.37
cpm
(x  s)
213482262
22976243
208212481
188951841
186842380
198052000
4d
inhibition
ratio (%)
-7.79
-16.01
-5.13
4.60
5.66
cpm
(x  s)
68051835
67252443
6256172
6176800
62231490
63631100
inhibition
ratio (%)
-6.94
-5.69
1.69
2.95
2.20
8d
cpm
(x  s)
70611058
74181029
7322867
88531168
94711748
113403474
inhibition
ratio (%)
37.74
34.58
35.43
21.94
16.48
Table 2. Effect of bellidifolin on HBsAg and HBeAg.
HBsAg
HBeAg
4d
concentration
(g/ml)
2.0
3.9
7.8
15.6
31.2
Control group
cpm
(x  s)
2785317
26391047
2769279
2704232
2112374*
2783102
8d
inhibition
ratio (%)
-0.07
5.15
0.48
2.84
24.11
cpm
(x  s)
6928302
53051008*
3341718**
3548675**
3272214**
96081650
4d
inhibition
ratio (%)
27.89
44.79
65.22
63.07
65.94
cpm
(x  s)
3681132
364727
376472
4361139
4237413
3891215
8d
inhibition
ratio (%)
5.41
6.28
3.28
-12.07
-8.89
cpm
(x  s)
5134431
5871500
4844229
52531264
59831191
63931584
inhibition
ratio (%)
19.70
8.18
24.23
17.83
6.42
**p < 0.01, *p < 0.05, compare with cell compare group,
DISCUSSION
Hepatitis B infection is a major health concern worldwide,
especially in Asia. As a consequence, there is an increasing
interest in the anti-HBV activities of natural products from
Chinese herbs. Swertia punicea Hemsl is a traditional
Chinese medicinal herb which has been used widely for
many diseases including hepatitis for a long time. In this
study, we isolated and identified two active xanthones from
Swertia punicea Hemsl, which showed significant
suppression effects on the expression of HBsAg or HBeAg in
human hepatocellular carcinoma HepG2 cells in culture.
These two xanthones were identified as swertianolin and
bellidifolin by analysis of the spectral data. Furthermore, we
show for the first time that these two natural products from
Swertia punicea Hemsl exhibit anti-HBV activities in vitro
and this property may partly explain the reported effects of
this medicinal plant in clinical application.9 Therefore, our
findings suggest that swertianolin and bellidifolin may
possess potential in the development of effective anti-HBV
drugs in the future.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
CONFLICT OF INTEREST
None.
12.
ACKNOWLEDGEMENTS
This work is supported by a grant from the National Natural Science
Foundation of China, NO: 30271590.
13.
14.
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AJ. Therapeutic strategies in the management of patients with chronic
hepatitis B virus infection. Lancet Infect Dis. 2008;8(3):167-178.
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Tan P, Liu YL, Hou CY. The structure of swertiapuniside from Swertia
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Tan P, Liu YL, Hou CY. The structure of swertiapunimarin from
Swertia punicea Hemsl. Yao Xue Xue Bao. 1993;28(7):522-525.
Liu GM, Yang YS, Dong GP. Isolation and identification of oleanolic
acid and mangiferin from Swertia punicea Hemsl. J. of DaLi Medical
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Qian JF, Liu GM, Huo M. Isolation and Identification of Xinthones
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Cai DY, Zhao G, Chen JC, et al. Therapeutic effect of Zijing capsule in
liver fibrositis rats. World J Gastroenterol. 1998;4(3):260-263.
Pang R, Tao J, Zhang S, et al. In vitro Anti-Hepatitis B Virus Effect of
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Wang WN, Yang XB, Liu HZ, Huang ZM, Wu GX. Effect of
Oenanthe javanica flavone on human and duck hepatitis B virus
infection. Acta Pharmacologica Sinica. 2005;26(5):587-592.
Lennette EH, Schmidt N. Diagnostic Procedures for Viral and
Rickettsial Infections. 5nd ed. New York, American Public Health
Association, Publishers; 1979.
Wan LS, Min QX, Wang YL, Yue YD, Chen JC. Xanthone glycoside
constituents of Swertia kouitchensis with α-glucosidase inhibitory
activity. J Nat Prod. 20013;76(7):1248-1253.
Tan GS, Xu PS, Tian HY, Xu KP, Dai ZY. Studies on the chemical
constituents of Swertia davidii. Chinese Pharmaceutical Journal.
2000;35(7):441-443.
Zhang XQ, Tian LY, Chen JC, Liu YW. Constituents from Swertia
Punicea Hemsl. Chin. Tradit. Herb Drugs. 2007;38(8):1153-1154.
North American Journal of Medicine and Science
Apr 2014 Vol 7 No.2
75
Original Research
Validity of Fine Needle Aspiration Cytology in Diagnosis
of Prostatic Lesions and Correlation with Trucut Biopsy
Judith J. Thangaiah, MD;1* Krishna Balachandran, MD;1 Usha Poothiode, MD;1 Suresh Bhat, MD2
1
Department of Pathology, Medical College Kottayam, Kerala, India
Department of Urology, Medical College Kottayam, Kerala, India
2
Prostate fine needle aspiration (FNA) is an easy-to-perform outpatient procedure requiring no expensive
equipment or anesthesia. The aim of this study was to analyze the cytomorphology of prostatic lesions and
to correlate the findings in cytology with that of the histopathological appearance. In doing so we also
assessed the diagnostic accuracy of fine needle aspiration cytology and identified possible pitfalls. The
study was carried out in 100 patients who underwent tru-cut biopsy in the department of Urology at
Kottayam Medical College, India during the period spanning from March 2010 to March 2011. Fine
needle aspiration cytology (FNAC) was done with Franzen needle and followed by tru-cut biopsy after
which the results of both were compared. FNAC gave a benign diagnosis in 64 cases and identified a
malignant pattern in 36 cases. The overall accuracy of FNAC in this series in diagnosing prostatic lesions
was 97% with a sensitivity of 100% and specificity of 95.5%. This shows that FNAC prostate is a reliable,
relatively painless tool, which can be used for the diagnosis of prostatic carcinoma, especially in patients
with high risk complications such as bleeding and infections in whom a tru-cut biopsy is more invasive. In
addition it is also cost-effective and may sample a larger area.
[N A J Med Sci. 2014;7(2):75-80. DOI: 10.7156/najms.2014.0702075]
Key Words: fine needle aspiration cytology, prostate, Franzen needle, correlation of cytology and biopsy
INTRODUCTION
Prostate cancer continues to be a major public health problem
in both industrialized and developing countries worldwide.
According to the World Health Organization, there are about
250,000 new cases of prostatic cancer every year. When
diagnosed in time, the disease has a cure rate of over 90%.
Elevated prostate specific antigen (PSA) levels suggest a
likelihood of malignant disease however such levels can
occur in benign prostatic diseases as well. Cytology and
histopathology have been the forefront of cancer detection
but how well these two correlate has been a content of
debate.
In 1930, Russell Ferguson reported that prostate cancer could
be diagnosed by transperineal fine needle aspiration (FNA);
however, it took three decades before Sixten Franzen
developed a trans-rectal approach to prostate biopsy and
applied prostate FNA to diagnostic uropathology. 1 The
development of a special instrument for prostatic aspiration
led to a painless quick method of cytologic sampling of the
prostate by trans-rectal FNA biopsy. In 1988, Benson2
recommended that prostate FNA should be encouraged as a
standard diagnostic tool that is performed by urologists,
taught to urology residents, and diagnosed by pathologists.
He mentioned that, while accuracy for cytodiagnosis was
similar to that of histopathologic diagnosis, fine-needle
Received: 03/30/2014; Revised: 04/12/2014; Accepted: 04/19/2014
*Corresponding Author: 400, Alberta Drive #2, Amherst, NY 14226.
(Email: [email protected])
aspiration was less traumatic and cost-effective compared to
more invasive histologic biopsy methods. However, some
pathologists find the core biopsies easier to interpret than
aspiration cytology, and hence underestimate the role of FNA
as a diagnostic tool. Whereas core biopsies offer the
advantage of a more precise localization of the lesions within
the target organ, FNA of the prostate offers its own unique
advantages. First, it is an outpatient procedure, well tolerated
by the patients because the discomfort and trauma from the
22-gauge needle are minimal.3,4 Second, sampling area is
larger and more representative5 than that of core biopsies.
Third, smears can be processed and interpreted rapidly.
Finally, it is accurate in experienced hands and has low risk
of complications6 and seeding of tumor cells.
METHODS
Our study was undertaken to elucidate the cytomorphological
features of prostatic lesions and explore the diagnostic
accuracy of FNA by comparing it with concurrent
histopathology. Since the procedure was recently adopted by
our institution, we standardized the technique by using
different needles. Initially we tried with intravenous needle.
It was technically easy because the needle is rigid but the
smears were bloody, obscuring the cells. Subsequently, we
used spinal needle. It was technically difficult because of the
flexibility of the needle and we could not assess the depth of
penetration. However, it gave better yield in the hands of
experts. Most recently, we started using Franzen needle
which is the recommended needle for prostatic FNAC. It was
76
Apr 2014 Vol 7 No.2
technically easy and gave high yield, and we could easily
assess the depth of penetration.
One hundred patients who were scheduled to undergo a trucut biopsy in the department of Urology during the study
period spanning from March 2010 to march 2011 were
selected for this study. Cases were chosen after proper
history-taking, physical and imaging examination (including
digital rectal examination, trans-rectal ultrasound) and
informed consent. A single dose of quinolone was given one
hour prior to the procedure. They were then subjected to
FNA by trans-rectal route with the Franzen needle before trucut biopsy. The aspiration was done by specially constructed
Franzen needle which is a 22-gauge, 20-cm in length flexible
needle that is slightly thicker and rigid in the proximal 5 cm.
The aspiration was performed with the patient in left lateral
position. Patients with inflamed hemorrhoids or anal fistula
were appropriately treated initially and received an anesthetic
jelly prior to the procedure.
The suspected area of the prostate was palpated with the
index finger of the non-dominant hand, after which the
needle was advanced into the lesion with the plunger of the
syringe down. When the needle entered the lesion, several
small amplitude to-and-fro movements of the needle were
performed to loosen the target tissue. Negative pressure was
obtained by pulling on the syringe plunger in order to
aspirate the material into the needle. Before withdrawing the
needle from the prostate, the negative pressure was released,
North American Journal of Medicine and Science
a most important step that will ensure that the aspirated
material remained in the needle and does not enter the barrel
of the syringe, where it would be irretrievably lost. If
aspiration of several areas of the prostate was needed, the
needle was withdrawn and replaced with another needle. It is
not advisable to attempt to change the direction of the needle
while being lodged in the target tissue, because of the risk of
hemorrhage and injury to the prostate. The smears were then
prepared from needle contents and processed as either airdried May-Grunwald-Giemsa (MGG)-stained or alcoholfixed Papanicolaou (PAP)-stained smears. The presence of
10-12 epithelial cell clusters were taken as adequate for
diagnosis. This was followed by the tru-cut biopsy performed
by the Urologist and was then subjected to histopathological
examination.
The smears were evaluated on the very same day the FNA
was done. The corresponding biopsy sections were studied
and reported by a different pathologist when the H&E
sections were ready after 2 days. The interpreters of the FNA
and core biopsy were blinded to each other.
RESULTS
Benign prostate lesions in smears are usually composed of
large clusters of normal, flat, non-stratified sheets of benign
epithelial cells with regular architecture and cells in honey
comb pattern (Figures 1a, 1b, 1c). Another pattern of benign
lesions is cell grouping as large multilayered plug of ductal
epithelial cells (Figure 1d).
Figure 1. Characteristic patterns of benign prostate lesions on FNA smears (MGG stain). 1a-1c. Large clusters of normal, flat, non-stratified
sheets of benign epithelial cells with regular architecture and cells in honey comb pattern in a clean background (a. magnification ×100; 1b.
magnification ×400; 1c. magnification ×400). 1d. Clusters of ductal epithelial cells with no definite outline, overlapping and mild variation in
size (magnification ×400).
North American Journal of Medicine and Science
Apr 2014 Vol 7 No.2
The cytological criteria for diagnosing prostatic carcinoma in
aspirates have been well-defined.7,8 In smears from low-grade
carcinoma (Grade I), sheets of cells in unicellular layer
resembling benign pattern under low power view and cells in
micro-adenomatous pattern can be seen (Figure 2a), and it
can be considered as a malignant lesion even without
significant nuclear polymorphism (Figures 2b and 2c). The
adequate aspiration smears in prostatic adenocarcinoma are
usually richer in cells than smears from benign conditions.
In moderately differentiated adenocarcinoma (Grade II), the
micro-adenomatous pattern is still evident but the component
77
cells are much larger (Figures 3a, 3b and 3c). However, the
smears are mainly composed of more solid groups of
malignant cells with significant nuclear polymorphism, large,
irregular nuclei and prominent nucleoli. In smears from
poorly differentiated prostatic cancers (Grade III), the
malignant cells are often dissociated and may be strikingly
polymorphic with bizarre forms and very large nuclei
(Figures 4a and 4b). In the anaplastic variant, the picture is
monotonous and may resemble the pattern of leukemia or
lymphoma. Clustering and micro-adenomatous complexes
are rare. This grading was applied to the FNA samples and
was compared with the tru-cut biopsy interpretation.
Figure 2. Grade I Adenocarcinoma. 2a. Cohesive clusters of cells in unicellular layer resembling benign pattern under low power view
(MGG stain, magnification ×100). 2b. Higher power view shows that the cells have minimal atypia (MGG stain, magnification ×400). 2c.
Some cells may show micro-adenomatous pattern (arrow, PAP stain, magnification ×1000). 2d. A concurrent tru-cut biopsy showing prostate
adenocarcinoma with a Gleason score of 2+2 (H&E stain, magnification ×400).
After data on 100 patients was collected the results were
analyzed. 64 cases were diagnosed as benign lesions and 36
were malignant as per cytology. On histological examination,
the 64 cases which were cytologically diagnosed as benign
were proven to be benign. This included 58 cases of benign
prostatic hyperplasia (BPH), 4 cases of BPH with chronic
prostatitis, and 2 cases of BPH with basal cell hyperplasia.
Of the 36 cases diagnosed cytologically as malignant, 33
cases were proven to be cancerous by histological
examination, and 3 cases were proven to be false positive
including 2 cases of basal cell hyperplasia and 1 case of
chronic prostatitis. Using Fisher’s exact test to correlate the
results of FNAC and tru-cut in diagnosing prostatic lesions,
the accuracy of FNAC was 97% with a sensitivity of 100%
and a specificity of 95.5%. The positive predictive value and
negative predictive value were 91.6% and 100%,
respectively.
Of the 33 malignant cases, 5 cases (15%) were Grade I by
cytology and correlated with Gleason score 2-5 in biopsy
(Figure 2d), 23 cases (70%) were Grade II by cytology and
correlated with Gleason score 6-8 in biopsy (Figure 3d), and
5 cases (15%) were Grade III by cytology and correlated with
Gleason score 9-10 in biopsy (Figures 4c and 4d).
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Apr 2014 Vol 7 No.2
North American Journal of Medicine and Science
Figure 3. Grade II Adenocarcinoma. 3a-3c. A case with predominantly micro-adenomatous pattern (arrows) and with solid cell clusters
(arrowhead) showing increased nuclear atypia (a. MGG stain, magnification ×100; 3b. magnification ×400; PAP stain, 3c. magnification
×1000). 3d. A concurrent tru-cut biopsy showing prostate adenocarcinoma with a Gleason’s score of 3+4 (H&E stain, ×1000 magnification).
Figure 4. Grade III Adenocarcinoma. 4a-4b. A case with mainly dissociated cells with marked atypia (MGG stain, a. magnification ×100;
4b. magnification ×400. 4c-4d. A concurrent tru-cut biopsy showing prostate adenocarcinoma with a Gleason score of 4+5 (H&E stain, 4c.
magnification ×400; 4d. magnification ×1000).
Apr 2014 Vol 7 No.2
North American Journal of Medicine and Science
79
Figure 5. Correlation of results of fine needle aspiration and tru-cut.
15%
15%
Grade 1- Gleason Score 2-5
Grade 2- Gleason score 6-8
Grade 3- Gleason Score 9-10
70%
Figure 6. Cytological Grade and correlation with Gleason score obtained by tru-cut biopsy.
DISCUSSION
Prostatic carcinoma is one of the most important causes of
mortality in elderly men mainly because of the late detection
despite of the fact that it is a potentially curable disease. As
FNA is painless, simple, low-cost, repeatable, with low risk
of complications, it can be employed to detect occult or early
prostatic carcinoma and in follow-up of confirmed cases.
However the usefulness of FNA and the robust supportive
data behind it in replacing or being an adjunct to tru-cut
biopsy is a matter of contention.
There has been numerous research supporting the use and
accuracy of FNA. In a study done by Saleh AF et al, 9 the
sensitivity was 88% and specificity was 93% with an
accuracy of 91.7%. FNA seems to be very effective in
identifying benign lesions as shown in another prospective
study by Singh et al where the accuracy for benign and
malignant lesions of prostate were 98.33% and 81.88%,
respectively.10 These facts argue that FNA could have a high
negative predictive value and be a useful tool in low
prevalence populations who may not need a more invasive
test.
By securing a larger sampling area FNA is less likely to miss
early malignant pocket of cells. Tru-cut biopsies with more
cores also carry higher rates of complications which can be
avoided by FNA. Polito M, et al showed that FNA had a
sensitivity of 98.2%, specificity of 98.1% and accuracy of
96% which is almost similar to our study. 11 A similar study
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Apr 2014 Vol 7 No.2
by Honig et al also showed that aspiration cytology of
prostate increased the incidence of finding adenocarcinoma
from 10% to 14% in patients undergoing transurethral
resection of the prostate (TURP).12 Klotz et al showed that all
patients with positive findings on aspiration also had positive
findings on core biopsy which could put the positive
predictive value for aspiration close to 100%.13 However, we
found 3 false-positive cases in our study. On further
investigation these three cases were found to be basal cell
hyperplasia (n = 2) and chronic prostatitis (n=1). In the two
cases of basal cell hyperplasia, smears showed high
cellularity with solid clusters and crowed cells with hyper
chromatic nucleus. In the case of chronic prostatitis, smears
showed inflammatory atypia with background inflammatory
cells. Though these diagnostic pitfalls afflict correct
interpretation, the high negative predictive value of prostatic
FNA is undeniable.
There is also an argument that cytological grade of FNA
prostate correlates well with the Gleason score in prostate
biopsy sections. In our study the correlation of cytological
grade with histological score was 100%. This is in
accordance to another study published by Willems et al. who
showed that cytological grading of prostatic carcinoma into
well, moderately, and poorly differentiated types had shown
significant correlation with not only to histopathological
grading, but also to clinical stage, response to hormonal
therapy and survival.7
Another research by Maksem JA showed that when
malignancies were classified as well differentiated,
moderately differentiated, or poorly differentiated, there was
84% agreement between histology and cytology.14 However
not all researchers agree on the correlation of this grading to
Gleason score. Hostetter AL showed a tendency toward
underestimation of both the extent and degree of
differentiation of the prostate carcinomas during cytological
examination,15 and Adolfsson J argued that core biopsies
were generally graded higher than fine needle aspirations.16
Since the technique of obtaining the sample and
interpretation of FNA are highly operator dependent and
relies heavily on the urologists’ and pathologists’ expertise,
the correlation of the samples may not be universally
concordant.
CONCLUSION
We conclude that FNAC prostate is a reliable tool for the
diagnosis of prostatic lesions, especially in patients with high
risk of complications, bleeding tendencies and in follow-up
of previously diagnosed cases.
The accuracy of FNAC depends largely upon the skill of the
examiner taking the cell samples and the alertness of the
North American Journal of Medicine and Science
cytopathologist for possible diagnostic pitfalls. The
procedure is quick, safe, and results are available the same
day. Several aspirations can be done even in outpatients with
minimal trauma. Complications are rare. Our results support
the use of needle aspiration as an initial diagnostic maneuver
especially in the low prevalence population.
CONFLICT OF INTEREST
The authors have no conflict of interest to disclose.
REFERENCES
1.
Franzen S, Giertz G, Zajicek J. Cytological diagnosis of prostatic
tumours by transrectal aspiration biopsy: a preliminary report. Br J
Urol. 1960;32:193-196.
2.
Benson MC. Fine-needle aspiration of the prostate. NCI monogr.
1988(7):19-24.
3.
Chodak GW, Bibbo M, Straus FH, 2nd, Wied GL. Transrectal
aspiration biopsy versus transperineal core biopsy for the diagnosis of
carcinoma of the prostate. J Urol. 1984;132(3):480-482.
4.
Maksem JA, Park CH, Johenning PW, Galang CF, Tannenbaum M.
Aspiration biopsy of the prostate gland. Urol Clin North Am.
1988;15(4):555-575.
5.
Cullmann HJ. [Current value of transrectal fine needle biopsy. High
predictive value in the diagnosis of prostate carcinoma, minimal
discomfort to the patient]. MMW Fortschr Med. 1991;109(26):518520.
6.
Andersson L, Hagmar B, Ljung BM, Skoog L. Fine needle aspiration
biopsy for diagnosis and follow-up of prostate cancer. Consensus
Conference on Diagnosis and Prognostic Parameters in Localized
Prostate Cancer. Scand J Urol Nephrol Suppl. 1994;162:43-49.
7.
Willems JS, Lowhagen T. Transrectal fine-needle aspiration biopsy for
cytologic diagnosis and grading of prostatic carcinoma. Prostate.
1981;2(4):381-395.
8.
Zattoni F, Pagano F, Rebuffi A, Costantin G. Transrectal thin-needle
aspiration biopsy of prostate: four years' experience. Urology.
1983;22(1):69-72.
9.
Saleh AF, Nahar Rahman AJ, Salam MA, Islam F. Role of fine needle
aspiration cytology (FNAC) in the diagnosis of prostatic lesions with
histologic correlation. Bangladesh Med Res Counc Bull.
2005;31(3):95-103.
10. Singh N, Shenoi UD, Raghuveer CV. FNAC and transabdominal
ultrasonography in the diagnosis of prostatomegaly. Indian J Pathol
Microbiol. 1997;40(4):473-479.
11. Polito M, Alberti R, Muzzonigro G, Baldi A, Diambrini M, Vecchi A.
Fine needle aspiration biopsy of the prostate gland: our experience
concerning 101 cases with histological follow-up. Prostate.
1990;17(2):85-94.
12. Honig SC, Stilmant MM, Klavans MS, Freedlund MC, Siroky MB.
The role of fine-needle aspiration biopsy of the prostate in staging
adenocarcinoma. Cancer. 1992;69(12):2978-2982.
13. Klotz LH, Shaw PA, Srigley JR. Transrectal fine-needle aspiration and
truecut needle biopsy of the prostate: a blinded comparison of
accuracy. Can J Surg. 1989;32(4):287-289.
14. Maksem JA, Johenning PW. Is cytology capable of adequately grading
prostate carcinoma? Matched series of 50 cases comparing cytologic
and histologic pattern diagnoses. Urology. 1988;31(5):437-444.
15. Hostetter AL, Pedersen KV, Gustafsson BL, Manson JC, Boeryd BR.
Diagnosis and localization of prostate carcinoma by fine-needle
aspiration cytology and correlation with histologic whole-organ
sections after radical prostatectomy. Am J Clin Pathol. 1990;94(6):693697.
16. Adolfsson J, Skoog L, Lowhagen T, Waisman J. Franzen transrectal
fine-needle biopsy versus ultrasound-guided transrectal core biopsy of
the prostate gland. Acta Oncol. 1991;30(2):159-160.
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Apr 2014 Vol 7 No.2
81
Case Report
Eosinophilic Esophagitis Presenting as Complete
Esophageal Desquamation: An Unusual Case of Chest Pain
Kheng-Jim Lim, MD;1* Lanjing Zhang, MD, MS;2,3 Anish Sheth, MD1,4
1
Department of Medicine, Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ
2
Department of Pathology, University Medical Center at Princeton, Plainsboro, NJ
3
Department of Pathology and Lab Medicine, Rutgers Robert Wood Johnson Medical
School and Rutgers Cancer Institute of New Jersey, New Brunswick, NJ
4
Department of Medicine, University Medical Center at Princeton, Plainsboro, NJ
The diagnosis of eosinophilic esophagitis has been steadily increasing with the increase usage of endoscopy
as a diagnostic tool. Here we present a case of complete esophageal desquamation visualized on endoscopy
without any evidence of caustic ingestion or any other potential disease process that would cause a similar
presentation. The diagnosis of eosinophilic esophagitis was established by significantly increased
intraepithelial eosinophils, eosinphilic micro-abscess and partially detached squamous epithelium on the
esophageal biopsy. There was complete resolution of symptoms with standard therapy for eosinophilic
esophagitis. To the best our knowledge, this is the first reported case in the English literature of
eosinophilic esophagitis that presents as complete esophageal desquamation.
[N A J Med Sci. 2014;7(2):81-83. DOI: 10.7156/najms.2014.0702081]
Key Words: eosinophilic esophagitis, desquamative esophagitis
INTRODUCTION
The incidence of Eosinophilic Esophagitis (EoE) has been
steadily increasing since it was first described in the late
1970’s. It is defined as “a chronic, immune/antigenmediated, esophageal disease characterized clinically by
symptoms related to esophageal dysfunction and
histologically by eosinophil-predominant inflammation.”1
Clinically these patients present with a variety of symptoms
including dysphagia, heart burn/chest pain and food
impactions.2
They would usually undergo an
esophagogastroduodenoscopy (EGD) as the next step in their
workup as they do not respond to empiric treatment with
proton pump inhibitors (PPI). Typically on endoscopy one
might see one or multiple of the following morphological
features which include rings, linear furrowing, white papules,
strictures, attenuation of the subepithelial vascular pattern
and a small caliber esophagus. These endoscopic findings
alone are not sufficient to make a diagnosis of EoE. To
confirm a diagnosis of EoE, biopsies must be taken and show
at least 15 eosinophils per high power field. We describe a
case with symptoms and histological confirmation of EoE
which presents a unique endoscopic finding mimicking
desquamative esophagus. The findings are consistent with the
endoscopic appearance of Esophagitis dissecans superficialis
(EDS).
CASE REPORT
The patient is a 43 year old female with a history of
depression who presented for epigastric/low chest pain and
Received on 03/07/2013. Revised 03/26/2014. Accepted 03/29/2014
*Corresponding Author: Internal Medicine Residency Program,
Department of Medicine, One Robert Wood Johnson Place, MEB 486, P.O.
Box 19, New Brunswick, NJ 08903-0019. Tel: 732-235-8377.
(Email: [email protected])
reduced appetite which started 3 days prior to her reduced
presentation. The pain was described as a dull discomfort
which was non-radiating, but worsened upon eating and deep
inspiration. She complained of odynophagia without any
complaints of dysphagia. She had a similar presentation 4
months earlier which lasted 4-5 days and spontaneously
resolved without any intervention. She additionally denied
nausea, vomiting and melena. However, she did endorse a
decrease in appetite. Her physical exam was unremarkable.
Due to the severity of her symptoms and with a decrease in
appetite an EGD was performed. The esophagus showed a
complete desquamation of the esophageal mucosa as well as
linear furrowing and rings. (Figure 1) Additionally, her
biopsy obtained from proximal, mid and distal esophagi
showed eosinophilic abscesses (Figure 2) and 15
eosinophils/High power field (HPF, 400x) on average,
ranging from 2 eosinophils/HPF to 40 eosinophils/HPF
(Figures 3, Figure 4) which confirmed the diagnosis of
Eosinophilic Esophagitis. The biopsy material was grossly
white-tan, and soft. The patient was started on 4 mg of
methylprednisolone which improved the complaints of
odynophagia. The patient was eventually transitioned to
swallowed fluticasone for maintenance therapy. At her 2month follow-up, the patient responded well to the
treatments, with no symptoms or signs, and unremarkable
endoscopic findings.
DISCUSSION
Although the diagnosis of EoE is fairly common the
endoscopic appearance of a completely desqamative process
is indeed a new and undocumented presentation of EoE.
Upon literature review we found that the clinical presentation
and endoscopic findings to be consistent with Esophagitis
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Apr 2014 Vol 7 No.2
North American Journal of Medicine and Science
dissecans superficialis (EDS), an endoscopic finding that is
described as sloughing of large fragment of esophageal
squamous mucosa.3 Although the pathogenesis of EDS is
unknown it has been associated with certain medications
(Bisphosphanates, NSAIDS and Potassium Chloride),
irritants ranging from hot beverages to corrosive irritants,
collagen vascular disorders and celiac disease. In the case
presented the patient was not on any medications that may
cause EDS or was there any history of ingestion of any
potential irritants. Additionally she did not have any history
suggestive of collagen vascular disorder or celiac disease.
Figures 1. Endoscopic view of the esophagus showing complete desquamation of the esophageal mucosa as well as linear furrowing and rings.
Figure 2. Eosinphilic miscro-abscess in the biopsy (400x).
Finally certain dermatological conditions with esophageal
involvement must be excluded that have similar
morphological appearance on EGD such as Pemphigus
vulgaris 4,5 and Lichen planus.6,7 Eosinophilic micro-abscess
and the presence of intraepithelial eosinophilia in the
mid esophagus are consistent with EoE. Clinically the patient
did not have systemic pemphigus and endoscopically the
lesions involved the whole esophagus while phemghigus is
typically more focal in nature.8 Additionally phemphigus
endoscopically and pathologically also shows bullae and
exfoliated erosions.5 On the other hand, lichen planus has
characteristic band-like or lichenoid lymphocytic infiltrate
and elongated rete pegs, which this case also lacks. 5 These
disease processes with esophageal involvement have been
described as being a separate entity from EDS. However, a
recent study reported that these dermatologic disorders and
others with esophageal involvement may be associated with
EDS.9 Ultimately the patient presented here did not have any
dermatological complaint or symptoms that would lead to
any diagnosis with a dermatological cause.
Figure 3. Esophageal biopsy at 200 x with H&E stain, showing
desquamation, focal eosinophilic abscess and increased eosinohpils in the
mid esophagus.
Figure 4. Esophageal biopsy at 400x with H&E stain showing 2 -40
eosinophils/HPF (Note: the micrograph only illustrates the cropped centralarea of the high power field, with only 21 of the 40 eosinophils shown here).
North American Journal of Medicine and Science
Apr 2014 Vol 7 No.2
In conclusion, this case to our best knowledge is the first
documented EoE with EDS endoscopic presentation.
Although EDS has been reported in association with certain
medications and esophageal strictures, the definitive etiology
is still unknown. The significance of this case report is that
clinicians should keep in mind to include the diagnosis of
EoE in the differential diagnoses of EDS. Given the
increasing diagnosis of EoE and the more frequent use of
EGD as a tool for diagnosing numerous upper
gastrointestinal pathologies, our case may represent the first
of many future presentations of EDS with a confirmed
diagnosis of EoE. Our patient had an excellent response to
steroid treatment. Our report also suggests that consideration
may be given to the cases with similar endoscopic
characteristics and biopsies, ideally from proximal, mid and
distal esophagi, must be taken for pathological confirmation.
CONFLICT OF INTEREST
The authors have no conflict to interest to disclose.
83
REFERENCES
1.
Liacouras CA, Furtua GT, Hirano I, et al. Eosinophilic esophagitis:
updated consensus recommendations for children and adults. J Allergy
Clin Immunol. 2011;128(1):3-20.
2.
Shahzad G, Mustacchia P, Frieri M. Role of mucosal inflammation in
eosinophilic esophagitis: Review of the literature. ISRN
Gastroenterology. 2011;2011:468073.
3.
Matunaga Y, Goto A, Fujii K, et al. Desquamative esophagitis due to
pemphigus vulgaris. Endoscopy. 2010;2(7):252-256.
4.
Fukuchi M, Otake S, Naitoh H, et al. A case of exfoliative esophagitis
with pemphigus vulgaris. Dis Esophagus. 2011;24(3):23-25.
5.
Chandan VS, Murray JA, Abraham SC. Esophageal lichen planus.
Arch Pathol Lab Med. 2008;132(6):1026-1029.
6.
Westbrook R, Riley S. Esophageal Lichen Planus: Case Report and
Literature Review. Dysphagia. 2008;23(3):331-334.
7.
Khamays I, Eliakim R. Esophageal pemphigus vulgaris: a rare
manifestation revisited. Gastroenterol Hepatol (NY). 2008;4(1):71-72.
8.
Kanbay M, Selcuk H, Gur G, et al. Involvement of the esophagus in a
patient with pemphigus vulgaris who was on immunosuppressive
therapy. J Natl Med Assoc. 2006;98(8):1369-1370.
9.
Hokama A, Yamamoto Y, Taira K, et al. Esophagitis dissecans
superficialis and autoimmune bullous dermatoses: A review. World J
Gastrointest Endosc. 2010;2(7):252-256.
Apr 2014 Vol 7 No.2
84
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Case Report
Furosemide Induced Bullous Pemphigoid
Associated with Antihistone Antibodies
Matthew F. Helm, BS;1* Lin Lin, MD, PhD;2 Peter Santalucia, MD;¹
Brummitte Dale Wilson, MD;¹RW Plunkett, PhD;2 Raminder Grover, MD2
1
B.D. Wilson & Associates Dermatology Center, SUNY at Buffalo, Buffalo, NY
2
Department of Dermatology, SUNY at Buffalo, Buffalo, NY
An 81 year old man developed tense blisters on his abdomen and thighs several months after starting oral
furosemide. Routine histologic studies revealed subepidermal bullae filled with eosinophils and neutrophils
typical of bullous pemphigoid. Direct immunofluoresence studies revealed weak linear deposits of IgG and
trace C3 along the dermal-epidermal junction along with a striking in vivo ANA reaction. ELISA studies
to BP180 and BP230 antigens were negative although a low titer of IgG4 was noted in the blister roof on
1M NaCl split skin. A homogenous pattern of ANA on Hep2 cells was detected at a titer of > 5120.
Antibodies to histone were very high when detected with ELISA. The clinical and pathologic findings are
consistent with drug induced bullous pemphigoid. The associated drug induced lupus erythematosus-like
immunopathologic findings are unusual and illustrate the broad range of changes that may occur.
Furosemide induced bullous pemphigoid and the significance of antihistone antibodies in drug induced
autoimmune disease will be reviewed.
[N A J Med Sci. 2014;7(2):84-86. DOI: 10.7156/najms.2014.0702084]
Key Words: bullous pemhigoid, drug reaction, furosemide, antihistone antibodies, antinuclear antibodies
INTRODUCTION
Cutaneous drug reactions are common and may present with
a wide variety of primary lesions ranging from erythematous
macules to extensive desquamation. As many as 11.6% of
patients in a medical intensive care may develop a cutaneous
drug reaction during the course of their hospitalization.1
Bullous and vesicular drug reactions are uncommon but well
recognized manifestations of drug reactions. Some blistering
eruptions are associated with well defined immunopathologic
changes. Vancomycin induced linear IgA disease is perhaps
one of the most widely recognized examples.2 Drug-induced
lupus also may have cutaneous involvement but has a lower
incidence of cutaneous involvement when compared to
idiopathic lupus erythematosus.3,4 When cutaneous lesions
occur in the setting of drug induced lupus, they are usually
comprised of erythematous macules, papules, and
papulosquamous lesions.
Characteristic serologic and
immunopathologic findings such as the presence of
anihistone antibodies and the presence of anti-ssDNA
antibodies aid in diagnosis.3,4,5
Bullous pemphigoid is an acquired subepidermal blistering
disease typically affecting older individuals that is associated
with antibodies directed at hemidesmosomal antigens BP230
Received: 03/07/2014; Revised: 03/26/2014; Accepted: 03/29/2014
*Corresponding Author: Buffalo Medical Group, Department of
Dermatopathology, 6225 Sheridan Drive, Ste. 208, Bldg. B, Williamsville,
NY 14221. Tel: 716-630-2582. Fax: 716-630-2594.
(Email: [email protected])
(BPag1) and BP180 (BPag2).6 Routine histologic evaluation
reveals a subepidermal blister filled with eosinophils and
neutrophils. Direct immunofluorescence (DIF) highlights
IgG, C3, and other immune components in a linear pattern
along
the
dermal-epidermal
junction.6
Indirect
immunofluorescence (IIF) on 1 M NaCl split skin reveals
immunoreactants on the blister roof.7
Drug-induced bullous pemphigoid was first reported in
association with salicylazosulfapyridine.8 Many drugs are
now known to be associated with bullous pemphigoid, and
furosemide is one of the most commonly encountered
culprits (Table 1).8-15 Antibodies are typically directed at the
same antigens associated with idiopathic bullous pemphigoid.
Table 1. Drugs that may induce Bullous Pemphigoid.
Adalimumab
Ampicillin
Bumetanide
Celecoxib
Ciprofloxacin
Enoxaparin
Furosemide
Iodine
Lisinopril
Penicillin
Psoralen UVA phototherapy (PUVA)
Serratiopeptidase
Terbinafine
Valsartan
Amoxicillin
Anti-TNF therapy
Captopril
Chloroquine
Enalapril
Fluorouracil
Ibuprofen
Levofloxacin
Penicillamine
Phenacetin
Salicylazosulfapyridine
Spironolactone
Tiobtarit
North American Journal of Medicine and Science
Apr 2014 Vol 7 No.2
85
Figure 2. Biopsy reveals a subepidermal bulla filled with
eosinophils. A few necrotic keratinocytes are noted and
numerous eosinophils are evident in the blister cavity
(Hematoxylin and eosin stained sections; original
magnification 200x).
Figure 1. Tense bullae are noted in the groin area.
Laboratory evaluation revealed a positive antinuclear
antibody (ANA) on HEp2 cells with a titer of > 5120 in a
homogenous pattern (Figure 4). Mitotics were noted.
Antibodies to histone were positive at 11.8 units on ELISA
testing, but evaluation for antibodies to Ro (SSA) and La
(SSB) were negative. Indirect immunofluorescence on
monkey esophagus substrate revealed an ANA titer of > 1:80.
Intercellular antibodies were negative.
CASE REPORT
An 81 year old man presented for evaluation of an
erythematous blistering eruption. His history was remarkable
for diabetes, atrial fibrillation, cardiomyopathy, chronic
obstructive pulmonary disease, chronic lymphocytic
leukemia, pacemaker placement, and hip replacement
surgery. His medications included warfarin sodium, losartan,
digoxin, levothyroxine sodium, finasteride, tiotropine
bromide inhaler, metformin, pantoprazole, atorvastatin,
doxazosin, a daily multivamin, iron supplementation, and
furosemide. He appeared ill, but was in no acute distress. He
complained of pruritic lesions on the scrotum, penis, and
chest. He had used nystatin cream for the lesions in the
genital area without benefit. His height was 5’7” and his
weight 77.7 kg. He was sent for dermatologic consultation
where vesicles and bullae were noted on the chest, thighs,
and groin area (Figure 1). Biopsy for routine histologic
studies revealed a subepidermal blister with eosinophilia in a
pattern typical of bullous pemphigoid (Figure 2). Therapy
with oral prednisone at a dose of 30 mg daily was initiated in
addition to epsom salt soaks.
Direct immunofluorescence revealed linear deposits of weak
IgG and trace C3 at the dermal-epidermal junction as well as
an in vivo ANA reaction, but a negative LE band test.
Indirect immunofluorescence for IgG4 revealed antibodies
binding to the epidermal roof of 1 M NaCl salt split skin
(Figure 3). The dermal floor of the salt split skin was
negative. Antibodies to BP180 were measured on an ELISA
unit of 5.8. Antibodies to BP230 had a level of 8.1. Both of
these were negative according to established controls in our
laboratory.
Figure 3. IgG4 split skin: Indirect immunofluorescence test for
IgG4 antibodies on 1.0 M NaCl split skin revealing reactions
with the epidermal roof. (original magnification 200x).
The clinical appearance of tense bullae on an erythematous
base supported a diagnosis of bullous pemphigoid. Treatment
with prednisone 30 mg daily for 6 days, and then 20 mg daily
thereafter was associated with improvement.
Apr 2014 Vol 7 No.2
86
North American Journal of Medicine and Science
methylation of gene promoters can impact how genes are
transcribed.17
The significance of the antihistone antibodies in our case is
uncertain. The recent finding that the deacteylase inhibitor
vironostat can be used to treat BP17 indicates that the role of
histone deacetylation and methylation of gene promoters may
offer important insights into the pathogenesis of BP. The
unusual findings noted indicate that additional immunologic
changes may be occurring in some cases and that further
study is needed to understand their significance.
CONFLICT OF INTEREST
There were no funding sources for this project.
Figure 4. ANA test on HEp2 cells revealing a homogeneous
pattern associated with mitotics. (original magnification 200x).
DISCUSSION
Drug induced bullous pemphigoid is associated with a variety
of
medications
(Table
1).
Although
direct
immunofluorescence findings are identical to those of
idiopathic BP, routine histology may reveal a few subtle
clues such as necrotic keratinocytes and increased
intraepidermal vesicle formation when compared to the
findings encountered in idiopathic BP.16 The clinical course
may vary. Some patients have their lesions resolve quickly
after removal of the offending drug, whereas others have a
protracted course that mimics idiopathic BP. Our patient
showed good response to treatment and discontinuation of
furosemide.
The striking ANA and high titer of antihistone antibodies
noted in our patient are unusual.
Although the
immunopathologic findings raise the possibility that our
patient might have drug induced lupus, his clinical picture,
clinical course, and findings noted on routine histologic
examination all indicate that he is best classified as having
drug induced BP. Drug induced lupus is not only associated
with antihistone antibodies and a positive ANA, typical
cutaneous changes may be noted. Cutaneous findings may
include leukocytoclastic vasculitis as well as a typical
distribution of papulosquamous or annular lesions.4 Drugs
metabolized by acetylation have been most closely associated
with drug induced lupus erythematosus.4
Histone
deacetylase inhibitors show promise as a treatment for lupus
erythematosus.17
The presence of IgG4 antibodies in our case is interesting in
the context of recent studies that illustrate type VII collagen
antibodies present in a wide variety of autoimmune
conditions.18 Although drug induced bullous pemphigoid has
typically been thought to exhibit identical immunopathologic
findings with idiopathic BP, drug reactions are often complex
and can be associated with varied autoimmune phenomena.
Drug induced lupus (DIL) is more common in women,
affects older individuals, and exhibits a predilection for
African Americans.4 Although ANA are often detected,
ANA in DIL are much less likely to display complement
fixing activity.19 Epigenetic changes may play a role in how
drugs impact the immune system. Histone acetylation and
REFERENCES
1.
Campos-Fernandez M, Ponce-Deleon-Rosales S, Archer-Dubon C,
Orozco-Topete R. Incidence and risk factors for cutaneous adverse
drug reactions in an intensive care unit. Revista de Investigacion
Clinica. 2005;57(6):770-774.
2.
Kuechle MK1, Stegemeir E, Maynard B, Gibson LE, Leiferman KM,
Peters MS. Drug-induced linear IgA bullous dermatosis: report of six
cases and review of the literature. J Am Acad Dermatol. 1994;30(2 Pt
1):187-192.
3.
Antonov D, Kazandjieva J, Etugov D, Gospodinov D, Tsankov N.
Drug-induced lupus erythematosus. Clin Dermatol. 2004;22(2):157166.
4.
Borchers AT, Keen CL, Gershwin ME. Drug-induced lupus. Ann N Y
Acad Sci. 2007;1108:166-182.
5.
Burlingame RW, Rubin RL. Drug-induced antihistone autoantibodies
display two patterns of reactivity with substructures of chromatin. J
Clin Invest. 1991;88(1):80-90.
6.
Lee JJ, Downham TF II. Furosemide-induced bullous pemphigoid:
case report and review of literature. J Drugs Dermatol. 2006;5(6):562564.
7.
Smith EP, Taylor TB, Meyer LJ, Zone JJ. Antigen identification in
drug-induced bullous pemphigoid. J Am Acad Dermatol. 1993;29(5 Pt
2):879-882.
8.
Bean F, Good RA, Windorst DB. Bullous pemphigoid in an eleven
year old boy. Arch Dermatol. 1970;102(2):205-208.
9.
Fellner MJ, Katz JM. Occurrence of bullous pemphigoid after
furosemide therapy. Arch Dermatol. 1976;112(1):75-77.
10. Ma HJ, Hu R, Jia CY, Yang Y, Song LJ. Case of drug-induced bullous
pemphigoid by levofloxacin. J Dermatol. 2012;39(12):1086-1087.
11. Kimyai-Asadi A, Usman A, Nousari HC. Ciprofloxacin-induced
bullous pemphigoid. J Am Acad Dermatol. 2000;42(5 Pt 1):847.
12. Dyson SW, Lin C, Jaworsky C. Enoxaparin sodium-induced bullous
pemphigoid-like eruption: a report of 2 cases. J Am Acad Dermatol.
2004;51(1):141-142.
13. Kalinska-Bienias A, Rogozinski TT, Wozniak K, Kowalewski C. Can
pemphigoid be provoked by lisinopril? Br J Dermatol.
2006;155(4):854-855.
14. Stausbol-Gron B, Deleuran M, Sommer Hansen E, Kragballe K.
Development of bullous pemphigoid during treatment of psoriasis with
adalimumab. Clin Exp Dermatol. 2009;34(7):e285-e286.
15. Femiano F. Mucocutaneous bullous pemphigoid induced by valsartan.
A clinical case. Minerva Stomatol. 2003;52(4):187-190.
16. Alcalay J, David M, Ingber A, Hazaz B, Sandbank M. Bullous
pemphigoid mimicking bullous erythema multiforme: an untoward side
effect of penicillins. J Am Acad Dermatol. 1988;18(2 Pt 1):345-349.
17. Gardner JM, Evans KG, Goldstein S, Kim EJ, Vittorio CC, Rook AH.
Vorinostat for the treatment of bullous pemphigoid in the setting of
advanced, refractory cutaneous T-cell lymphoma. Arch Dermatol.
2009;145(9):985-988.
18. Licarete E, Ganz S, Recknagel MJ, et al. Prevalence of collagen VIIspecific autoantibodies in patients with autoimmune and inflammatory
diseases. BMC Immunology. 2012:13:16.
19. Rubin RL, Teodorescu M, Beutner EH, Plunkett RW. Complementfixing properties of antinuclear antibodies distinguish drug-induced
lupus from systemic lupus erythematosus. Lupus. 2004;13(4):249-256.
20. Vaissiere T, sawan C, Herceg Z. Epigenetic interplay between histone
modifications and DNA methylation in gene silencing. Mutat Res.
2008;659(1-2):40-48.
North American Journal of Medicine and Science
Apr 2014 Vol 7 No.2
87
Review
Sebaceous Tumors of the Skin and Muir
Torre Syndrome – A Mini-Review
Ayesha Arshad, MD; Christopher A. D’Angelis, MD, PhD*
Veteran Affairs Medical Center, Buffalo, NY
Sebaceous glands develop along with hair follicles as part of the folliculo-sebaceous unit and are present in
all hair-bearing skin. Sebum, the product of sebaceous glands, participates in the overall barrier function
skin and its production appears in large part is regulated by hormonal levels. Common tumors derived
from sebocytes range from hyperplasia to benign neoplasms to invasive malignancies with metastatic
potential. Additionally, sebaceous neoplasms with unique clinical and histologic features are now
recognized as part of the phenotype of the autosomal dominant cancer pre-disposition syndrome, MuirTorre Syndrome. Here we review the clinico-pathologic features of benign and malignant tumor of
sebaceous origin including an overview of the current molecular basis and clinical management of MuirTorre Syndrome.
[N A J Med Sci. 2014;7(2):87-92. DOI: 10.7156/najms.2014.0702087]
Key Words: sebaceous adenoma, sebaceous carcinoma, sebaceoma, Muir-Torre Syndrome
INTRODUCTION
Sebaceous glands are comprised of lobular collections of
sebocytes which develop from and remain in close
association with hair follicles, together referred to as the
folliculo-sebaceous unit (Figure 1A). Sebum, produced by
sebaceous glands is a complex mixture of lipid compounds,
secreted into the follicular isthmus by the disintegration of
sebocytes (holocine secretion). Ultimately, sebum is carried
through the follicular infundibulum along with the hair shaft
to the skin surface where it contributes to thermoregulation
and barrier function.1 Sebaceous glands are found in all hairbearing skin with the highest concentrations present on the
face, upper chest and back. Given the close anatomic and
embryologic association between follicular and sebaceous
structures many "primary follicular" tumors display
significant sebaceous differentiation as well (e.g., nevus
sebaceous, folliculosebaceous cystic hamartoma, sebaceous
trichoepithelioma, etc). Discussion of these mixed folliculosebaceous lesions is beyond the scope of this brief review.
Herein we focus on tumors which classically are defined as
sebaceous, keeping in mind that expression of follicular
epithelial elements within sebaceous neoplasms is not
uncommon. Further, we review the histopathology and
diagnostic work-up of sebaceous tumors presenting as a
component of the micro-satellite instability cancer syndrome
Muir-Torre syndrome (MTS).
SEBACEOUS HYPERPLASIA
Sebaceous hyperplasia presents as asymptomatic subcentimeter papules, most commonly on the face or upper
*Corresponding Author: VAMC Buffalo NY, Department of Pathology.
3495 Bailey Ave, Buffalo, NY 14215.
(Email: Christopher.d’[email protected])
trunk of middle aged and older individuals.
Normal
development and function of sebaceous glands are believed
to be in large part, influenced by circulating androgen levels, 2
implicating altered androgen levels common in older
individuals as a prime pathogenic mechanism for sebaceous
hyperplasia. Although numerous in vitro studies have
demonstrated this relationship, no large scale in vivo studies
corroborating the association between androgen levels and
clinical sebaceous hyperplasia could be found in the
literature. Similarly, altered hormonal levels normally
occurring during the perinatal period are postulated to be
responsible for sebaceous hyperplasia commonly seen in
newborns.3 The clinical significance of sebaceous
hyperplasia in older individuals arises from its anatomical
and morphologic overlap with early sun-induced skin
cancers, primarily basal cell carcinoma. Although careful
inspection will often reveal characteristic features of
sebaceous hyperplasia, such as yellowish-white color and a
central dell (corresponding to a dilated follicular ostium)
occasionally, definitive distinction from carcinoma requires
biopsy. Histologically, sebaceous hyperplasia is seen as
superficial, enlarged sebaceous lobules arranged around a
dilated hair follicle. Beyond their increased volume,
hyperplastic glands appear otherwise normal (Figure 1B1C). At certain anatomic sites, sebaceous hyperplasia can
occur within non-follicular associated sebaceous glands (i.e.,
glands emptying directly to skin or mucosal surfaces)
common sites include the areola (Montgomery tubercles) and
Fordyce spots occurring on the oral-labial mucosa, penis and
genital labia. An additional interesting presentation of
sebaceous hyperplasia occurs as linearly arranged papules
along the lower neck termed juxta-clavicular beaded lines.
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Apr 2014 Vol 7 No.2
North American Journal of Medicine and Science
Figure 1. A. A Normal folliculo-sebaceous unit. Lobules of sebocytes empty into the follicular isthmus. Basal germinative sebocytes form an
inconspicuous layer at the periphery of the lobule. B. Sebaceous hyperplasia. Enlarged lobules of sebocytes form a nodule on the skin
surface. C. Sebocytes in sebaceous hyperplasia appear otherwise normal.
Figure 2. A & B. Sebaceous adenoma. Enlarged lobules of clear sebocytes and germinative cells with prominent nuclei. The overall lobular
architecture is retained. C & D. Sebaceous carcinoma. Infiltrating cells with some identifiable sebaceous differentiation are seen as clear
cells in the left side of the field in C. The sebaceous carcinoma shown in D. is so poorly differentiated that the initial impression was a
melanoma. Immunotains specific for sebaceous differentiation were positive (not shown).
North American Journal of Medicine and Science
Apr 2014 Vol 7 No.2
SEBACEOUS ADENOMA
Sebaceous adenomas are benign sebaceous neoplasms which
occur in association with MTS and as sporadic non-MTS
associated forms. The characteristic clinical and histologic
features of MTS-associated sebaceous adenomas are
discussed in depth below. Similar to sebaceous hyperplasia,
sebaceous adenomas (more so non-MTS associated forms)
occur commonly at sites with high concentrations of
sebaceous glands - the face, back and chest. In comparison
to hyperplasia, adenomas present as larger, more deep-seated
lesions, often requiring biopsy to rule out malignancy. At
low power magnification, sebaceous adenomas appear more
basophilic and cellular than hyperplasia, due to the increased
nuclear size and prominence of germinative sebocytes. In
spite of this increased cellularity, a benign well-circuscribed
lobular architecture is retained (Figure 2A-2B). Adenomas
with a high proportion of germinative cells, by convention
greater than 50%, are often referred to as sebaceomas, also
with a benign course. Beyond its distinction form carcinoma,
examination of sebaceous adenomas should include an
assessment for characteristic microscopic and clinical
features indicative of MTS association (see below).
SEBACEOUS CARCINOMA
Sebaceous carcinoma is a relatively rare cutaneous
malignancy, occurring primarily in the head neck with
distinct a predilection for the eyelid. 4 Approximately, 75% of
all sebaceous carcinomas occur on the eyelid, only basal cell
89
and squamous carcinoma occur with greater frequency on
peri-ocular skin.5 Initial studies demonstrated an overall
worse prognosis for peri-ocular sebaceous carcinomas in
comparison to other anatomic sites, however, more recent
studies have not supported this finding.4 Interestingly, studies
have not demonstrated a link between peri-ocular sebaceous
carcinoma and the development of additional visceral
malignancies. This is in contrast to the increased incidence of
MTS reported in patients with extra-ocular sebaceous
carcinoma.6,23 Clinically, sebaceous carcinomas tend to
present as slow growing nodules, often with ulceration. On
the eyelid, an early sebaceous carcinoma can be clinically
indistinguishable from a chalazion, often resulting in delay in
definitive diagnosis and treatment.
The microscopic
presentation of sebaceous carcinoma can range from invasive
lobules, to nests and even infiltrative single atypical
sebocytes (Figure 2C-2D). Upward (pagetoid) migration of
single malignant sebocytes into the overlying epidermis can
occur, similar to that seen in malignant melanoma and (extra)mammary Paget disease. In poorly differentiated sebaceous
carcinomas, recognizable clear bubbly sebocytes may be
absent, prompting misdiagnosis as other entities such as basal
cell carcinoma, merkel cell carcinoma, (extra-)mammary
Paget disease and melanoma. In such cases, specific
immunohistochemical markers with specificity for sebocytes
are necessary such as epithelial membrane antigen,
adipophilin and androgen receptor.
Figure 3. A-D. Unusual sebaceous neoplasms associated with Muir-Torre syndrome. A. Endophytic neoplasm showing features of a
keratoacanthoma and sebaceous adenoma. B-D. Well-circumscribed cystic sebaceous neoplasms.
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North American Journal of Medicine and Science
Figure 4. A - D. Immunohistochemical staining for markers of microsatellite instability. The loss of nuclear staining in neoplastic sebocytes
is strongly suggestive of MTS-related mutations. In this case loss of staining for PMS2 and MSH6 are noted; MSH2 and MLH1 staining is
preserved. This patient had several cystic sebaceous tumors of the trunk.
MUIR TORRE SYNDROME AND SEBACEOUS
NEOPLASMS
Muir Torre Syndrome (MTS) was initially described more
than four decades ago by Muir and Torre as a rare Autosomal
dominant genodermatosis with a high degree of penetrance
and variable expressivity.13 MTS is recognized clinically by
the co-incident occurrence of one or more skin tumors,
characteristically a Sebaceous neoplasm or Keratoacanthoma
and at least one internal malignancy most commonly
colorectal cancer.7,23,25 Cutaneous neoplasia more commonly
associated with MTS are sebaceous adenoma, sebaceoma and
sebaceous carcinoma, especially, when occurring in an
unusual distribution such as the trunk and extremities and as
multiple lesions.7,10,12,13,18 Keratoacanthomas, notably those
with prominent sebaceous differentiation and cystic
sebaceous neoplasms (Figure 3A-3D) are reportedly marker
lesions of MTS.13,18,23 Cystic sebaceous neoplasms in MTS
arise in deep dermis and subcutis and are composed of
convoluted glands, often with a large germinative cell
component. Cysts are usually surrounded by a thick capsule
and filled with eosinophilic material. Mature sebocytes are
only found randomly interspersed between the germinative
cells and towards the center of the lesions. Some cellular
atypia and scattered mitoses can be encountered within a
morphologic spectrum of tumors ranging from benign cystic
to proliferative cystic. Their biologic behavior is unknown
however there have been no reports of recurrences or
metastases. Complete excision of such lesions is
recommended, however further aggressive therapy is not
required. Cases where multiple lesions were erroneously
diagnosed as metastatic sebaceous carcinoma have been
reported.8,9
Muir-Torre syndrome is now regarded as a small subset of
Hereditary Non Polyposis Colorectal Cancer (HNPCC)
Syndrome and postulated as encompassing the full
phenotypic expression of HNPCC. Specific association
between Muir-Torre Syndrome and HNPCC or Lynch
Syndrome was not discovered until 1981 when Lynch et al.,
reported the occurrence of Sebaceous Tumors. 6,14,18-20,22
HNPCC is an autosomal dominant cancer pre-disposition
syndrome due to inherited (germ-line) mutations in the
mismatch repair genes. Mismatch repair proteins encoded by
genes MLH1, MSH2, MSH3, MLH3, MSH6, PMS1 and
PMS2 function in detection and repair of single base errors
that occur during DNA sequence replication particularly in
regions of repetitive DNA called microsatellites, acting
conceptually like tumor suppressor genes. 7-9,16,17,21
Microsatellites are natural and commonly repeated sequences
of DNA of 1-6 base pairs in length. These are of constant
length in a given individual but vary from person to person.
Mutations in DNA repair genes result in the accumulation of
North American Journal of Medicine and Science
Apr 2014 Vol 7 No.2
errors in the microsatellite sequences so that they become
either longer or shorter, referred to as microsatellite
instabilities (MSI). The mechanism of tumorigenesis follows
Knudson’s two hit hypothesis in which one mutant allele is
inherited and the remaining functional wild type allele
undergoes somatic mutation. The vast majority of mutations
occur in MSH2 and MLH1 genes involved in DNA mismatch
repair and less commonly MSH6, PMS1 and PMS2. 8,12,13
Loss of MLH1 is usually coupled with loss of PMS2 and loss
of MSH2 is coupled with loss of MSH 6. See Figure 4.
However loss of PMS2 or MSH6 can occur without loss of
91
MLH1 and MSH2, resulting in a weaker phenotype. 13 The
currently accepted method to evaluate for the functional
status of mismatch repair proteins within suspect sebaceous
tumors or visceral malignancies is illustrated in Figure 5.13,14
Five makers also termed Bethesda markers have been
recommended as the standard screen for assessing MSI in
tumors in patients suspected of having HNPCC Syndrome or
MTS. Detection of MSI in any two of the five MMR protein
markers is considered a positive result and indicative of a
high probability of MSI (MSI-H).13
Figure 5. Diagnostic Algorithm for patients presenting with an unselected Sebaceous Neoplasm. 13
Table 1. Screening recommendations for patients with Muir–Torre syndrome and their first-degree relatives.3,11-14,16
CA-125, carbohydrate antigen 125; CEA, carcinoembryonic antigen; CRC, colorectal cancer; FBC, full blood count; FOB, fecal
occult blood; USS, ultrasound scan.
Physical exam: Yearly including breast in women, testicular and prostate in men and laboratory tests including FBC, CA-125,
CEA, FOB, urinalysis.
Colonoscopy: Every 1–2 years from age 25 years or 5 years before the youngest age of diagnosis of CRC in family, and annually
from age 40 years.
Pelvic examination: Annually in women with transvaginal USS, and endometrial biopsy in patients with a gene mutation, from
age 25 years.
Consider prophylactic colectomy in patients with a gene mutation.
Consider gastroscopy every 1–2 years in families with history of gastric cancer.
Consider renal USS every 1–2 years in families with history of renal tract cancer.
Visceral malignancies commonly associated with MTS or
HNPCC Syndromes include colorectal, genitourinary, head
and neck, hematologic and small bowel. Additional
histologic and clinical features include increased intratumoral
and peritumoral lymphocytes, presentation at a younger age
(on average 10-11 years earlier than tumors with somatic
mutations) and a higher prevalence in males. 15,18 Both
cutaneous and visceral malignancies in patients with MTS
behave less aggressively as compared to their somatic
counterparts and are more responsive to immunomodulating
therapies.6
A vast majority of patients with multiple sebaceous
adenomas/ sebaceomas and almost all patients with cystic
sebaceous neoplasms exhibit MSI-H and are thus at risk for
an underlying inherited DNA MMR defect. MSI-H is also
caused by a somatic inactivation of both alleles of a DNA
MMR gene however, the incidence of such mutations are
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Apr 2014 Vol 7 No.2
relatively low. Reportedly at least two-thirds of MTS
patients with MSI-H sebaceous tumors harbor a germline
mutation in one of the DNA MMR genes most frequently
MSH2 (>90%) and less frequently MLH1.7-9 Sebaceous
neoplasms may precede the visceral malignancies by decades
and thus the full clinical phenotype of MTS may not be
established till the visceral malignancy appears and even then
it may go unrecognized. Patients fulfilling the Amsterdam
and Bethesda clinical criteria for MTS and HNPCC but
lacking demonstrable MMR deficiency or MSI have been
reported. Currently, the molecular mechanisms underlying
such cases are unknown.12
In Summary, patients with sebaceous neoplasms with
particular histologic features and presenting at unusual sites,
even in the absence of family history satisfying Amsterdam
or Bethesda criteria, should still be evaluated for MMR
deficiency by IHC or MSI testing so that affected patients
can be placed on appropriate cancer screening programs
based on guidelines issued by the International Collaborative
group on HNPCC or MTS (Table 1). Formal genetic
counseling for family members can be instituted and
prophylactic measures and psychologic support offered to
patients and family members. The histopathologists role in
recognizing suspect MTS-associated lesions and alerting the
appropriate healthcare personnel is thus integral in the team
approach required for managing genodermatosis.9
CONFLICT OF INTEREST
None.
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Harvard Medical School [email protected]
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