Download Lynch Syndrome Presenting as Endometrial Cancer Reviews

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Clinical Chemistry 60:1
111–121 (2014)
Lynch Syndrome Presenting as Endometrial Cancer
Laura J. Tafe,1 Eleanor R. Riggs,2 and Gregory J. Tsongalis1*
BACKGROUND: Lynch syndrome (LS) is the most common form of the hereditary colon cancer syndromes.
Because of its high prevalence, a nationwide campaign
has begun to screen all colorectal cancers for the genetic
abnormalities associated with LS.
CONTENT: Next to colorectal cancer, endometrial cancer is the most common form of malignancy found in
women with LS. Identifying individuals who harbor
the well-characterized mismatch-repair gene mutations via immunohistochemistry, microsatellite instability analysis, or direct gene sequencing is critical to
managing the LS patient and to surveillance for the
development of other associated tumor types.
SUMMARY: Although many institutions have begun
screening all colorectal tumors for LS, the evidence is
sufficient to warrant the testing of all endometrial cancers for LS as well. Various testing algorithms, along
with genetic-counseling efforts, can lead to a costefficient and beneficial screening program.
© 2013 American Association for Clinical Chemistry
In 2013, more than 1.6 million new cases of cancer will
be diagnosed in the US, and more than 500 000 individuals will succumb to their disease (1 ). Colorectal
cancer (CRC)3 and endometrial cancer (EC) represent
100 000 and 50 000 new cases, respectively, with corresponding 50 000 and 8000 cancer-related deaths (1 ).
CRC is the second leading cause of cancer-related mortality in the US (2 ). Up to 30% of CRCs are recognized
as having a familial component that can manifest as
one of the well-characterized hereditary colon cancer
susceptibility syndromes. One of these diseases, Lynch
1
Departments of Pathology and 2 Medicine, Geisel School of Medicine at Dartmouth, Hanover, NH, and Dartmouth-Hitchcock Medical Center and Norris
Cotton Cancer Center, Lebanon, NH.
* Address correspondence to this author at: Geisel School of Medicine at Dartmouth and Dartmouth-Hitchcock Medical Center, One Medical Center Dr.,
Lebanon, NH 03756. Fax 603-650-6120; e-mail gregory.j.tsongalis@
hitchcock.org.
Received June 26, 2013; accepted October 17, 2013.
Previously published online at DOI: 10.1373/clinchem.2013.206888
3
Nonstandard abbreviations: CRC, colorectal cancer; EC, endometrial cancer; LS,
Lynch syndrome; MMR, mismatch repair; MSI, microsatellite instability; MSI-H,
high MSI; FIGO, Fédération Internationale de Gynécologie et d’Obstétrique
(score); IHC, immunohistochemistry; NCCN, National Comprehensive Cancer
Network; PGM™, Ion Torrent Personal Genome Machine.
syndrome (LS), accounts for 3%–5% of all CRCs (2– 4 )
and 2%–3% of ECs (5, 6 ).
LS, also known as hereditary nonpolyposis colorectal cancer, was originally described by Warthin in
1913 in a kindred showing predisposition to cancers of
the colon, stomach, and endometrium (7 ). In 1966,
Lynch described similar findings for 2 large kindreds
(8 ). LS is an autosomal dominant disorder caused by
germ line–inactivating mutations in one of the 4 DNA
mismatch-repair (MMR) genes: MLH14 (mutL homolog 1), MSH2 (mutS homolog 2), MSH6 (mutS homolog 6), and PMS2 [postmeiotic segregation increased 2 (S. cerevisiae)] (9, 10 ). Deletion of the 3⬘ end
of the EPCAM (epithelial cell adhesion molecule) gene,
which resides upstream of MSH2, is also thought to
cause LS, through epigenetic silencing of MSH2 (3, 4 ).
Lack of or faulty MMR mechanisms can lead to increasing microsatellite instability (MSI) due to errorprone DNA replication.
CRC is the most common form of cancer seen in
LS patients, and the high prevalence has prompted universal screening of all CRCs for LS (10 ). LS is also characterized by extracolonic cancers, of which cancers of
the endometrium and ovary are the most common
(3, 10 ). Women with LS have a 40%– 60% chance of
developing EC as their first malignancy and have a
greater overall lifetime risk of developing EC than CRC
(9 –11 ). The incidence of LS in unselected populations
of EC patients is 2%–3%; however, that is possibly an
underestimate, because some populations may have
different mutation frequencies and because the clinical
importance of some mutations remains uncertain (5 ).
The frequency of mutations in MMR genes for EC is
50%– 66% in MSH2, 24%– 40% in MLH1, 10%–13%
in MSH6, and ⬍5% in PMS2 (10 ). This high prevalence, along with the implementation of universal
screening of CRCs for LS, has heightened awareness of
the consequences of identifying EC as an LS-related
cancer. We review the pathologic features, screening
approaches, and genetic-counseling aspects of LSrelated EC.
4
Human genes: MLH1, mutL homolog 1; MSH2, mutS homolog 2; MSH6, mutS
homolog 6; PMS2, postmeiotic segregation increased 2 (S. cerevisiae); EPCAM,
epithelial cell adhesion molecule; BRAF (v-raf murine sarcoma viral oncogene
homolog B; PMS2CL, PMS2 C-terminal like pseudogene.
111
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Histopathology of EC in LS
CRC in LS patients is well recognized to be associated
with several histopathologic features, including an origin in the right colon, a poorly differentiated or medullary histology with mucinous or signet ring cells,
abundant tumor-infiltrating lymphocytes, and peritumoral lymphoid follicles—the so-called Crohn-like
lymphocytic reaction (12 ). Screening algorithms based
on histologic observations (which are susceptible to intraobserver variability) and family/clinical history
alone are not sufficient, however, to identify patients
who should undergo further testing for LS. For this
reason, several organizations have recently proposed
recommendations for universal screening of CRC patients (13–16 ). In a 2005 series of 117 women with LS
and a personal history of CRC and either EC or ovarian
cancer, 14% had synchronous cancers at diagnosis. Of
the remaining patients, 51% were diagnosed first with a
gynecologic malignancy (17 ).
The histopathologic features of EC associated with
LS are less well characterized than for CRC. In 2006,
Broaddus et al. published their investigation of the
pathologic features of ECs (18 ). They compared 50
women with LS (all had confirmed mutations in either
MLH1 or MSH2) with 2 groups of patients with sporadic disease (confirmed negative MLH1 and MSH2
mutations): (a) women ⬍50 years of age (42 patients)
and (b) women of all ages with MSI-high (MSI-H) tumors secondary to MLH1 methylation (26 patients).
Ninety-four percent of the patients were carriers of
MSH2 mutations. The mean age at diagnosis for LS
patients was 46.8 years, vs. 39.9 years for the group ⬍50
years of age and 61.1 years for the MLH1-methylation
group. The majority of tumors had an endometrioid
histology, either pure or mixed, and 14% of the LS patients had nonendometrioid tumors (3 patients with
clear cell carcinoma, 3 with uterine papillary serous
carcinomas with a clear cell component, and 1 with
malignant mixed Müllerian tumor), all of which had
MSH2 mutations. Nonendometrioid tumors were very
rare in the 2 sporadic groups (2.4% and 3.8%, respectively). Compared with the other 2 groups, the MLH1methylated group was associated with a higher-grade
endometrioid tumor with lymphovascular invasion,
and 19% had the distinctive undifferentiated histology.
The 3 groups were not significantly different with respect to pathologic stage, but there was a trend toward
a more advanced stage (stage III or IV) in the groups
with sporadic disease, compared with the LS group
(18 ). One shortcoming of this study is that it did not
examine for pathogenic DNA alterations in the PMS2
and MSH6 genes; carriers of mutations in these 2 genes
might have fallen into their ⬍50-years-old category.
112 Clinical Chemistry 60:1 (2014)
Women with LS also have a 10%–12% lifetime risk
of developing ovarian cancer, and recent studies have
shown that endometriosis-associated ovarian cancers
(clear cell, endometrioid, undifferentiated, or mixed)
are more frequently associated with MMR abnormalities or are MSI-H, compared with high-grade serous
carcinomas and other histologic subtypes. Mutational
status was not confirmed in these patients, however
(17, 19 –21 ).
Several studies have attempted to characterize the
EC morphology suggestive of MMR deficiency or
the MSI-H phenotype. The host inflammatory response seems to be the most important feature. The
response includes (a) peritumoral lymphocytes, defined as lymphocytic aggregates apparent at a scanning
magnification, and (b) the presence of tumorinfiltrating lymphocytes, defined as lymphocytes
within tumor cell nests or glands (a score of ⱖ40 lymphocytes per 10 high-power fields indicates a positive
finding). An additional feature is tumor heterogeneity,
defined as 2 morphologically distinct populations of
tumor cells that constitute ⱖ10% of the tumor and are
juxtaposed but not admixed with one another. A tumor with this feature is often dedifferentiated EC,
which comprises FIGO (Fédération Internationale de
Gynécologie et d’Obstétrique) grade 1 or 2 endometrioid carcinomas and undifferentiated carcinomas
(19, 22 ). The undifferentiated histology is characterized by round or polygonal cells with vesicular nuclei
that grow in solid, discohesive sheets without a pattern
or gland formation. A variably myxoid matrix and
rhabdoid-like cells have also been described. Most undifferentiated carcinomas show focal but strong staining for cytokeratins or epithelial membrane antigen,
with cytokeratin 18 being the most frequently produced keratin. In contrast to better-differentiated
components, the undifferentiated component is usually histologically negative for estrogen receptor and
progesterone receptor (23 ). This histology is associated
with a particularly aggressive clinical course (23, 24 ).
In their study of MMR proteins produced in EC, Garg
et al. found that 7 of 8 dedifferentiated ECs occurred in
the group with abnormal immunohistochemistry
(IHC) characteristics. Of these 7 patients, 5 had associated MLH1/PMS2 abnormalities, and the other 2 patients had loss of MSH2 and MSH6 (19 ). The genotypic
and MLH1-methylation data available for these patients is limited; however, incorporating the features
discussed above into a screening algorithm might better define a target EC population for screening (25 ).
A recent report by Ryan et al. described a study of
76 women with mutation-confirmed LS and EC. Fifty
(66%) of these patients had MSH2 mutations: 18
(24%) had MLH1 mutations, and 8 (10%) had MSH6
mutations. The original histology results for 38 EC pa-
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Lynch Syndrome and Endometrial Cancer
tients were available for review. These investigators
identified a correlation between the anatomic origin of
the cancer and individuals with LS: Up to 29% of patients with ECs centered in the lower uterine segment
had LS (26 ). In another study, the actual incidence of
involvement of the lower uterine segment in LS patients was 5.3% (27 ). The study of Ryan et al. found
that 76% of the tumors were low-grade (grade 1 or 2)
endometrioid carcinomas. The remaining tumors were
of high grade (2 dedifferentiated carcinomas, 2 serous
carcinomas, 1 clear cell carcinoma, and 4 mixed/other
carcinomas, including malignant mixed Müllerian tumor). The morphologic review also focused on evaluating 4 features associated with MSI-H, including
lower uterine segment involvement, tumor heterogeneity (dedifferentiation), peritumoral lymphocytes,
and ⬎40 tumor-infiltrating lymphocytes per 10 highpower fields. Only 42% of tumors had ⱖ1 of these features without any significant difference in genotype,
suggesting that although these features may be helpful
for identifying patients with MSI-H, they are not specific for LS (26 ).
Screening for LS in EC
National guidelines have been created for both IHC
and MSI testing of CRC tumors to screen for potential
cases of LS (28 ). The main rationale for screening is the
opportunity for timely initiation of risk-appropriate
screening and prevention interventions for at-risk patients and their families. Historically, the revised
Bethesda criteria have been the most frequently used
guidelines, with the major indication for tumor testing
being CRC diagnosis before 50 years of age. The National Comprehensive Cancer Network (NCCN)
guidelines also support the use of IHC and MSI testing
for LS in individuals who have CRC diagnosed before
50 years of age (29 ). Although MSI is characteristic of
LS, it can also occur in up to 15% of sporadic CRCs.
The reported diagnostic sensitivity of MSI is 89% for
defective MLH1/MSH2 genes and 77% for MSH6 (28 ).
The NCCN practice guidelines recommend screening for LS in EC patients ⱕ50 years of age, but because of the high prevalence of EC in patients with
LS, including those older than 50 years, many institutions have adopted the practice of screening all
ECs for the genetic abnormalities associated with LS
(5, 30 ). More than 90% of LS-associated EC patients
show MSI.
IHC SCREENING FOR LS IN EC
The MMR proteins form heterodimers as part of the
base-excision repair complex, with MLH1 pairing with
PMS2 and MSH2 pairing with MSH6. Therefore, loss
of MLH1 expression is almost always coupled with loss
Table 1. Genetic defect in one of the 4 MMR genes
and the corresponding IHC staining patterns
expected.a
Gene with defect
IHC staining pattern
MLH1
MLH1 ⫺/⫹,b PMS2 ⫺
PMS2
MLH1 ⫹/⫺, PMS2 ⫺
MSH2
MSH2 ⫺, MSH6 ⫺
MSH6
MSH2 ⫹, MSH6 ⫺
a
Germ line mutation— or, in the case of MLH1, gene silencing by somatic
promoter hypermethylation in addition to germ line mutation—leads to
loss of the protein.
b
Occasionally, interpretation of IHC analysis can be problematic; abnormal
methylation or some MLH1 mutations may produce false-normal MLH1
IHC staining [Umar et al. (28 )].
of PMS2, and loss of MSH2 expression is accompanied
by MSH6 loss (Table 1) (31 ). For the IHC studies,
complete loss of expression of these genes in the setting
of a positive internal control (often stromal cells, lymphocytes, or nonneoplastic endometrium) is interpreted as a positive result (Fig. 1). IHC analysis for
MMR proteins is a convenient test and is a diagnostically sensitive and specific method for detecting MSI.
Modica et al. showed that testing with a panel of 4 antibodies (to MLH1, MSH2, MSH6, and PMS2) has a
91% diagnostic sensitivity and an 83% specificity for
detecting MSI-H (32 ). In addition, the staining pattern
is very useful, because it can guide subsequent genetic
testing for specific MMR gene sequencing or MHL1methylation studies.
MSI SCREENING FOR LS IN EC
MMR is one of the postreplication DNA-repair systems that correct errors made by the polymerase during DNA replication (33 ). Base– base mismatches and
insertion/deletion loops created by polymerase slippage within sequence repeats (microsatellites) are repaired by this MMR system. Lack of proper MMR
function leads to the accumulation of mutations and
genomic instability that can manifest as MSI. MSI refers to the altered length of somatic oligonucleotide
repeat sequences (short tandem repeats of up to 6 bp)
within the genome. MSI can be useful as a surrogate
marker for defective MMR when the presence of MSI
in the tumor suggests an MMR defect. MSI is detected
as either increases or decreases in the number of repeats
in these short tandem repeat sequences (29 ).
The standard method for detecting MSI is to use
the PCR and fluorescently labeled primers to amplify
microsatellite repeat sequences in samples of tumor
and normal tissues from the same patient. After the
PCR, fragment size is typically analyzed with capillary
Clinical Chemistry 60:1 (2014) 113
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A
B
C
D
Fig. 1. Immunohistochemistry of the 4 MMR proteins: MLH1, MSH2, MSH6, and PMS2.
This tumor displays loss of nuclear staining of MSH6 (A), whereas staining is retained for MSH2 (B), MLH1 (C), and PMS2 (D)
(magnification, ⫻20).
electrophoresis to identify repeat-sequence PCR products of different sizes from tumor tissue, which is accomplished by comparing the results with those for
normal tissue. The detection in tumor tissue of fragments of additional sizes, which are more often smaller
than the unmutated alleles, suggests defective MMR
(Fig. 2) (28 ).
A panel of 5 microsatellite markers had originally
been recommended in 1997 by a National Cancer Institute workshop and was embedded in the Bethesda
guidelines. This panel consisted of 2 mononucleotide
repeats (BAT-25, BAT-26) and 3 dinucleotide repeats
(D2S123, D5S346, D17S250) (34 ). Several other panels
that have since been described include the moresensitive mononucleotide markers (35 ). Many laboratories currently use a commercially available panel
(Promega Corporation) that includes 5 mononucleotide markers (BAT-25, BAT-26, MONO-27, NR-21,
NR-24) and 2 pentanucleotide markers (Penta C,
114 Clinical Chemistry 60:1 (2014)
Penta D) to identify the tumor and healthy tissue as
arising from the same individual. If instability in ⱖ2 of
the 5 markers is detected, the tumor is classified as
MSI-H. Instability in only 1 of the 5 markers is considered MSI low, and instability in none of the markers is
considered MSI stable. The newest panel contains
quasimonomorphic mononucleotide repeats with an
increased diagnostic sensitivity, which allows for the
detection of MSI in tumor tissue without the need for
testing healthy tissue; however, this practice has not
been widely adopted (36, 37 ).
If only a single modality is to be chosen, there are 2
main reasons why screening with IHC might be preferred to MSI testing. First, MSH6 mutations may not
show MSI-H; therefore, these cases would not be
flagged for additional testing. Of 6 patients with MSH6
mutations in a study by Hampel et al., 1 was MSI stable,
2 were MSI low, and 3 were MSI-H (5 ). This result is
particularly relevant, because the likelihood of an EC
Lynch Syndrome and Endometrial Cancer
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Fig. 2. In MSI testing, control genomic DNA from a blood sample is compared with genomic DNA from a patient’s
tumor.
Seven MSI markers are evaluated in this assay (5 mononucleotides on the left and 2 pentanucleotides on the right); the arrow
indicates markers that show instability when comparing the 2 samples. MSI at ⱖ2 of the 5 mononucleotide markers indicates
MSI-H.
patient having an MSH6 mutation is 5-fold higher than
that of a CRC patient and because the lifetime risk for
EC may be as high as 70% in patients with MSH6 mutations. Additionally, these patients often have an older
age at presentation (5, 38 ). The second reason is that
although in CRC a somatic BRAF (v-raf murine sarcoma viral oncogene homolog B) p.V600E mutation is
present in about 64% of sporadic tumors with a loss of
MLH1 expression as detected by IHC (likely due to
MHL1 methylation), a similar test is not currently
available for LS-associated EC (39 ). Djordjevic et al.
recently showed that positive IHC results for PTEN in
nonendometrioid (but not endometrioid) carcinomas
is associated with a high likelihood of intact MMR (as
detected by IHC staining), suggesting that PTEN IHC
positivity combined with histology data may also become clinically useful for detecting sporadic nonendometrioid EC (40 ).
MLH1-METHYLATION TESTING
MSI associated with LS is caused mainly by inherited
mutations in MMR genes, but somatic hypermethyl-
ation of the MLH1 promoter also leads to MSI in sporadic tumors. MLH1 methylation is found in about
15%–30% of all ECs (75% of MSI-H cases) (25 ). The
majority of these tumors will also test negative for
MLH1 and PMS2 by IHC analysis, a result that is indicative of a silenced MLH1 gene. Testing for MLH1
promoter hypermethylation can be used to distinguish
sporadic tumors from those that are likely to be associated with LS and require additional gene sequencing.
Methylation occurs on cytosine residues of CpG
islands in the promoter region of the MLH1 gene. Promoter methylation is one form of epigenetic gene regulation. The methyl groups on the cytosine residues
protrude into the major groove of the DNA molecule
and sterically hinder the ability of transcription factors
to anneal to the DNA, thereby blocking DNA transcription. Methylation also attracts methyl-binding
proteins, which cause chromatin compaction and gene
silencing (41 ).
The majority of methods for MLH1-methylation
analysis begin with the conversion of unmethylated cytosine to uracil by sodium bisulfite. The methylated
Clinical Chemistry 60:1 (2014) 115
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cytosine is left intact and can be detected with a variety
of methods. One common method is the MethyLight
assay, a quantitative real-time PCR assay for DNA
methylation. A patient’s sample is amplified with specific primers for MLH1 and a control gene and then
compared with a methylated control sample to determine the percent methylation (42 ). Additional detection techniques include methylation-specific multiplex
ligation-dependent probe amplification, methylationspecific melt curve analysis, pyrosequencing, and
methylation-specific PCR followed by capillary electrophoresis (40, 42 ). A positive MLH1-methylation result indicates MLH1 silencing via sporadic promoter
hypermethylation.
DNA SEQUENCING FOR SCREENING OF LS IN EC
Current testing practices in the setting of LS include
screening assays that use IHC and MSI analysis, which
may suggest the presence of a germ line mutation.
These assays are typically followed by Sanger sequencing of the MMR genes to identify specific mutations.
This type of testing algorithm has been developed because of the high cost of routine germ line screening via
Sanger sequencing.
When used in combination, these former assays
can be effective in identifying probable LS-related tumors. These tests have certain limitations, however,
that make alternative testing modalities desirable. IHC
and MSI testing have a demonstrated false-negative
rate of 5%–10%, and MSI is present in about 10% of all
CRCs (22, 30 ). IHC analysis can be limited by variation
in staining patterns, which can lead not only to uncertain interpretation but also to decreased efficacy with
small biopsy samples, for which staining may be difficult to visualize. MLH1 gene expression can also be
problematic for IHC testing, because ⬎33% of mutations in this gene are missense mutations that encode
proteins that are both functionally inactive and antigenically intact (therefore producing a normal staining
pattern). Finally, neither of these tests is capable of
identifying the specific germ line mutation; thus, additional testing is required for diagnostic and counseling
purposes.
Typical genetic analysis of the MMR genes would
consist of single-gene (Sanger) sequencing to identify
potential mutations. As a screening approach, Sanger
sequencing would not be cost-effective or timely for
identifying MMR gene mutations. On the other hand,
massively parallel sequencing or next-generation sequencing has great potential as a diagnostic or screening assay for directly identifying MMR gene mutations
(43 ). Use of this new molecular technique allows simultaneous sequencing of multiple genes and multiple
patient samples to identify mutations of interest (44 ).
116 Clinical Chemistry 60:1 (2014)
Whereas Sanger sequencing was previously reserved as a final confirmatory step in the testing protocol, but owing to its cost and long turnaround time, the
advent of next-generation sequencing technology has
made it possible and affordable to sequence all of the
LS-causative genes in parallel. It is also possible to bar
code and sequence multiple patient samples simultaneously, which would further reduce the costs and time
necessary to deliver actionable clinical data (43 ).
As more laboratories move testing to nextgeneration sequencing platforms, we will experience a
substantial increase in throughput and a decrease in
costs compared with traditional Sanger-sequencing
methods. Using instruments such as the Illumina
MiSeq and the Ion Torrent Personal Genome Machine
(PGM™), laboratories have already designed and
implemented next-generation sequencing assays for
the MMR genes, which have led to a cost- and
performance-competitive analysis scheme (43 ). We
have developed a next-generation sequencing assay
that uses the Ion Torrent PGM. The design of a custom
panel allows the greatest possible coverage of the region
to be sequenced. PGM custom panels can cover up to 1
Mb of genomic targets, and amplicons can be optimized for formalin-fixed paraffin-embedded samples.
The primers for these types of sequencing reactions can
be padded to increase the coverage of the exon regions
for the gene(s) of interest. Our assay design produced
an MMR panel consisting of 2 primer pools that produce 85 and 87 amplicons. This design approach allowed us to achieve a predicted coverage of the coding
regions for the 5 MMR genes of ⬎92%. Our ability to
sequence all of the MMR genes of interest and to combine bar-coded patient samples into a single sequencing run makes it conceivable for such an assay to replace much of the IHC and MSI testing in LS.
Genetic Counseling and Hereditary
Cancer Syndromes
Approximately 2.5% of all ECs are due to LS (35 ). LS is
inherited in an autosomal dominant fashion, meaning
that an individual who has this condition has a 50%
chance of passing it on to each of their offspring. In
addition, most individuals with LS have inherited the
condition from a parent (45 ). Families with LS, as with
other hereditary cancer syndromes, typically exhibit
the following key features: (a) an early age of cancer
diagnosis, (b) an autosomal dominant inheritance pattern of the cancer, (c) multiple relatives within 1 generation affected with similar types of cancers, (d) an
excess of multiple primary cancers, (e) the presence of
other nonmalignant features, and (f) the occurrence of
rare cancers (45 ).
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Lynch Syndrome and Endometrial Cancer
The process of genetic counseling and risk assessment for LS uses a variety of tools to help identify
whether an individual is at high risk. These tools include pedigree (family history) analysis, risk modeling,
genetic testing, biochemical testing, imaging, and, in
some cases, evaluation of physical features (46 ). If a
patient is identified as an appropriate candidate for genetic testing, the testing process is discussed with the
patient in greater depth. Such discussions include a review of the possible test results, as well as other issues:
the risk of uncovering a genetic variant of unknown
clinical significance; the risks, benefits, and limitations
of testing; the implications for medical management
based on the possible test results; and the potential psychosocial implications of testing (45 ). Ultimately, all of
the information gathered, including the results of genetic testing if pursued, is combined to develop a personalized management plan for cancer screening, risk
reduction, and prevention (46 ). Furthermore, when an
individual is found to have LS, the identification and
notification of at-risk relatives often becomes an integral extension of the genetic-counseling process.
Multiple publications have detailed when to refer patients for genetic counseling in the context of hereditary
cancer syndromes. Regarding LS specifically, a brief overview of appropriate referrals includes (a) individuals
meeting the revised Bethesda criteria, (b) individuals
from a family meeting the Amsterdam criteria, (c)
individuals with ⬎10 adenomas, or (d) individuals
from a family with a known LS mutation. The 2012
NCCN guidelines (47 ) provide a more comprehensive overview of appropriate referral indications.
PREVENTIVE SCREENING
The main cancer risks for women with LS include a
40%– 80% risk for colon cancer, a 25%– 60% risk for
EC, and a 10%–12% risk for ovarian cancer (48 –51 ). A
smaller— but still increased—risk of urinary tract,
small bowel, stomach, biliary tract, and brain tumors
also exists (48 –50 ). For ECs specifically, the median
age of diagnosis is approximately 48 – 62 years of age
(51, 52 ).
Screening for the gynecologic malignancies associated with LS is less well established than the screening
recommendations for colon cancer. A few published
studies have investigated screening for EC in LS patients, but the results have been conflicting. One study
showed that annual transvaginal ultrasound followed
by endometrial biopsy in the setting of an abnormal
ultrasound result detected 3 premalignant lesions;
however, this method failed to detect an actual EC that
was ultimately detected from symptoms (53 ). A second
study showed that a transvaginal ultrasound examination every 1 to 2 years failed to detect 2 ECs (54 ). In
contrast, a Finnish study showed that the use of endo-
metrial biopsy and transvaginal ultrasound together
detected EC better than transvaginal ultrasound alone.
Eleven of 14 ECs were diagnosed via endometrial biopsy, compared with 8 of 14 ECs diagnosed via ultrasound alone (55 ). Because many early-stage ECs present with such symptoms as abnormal vaginal bleeding,
however, it is still not clear whether heightened surveillance ultimately improves cancer detection and survival (56 ). The NCCN Clinical Practice Guidelines in
Oncology (47 ) recommend that female LS carriers be
educated about the signs and symptoms of EC, as well
as the importance of seeking prompt medical attention
should any of these symptoms present.
Several groups have proposed possible ECscreening protocols for LS carriers that could include
the following: referral to a gynecologic oncologist for
screening, total hysterectomy and bilateral salpingooophorectomy, transvaginal ultrasound beginning at
30 –35 years of age, and endometrial biopsy, if indicated (47, 57–59 ).
With regard to the other cancers associated with
LS, increased surveillance via colonoscopy screening
and subsequent removal of polyps have been shown to
be effective in reducing the incidence of colon cancer in
carriers (47, 56 ). The effectiveness of screening for the
extracolonic cancers has yet to be determined, however. Information on screening for both the colon and
extracolonic cancers associated with LS is available
(47, 57, 60 ).
GENETIC-COUNSELING COMPLEXITIES
Historically, genetic testing for LS has been based on
the fulfillment of specific clinical criteria. The original
clinical guidelines, known as the Amsterdam criteria,
were proposed by the International Collaborative
Group on HNPCC in 1991. These guidelines were subsequently revised to the Amsterdam II criteria (61 ).
Although they were the first guidelines to define LS
with the hope of determining the underlying genetic
etiology, they had low diagnostic sensitivity and limited
applicability in clinical practice. Further delineation of
the clinical criteria by the National Cancer Institute
produced the Bethesda guidelines, which helped establish which patients should pursue tumor testing via
MSI analysis (28 ). Not all individuals with LS will fulfill
these criteria, however, and the guidelines are somewhat cumbersome to use in clinical practice (62 ). That
is particularly true for carriers of PMS2 mutations, because the penetrance and lifetime cancer risks associated with mutations in this gene are much lower than
the cancer risks associated with the MLH1 and MSH2
genes (63 ).
Recently, there has been a push toward universal
screening via tumor testing for LS, because the vast
majority of these cancers exhibit MMR deficiency
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Blue line indicates paent-reported cancer history at inial genec-counseling session
Red line indicates paent-reported cancer history aer gathering more family history informaon
Black line indicates no cancer history
Colon cancer
Endometrial cancer
Colon polyps
Non-LS cancer
Fig. 3. A 3-generation pedigree for the family of a 64-year-old woman who was referred to our Familial Cancer
Program because of a history of EC.
and thus can be detected via either MSI or IHC approaches (discussed above) (64 ). Furthermore, the
current clinical guidelines (both Revised Bethesda
and Amsterdam II) will miss a substantial number of
LS cases (65 ).
The following case report illustrates some of the
abovementioned complexities associated with genetic
counseling for LS. It also addresses an additional issue
unique to PMS2—the PMS2CL (PMS2 C-terminal like
pseudogene) pseudogene (66 ).
A 64-year-old woman was referred to our Familial
Cancer Program because of a history of EC, diagnosed
at 61 years of age. She also had a history of basal cell
carcinoma, which was diagnosed at age 60 years. A
3-generation pedigree was obtained at her initial
genetic-counseling visit; however, she noted that she
did not have much information regarding her family
history of cancer and that she had a very large extended family (Fig. 3). The family history was notable for the following: a sister with cervical cancer,
diagnosed at age 28 years; a brother with basal cell
118 Clinical Chemistry 60:1 (2014)
carcinoma, diagnosed at age 58 years; and a nephew
with colon cancer, diagnosed at age 38 years. The
maternal family history was notable for the following: a maternal uncle with liver cancer, diagnosed at
age 76 years; 2 maternal aunts with skin cancer, diagnosed at ⬎70 years of age; a maternal aunt with
lung cancer, diagnosed in her late 30s; and a maternal great-grandmother diagnosed with uterine cancer in her 40s. The paternal family history was notable for the following: her father, who was diagnosed
with skin cancer at age 90 years; a paternal aunt,
diagnosed with leukemia in her 30s; and a paternal
grandmother, who was diagnosed with colon cancer
in her 50s and breast cancer at age 59 years.
On review of this pedigree, the patient’s family did
not meet the Amsterdam II criteria, because there was
no Lynch-associated cancer in her father’s generation.
Our patient did not fulfill any of the revised Bethesda
guidelines either, but her nephew did. At the time of
her initial visit, no additional testing was indicated;
however, after learning about LS and the importance of
Reviews
Lynch Syndrome and Endometrial Cancer
an accurate family history, the patient became very motivated and reached out to family members for any relevant cancer history information that she lacked. The
patient recontacted the genetic counselor after obtaining a much more comprehensive paternal family history. The following additional information was revealed: 2 sisters with multiple adenomatous colon
polyps; a paternal uncle with colon cancer, diagnosed
around the age of 70 years; a paternal aunt with colon
cancer, diagnosed in her late 60s/early 70s; a second
paternal aunt with colon cancer, diagnosed in her late
60s/early 70s; a paternal aunt with a history of colon
polyps; her paternal grandfather with colon and stomach cancer, diagnosed at approximately 70 years of age;
and finally, a paternal first cousin with colon cancer,
diagnosed at 61 years.
The additional family history markedly changed
the interpretation of this patient’s risk of being an LS
carrier. Indeed, her family now fulfilled the Amsterdam II criteria, and further testing was very much warranted. An IHC analysis of her EC sample revealed
complete loss of the PMS2 protein. Her blood sample
was then sent out for PMS2 gene sequencing and deletion/duplication analysis. This testing was performed
at a CLIA-approved commercial laboratory in the fall
of 2010. The initial results of the testing were inconclusive. After speaking with the laboratory, we determined
that the patient likely had a pathogenic mutation
within the PMS2 gene, specifically deletion of exons 14
and 15; however, this part of her testing had been done
in the research section of the laboratory. The laboratory was currently in the process of clinically validating
the methodology, which would be able to detect such
large deletions within PMS2; therefore, the laboratory
could issue only an inconclusive result. Part of the issue
with the clinical validation stemmed from the fact that
this region of the PMS2 gene contains the PMS2CL
pseudogene, which complicates an accurate analysis.
After an additional 9 months, the patient’s report was
amended to show that she carried a deleterious mutation in the PMS2 gene (i.e., deletion of exons 14 and
15), as expected. Clinical validation of this mutation
has subsequently allowed us to test other family members. To date, 2 additional family members have been
tested: One has tested positive for the known mutation,
and the other is a true negative.
This case highlights some of the important issues
surrounding genetic counseling for LS. First, it shows
how critical an accurate family history is for a precise
risk assessment (46 ). The 3-generation pedigree is the
foundation that guides the majority of the discussion in
counseling patients about hereditary cancer syndromes (46 ). This case nicely demonstrates how the
interpretation of an individual’s risk of being a carrier
can change dramatically, depending on the patient’s
knowledge of the pertinent family history. Initially, we
were not too concerned about the possibility for LS, but
the likelihood for LS became much greater after we
acquired the additional information.
Second, testing for LS is unique, in that 4 separate
genes can produce a similar clinical phenotype. Use of
IHC analysis can often narrow down the choices to 1 or
2 genes, thus allowing tailoring of the testing strategy
and eliminating unnecessary and costly testing (62 ). As
mentioned above, there has been a recent push to institute universal tumor testing via IHC or MSI analysis
(64 ). Such testing is increasingly commonplace in the
setting of CRC; however, it remains less routine in the
setting of EC (66 ). Had universal tumor testing of ECs
been implemented at the time our patient was diagnosed, the loss of PMS2 would have been detected, and
the patient’s clinicians would have been alerted to her
increased risk of LS early on in her cancer treatment.
Additionally, had this patient not been motivated to
obtain more of her family history, she likely would have
gone undiagnosed, perhaps until she developed a second LS-associated cancer. Universal tumor testing
would allow an alternative means for identifying LS
patients, especially in the setting in which the patient
does not have the means to obtain additional family
history information.
Finally, this case illustrates the complexities of
gene mutation analysis and how genetic technology is
constantly changing. Analysis of the PMS2 gene has
historically been hampered by the presence of 15 pseudogenes located on the same chromosome (65 ). One
pseudogene in particular, PMS2CL, contains 6 of the
PMS2 exons (9, 11–15, 67 ). Owing to the frequency of
recombination and/or gene conversion between PMS2
and PMS2CL, there is a high rate of producing hybrid
PMS2/PMS2CL alleles, which makes reliable analysis
very difficult, especially in the 3⬘ region of the gene
(68 ).
Conclusion
LS is the most common form of hereditary CRC, and
universal screening programs for all CRCs are being
implemented nationwide. Women with LS have a substantial risk of developing EC as their first tumor type
and therefore require testing to determine if the EC is
LS related. Identifying the EC as LS related can lead to
screening, early detection, and prevention of other LSrelated tumors, including CRC. A better understanding of the role and mutation spectrum of MMR genes
and the advent of newer technologies, such as massively parallel sequencing, could improve screening
for LS via more-direct and cost-effective mutation
analysis.
Clinical Chemistry 60:1 (2014) 119
Reviews
Author Contributions: All authors confirmed they have contributed to
the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design,
acquisition of data, or analysis and interpretation of data; (b) drafting
or revising the article for intellectual content; and (c) final approval of
the published article.
Authors’ Disclosures or Potential Conflicts of Interest: No authors
declared any potential conflicts of interest.
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