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Project plan
Unifocal, multifocal and diffuse growth pattern in breast cancer:
Histopathology, genetics and combinations of tumor markers; prognosis
and differences
Dr Gyula Pekar, Department of Pathology and Clinical Cytology, Falun
Prof Dan Hellberg, Department of Women´s and Children´s Health,
Uppsala University, Uppsala, and Center for clinical research, Falun
Ass. prof Tibor Tot, Department of Pathology and Clinical Cytology, Falun
Hypotheses and aims of the study
1. The use of molecular markers, tumor markers, in addition to E-cadherin, as well as tracing
genetic alterations in breast cancer tissue will add information on the biological differences
between cancer subtypes, in particular between invasive lobular and ductal cancer and in
relation to their distribution within the breast tissue.
2. Breast cancer is a disease of an entire breast lobe. Tumor marker expression in apparently
normal epithelium will provide evidence for this theory.
3. Studying epithelial – stromal interaction in different tumor subtypes may provide additional
information on differences in tumor growth pattern
4. Combinations of expression of tumor markers with prognostic significance individually
will refine prognosis prediction.
5. Serum levels of estradiol, progesterone and androstenedione influence the expression of
tumor markers in breast cancer.
6. Genetic alterations and fingerprints will add information on prognosis prediction,
correlation to tissue tumor marker expression and diagnostics.
Introduction
Breast cancer is by far the most common cancer in women, comprising more than 20% of all
female cancers. With more than 150,000 research articles listed in medline, it is also one of
the most studied cancer types. It has been estimated that there are more than 1,000000 new
cases per year worldwide. Breast cancer is more common in the developed world with age
standardised incidence rates of approximately 100 cases per 100,000 women, compared to
about 20/100,000 in many developing countries. Breast cancer rates are steadily increasing in
many parts of the world (1). In Sweden, more than 7,000 new cases per year are diagnosed
(2). Mortality in the developed countries is less than 20%. Increasing incidence has been
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explained by an increase of exposure to risk factors such as decreased childbearing and
breastfeeding, increased exogenous hormone exposure, and detrimental dietary and lifestyle
changes, including obesity and less physical activity.
Three major growth patterns have been identified with histopathological diagnostics. These
are one solitary tumor, unifocal breast cancer. Two or more independent tumors, multifocal
breast cancer, or diffuse growth characterize the other growth patterns. The two major
subtypes of breast cancer are ductal and lobular cancer, with the former beeing most common
(ca 80%), while the latter represents 5-15% of cases (3).
According to the theory of the ´sick lobe’, breast cancer is a lobar disease, where genetic
malformations in early embryonic life and/or increased pool of cancer stem cells. In a large
study on breast cancer, 34% was cathegorized as unifocal, 20% were multifocal, and 4%
diffuse. The remaining tumors had an in situ component. Lymph node metastases, the
endpoint, were significantly more common in multifocal (48%) and diffuse (60%) than in
unifocal (28%) cancer (4).
In lobular cancer, it was reported a death rate of 6-7% for unifocal and multifocal tumors,
while the corresponding figure for diffuse tumors was 25%. The findings of unifocal,
multifocal and diffuse tumors were 39%, 12% and 28%, respectively, while the remaining
cases were combined or not classified (3). Diffuse cancer growth seems to correlate to an
aggressive behavior, although the matter is not yet established.
Clinically, some of the other factors indicating a poor prognosis in breast cancer are
histological type (some rare types, like invasive micropapillary carcinoma), high histological
tumor grade, large tumor size and metastases in lymph nodes and elsewhere.
High molecular cytokeratins, eg. CK 5/6, 10 and 14, show strong antibody staining in benign
“usual” hyperplasia, but are almost absent in DCIS, while CK 5/6 is absent in LCIS, aiding in
this sometimes difficult differential diagnosis. Lymph node metastasis are generally
diagnosed with routine haematoxylin and eosin-stained sections. Micrometastases and
isolated cancer cells / cellgroups will, however, be easier detected after cytokeratin staining.
Immunohistochemistry for detecting proteins involved in diagnosis and prognosis has been
increasingly used during the last decades, and an ever-increasing number of those are
commercially available. Estrogen and progesterone receptors have become standard in breast
tumor specimen investigations. Immunohistochemistry is useful in determination of stromal
invasion, distinction between (IDC) ductal and lobular cancer (ILC), and evaluation of lymph
node metastases including sentinel lymph nodes. In contrast to and IDC, membraneous
staining of E-cadherin is nearly always lost in and ILC. E-cadherin is thus a valuable tool to
differentiate between these two types of breast cancer (5). Tumor markers, relevant and
potentially useful in breast cancer are given in a separate literature review.
There has been an increased interest in gene-expression profiles of the entire genome, and
correlation to prognosis in cancer. Recently, gene expression in the minority of breast tumors
characterized by CD44 but not CD24 expression was compared with normal breast
epithelium. The former are known to have a higher tumorigenic capacity than other subtypes
of cancer cells. A genetic signature of 186 genes was generated. A group of patients had been
delineated having a genetic profile associated with poorer prognosis as compared with other
genotypes of breast cancer. The method has, however, been criticised. Where several genetic
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studies are compared, the gene sets are largely non-overlapping. Tumor types, which might be
rare, and that are known to be aggressive are selectively studied.
Material and Methods
a) Material.
During 1996 to 2007 data on 2304 women with breast cancer have been computerized at the
Department of Pathology and Clinical Cytology at Falun Hospital.
Growth pattern will be revised. Approximately 70% of the tumors were of ductal subtype,
15% of lobular subtype and the remaining 15% were of other subtypes.
The entire study material will be characterized using the following data set:
Clinics
All patient records will be rewieved with a detailed structured questionnaire. It will include
patient history, symptoms, diagnostics, treatment and follow-up.
Histopathology
Personal identification code
Preoperative cytology
Tumor size
Tumor growth pattern (unifocal, multifocal or diffuse)
In situ component (type, grade size)
Location of tumor
Histological subtype (invasive component)
Differentiation, ploidy
S-phase fraction
Lymph node metastases
Distant metastases
The histopathological findings will be analysed separately and in combinations to improve
prognosis prediction.
Expression of 32 tumor markers (21-29) relevant for breast cancer will be studied in 700 cases
of breast cancer and grouped into unifocal and multifocal cancers
1. Estrogenreceptor ,
2. Progesteronreceptor,
3. c-erb-2/her-2,
4. E-cadherin (cell-cell adhesion),
5. Cytokeratin 5/6 (cytoskelett),
6. Cytokeratin 14 (cytoskelett),
7. Epidermal Growth Factor Receptor (proliferation)
8. Smooth muscle actin (stroma),
9. Tenascin C (stroma),
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10. Collagen 4 (stroma),
11-13. 4-6. CD 3, CD 4, CD 8, CD 20, CD1a, CD68 and CD138 (3-4 av dessa
immunologiiska markörer kommer att väljas),
12. CD 44v5 (cell-cell adhesion),
13. Ki-67/MIB1 (proliferation),
14. Cyclin E (cell cykel reglerare),
15. P53 (tumör suppressor),
16. Ski-related novel protein (SnoN) (proliferation),
17. Fragile histidine triad (FHIT) (tumör suppressor),
18. Bcl-2 (apoptosis suppressor),
19. COX-2 (multiple functions),
20. Telomerase (hTERT) (cancercellens odödlighet)
21. PARP-1 (predictive marker)
22. c-kit (predicitve marker)
23. OX40 (malignant transformation)
24. Tumor-necrosis-factor-receptor
25. Protein kinase C (potential target for anticancer therapy)
26. 21 integrin (metastasis (suppressor).
27. Mammaglobin (marker for breast cancer).
28. S100 (tumor uppression)
29. MCF-1-14 (invasion and metastasis).
Tissue micro array (TMA) will be made on 350 cases of each ductal and lobular tumors (cases
matched according to tumor stage and size) from a representative area of the original blocks.
Each TMA-block will include 24 two-mm biopsies, to cut down costs for antibodies and
diagnostics. In cases with local recurrence or metastasis the recurrent or metastatic invasive
tumor will also be sampled to TMA. Diagnostics will be made by a experienced pathologist
familiar with immunohistochemistry and blinded for all clinical and histopathological details
Tumor marker expression will be analysed separately, but in partcular if combinations will aid
in prognosis prediction.
Genetics
Genetic analyses will be performed and mutations, overexpression and underexpression will
be analysed and compared from different areas of the tumor, lymphoglandulae, blood,
surrounding normal breast tissue, normal and tissue as far away from the tunor as possible. In
addition blood samples will be taken. This prospective study will include 400 consecutive
patients.
Genetic fingerprints and gene expression will be analysed separately, and interactions with
tissue tumor marker expression, histopathological and clinical data.
The prognostic significance of all variables will be tested in relation to diagnostics, treatment,
overall survival and disease free survival.
Studies
Study 1.
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The entire study material of 2304 cases will be characterized regarding the above listed
clinical and basic histopathology dataset. This will allow delineating the cases of lobular
carcinomas and forming a corresponding group of ductal carcinomas of matching clinical
characteristics and basic morphologic parameters. Unifocal and multifocal growth will be
diagnosed and studied in 700 patients. Five year overall survival will be used as outcome to
test the significance of the listed parameters, which will be necessary for comparison when
the significance of biomarkers will be tested.
Study 2.
Initially, correlation between expression of the individual tumor markers and prognosis, other
clinical characteristics, and growth pattern will be investigated. The aim is then to find
combinations of individually significant tumor markers that could identify aggressive tumors
and strengthen prognosis prediction.
Comparisons of different histological subtypes of the tumor and their expression of tumor
markers will provide evidence if these are different cancers that share in common that they
affect the breast, or that different subtypes show many similarities and are only variants of the
same cancer. Lack of E-cadherin expression in lobular cancer indicates that subtypes are at
least partly different cancers.
The search for expression of tumor markers in normal epithelium of the same breast lobe and
in a normal lobe is made to investigate the theory of the ‘sick lobe’. Findings of expression of
one or more tumor markers simultaneously in the tumor and the lobe, but no expression in a
healthy breast lobe will support this theory.
Study 3.
In sharp contrast to expression of estrogen- and progesterone receptors, analyses of serum sex
steroid hormone levels and their association with tumor marker expression in breast cancer
tissue is an area of investigation that has been done sparsely. The aim of this study is mainly
to increase the knowledge about breast cancer mechanisms. The biological roles of androgens
in breast cancer are virtually unknown. Serum androstenedione might be a better marker of
androgenicity in women, but testosterone could be added to the analyses. One could speculate
that finding a specific role of androgens in breast cancer might open new ideas of
pharmaceutical treatment as a supplement to tamoxifen.
Study 4
Genetic analyses will be made in several foci of the multifocal tumors. In addition genetic
analyses from histologically normal breast tissue adjacent and as far as possible from the
tumor, in posititive lymphogladules and in blood will be made.
Purpose study 1:
The 13 histopathological variables will be compared and and sensitivity, specificity, and
positive and negative predictive value will be compared for prognosis. Unifocal, multifocal
and diffuse tumor growth will be analysed separately and as one cancertype. Ductal and
lobular cancer will be analysed accordingly. Panels of tumor markers will be investigated
with the aim to improve prognosis prediction.
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Purpose study 2:
Expression of each of the 32 tumor marker, or panels of tumor markers, will be investigated
for correlation to:
1. Unifocal growth pattern
2. Multifocal growth pattern
3. Diffuse growth pattern
4. Clinical and histopathological variables and relation to tumor growth.
Purpose study 3:
A panel of the 13 clinico-pathologically variables and 29 tumor markers as above will be
searched for, with the aim to further improve prognosis prediction with unifocal, multifocal
and diffuse growth pattern. Ductal and lobular cancer will be analysed separately and as one
group. Sensitivity, specificity, and positive and negative predictive value will be used to
estimate optimal clinical usefullness, in contrast to only statistical corelations.
Purpose study 4:
Two main questions to be answered are confirmation of similar genetic alterations in tumor
and normal tissue, the hypothesis is the ‘sick lobe’. Comparison will be made with genetic
alterations and the cellular target proteins, tumor markers. The role of genetic ‘fingerprints’
for prognostisation will be analysed.
Time schedule
End-point of the study is ten-year survival. Collection of data will thus close at the end of
2017. Final analyses of data and preparation of articles will be made before the end of 2020.
Five-year survival will be used for intermediate analyses and articles for women with their
diagnosis before 2003, and will be established through the patient records and the National
Register of Deaths, National Board of Health and Welfare, Sweden.
Analyses of distribution of tumor markers according to histopathological variables, subtype,
growth pattern etc. will start immediately. Tissue micro array (TMA) will be made on a
subgroup of 700 cases with an equal number of ductal and lobular tumors, and distributed on
unifocal, multifocal and diffuse growth pattern (matching will be made according to tumor
stage and size) from a representative area of the original blocks.
The first publication(s) will be made in 2012.
Results
First project.
The entire study material of 2304 cases will be characterized regarding the above listed
clinical and basic histopathology dataset. This will allow for a suffient number of tumors with
diffuse growth pattern. It will also allow selection of more than 350 cases of lobular cancer
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with a corresponding group of ductal carcinomas matched for clinical characteristics and
basic morphologic parameters. Five year overall survival will initially be used as outcome to
test the significance of the listed parameters in relation to outcome, which will be necessary
for comparison when the biomarkers will be tested.
Second project.
Initially, correlation between expression of the individual tumor markers and prognosis, other
clinical characteristics in tumors with different growth pattern will be investigated. The end
point will be to find combinations of individually significant tumor markers and
histopathological variables that could identify aggressive tumor growth pattern and strengthen
prognosis prediction.
Comparisons of different tumor growth patterns and their expression of tumor markers will
provide evidence if these are different cancers that share in common that they affect the
breast, or that they show most similarities and are only variants of the same cancer type.
Third project.
Histopathological nomograms, panels of different variables have been used successfully in
other cancer types, e.g. prostate cancer. This is poorly studied in breast cancer. Rarely,
nomograms including both histopathological findings and tumor markers have been studied in
cancer types. This is also true in breast cancer. Nomograms, created for unifocal, multifocal
and diffuse tumor groups might be a further step in prognosis prediction.
Fourth project:
Genetic analyses of large materials as adjunct for prognostisation and treatment have been
poorly investigated. If the sick lobe can be shown, it could mean changes in the therapy, not
the least surgical for selected patients. Comparison of tumor marker proteins and genetic
signatures could direct future choice of analyses for prognostisation that will also be an
adjunct for the aggressiveness of treatment. Genetic fingerprints is still hampered by small
study populations as compared to the present study.
Statistics
Each of the clinico-histological variables and tumor markers will be analysed separately for
correlation to survival with respect to growth pattern
A chi2 test (likelihood ratio) will be used for crude comparisons of dichotomous, categorical
variables. T-test will be used for continuous variables, such as age. Multifactorial analyses by
logistic regression (loglikelihood test) will be used for adjustment for possible confounding
factors, and for estimation of odds ratios (OR) and 95% confidence intervals (95% CI). Cox
regression (proportian hazard) will be used in multivariate analyses on survival, such as
inclusion of a number of variables included in the study. Kaplan Meier survival curves will be
used for graphics. Sensitivity, specificity, and positive and negative predictive value will be
included.
Power
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Based on 75% survival with expression of a tumor marker in one group of women and 85%
survival in the remaining women, 270 subjects will be required in each group to achieve
significance with 80% power.
Ethical approval
The study was approved from the Research Ethical Committee, Uppsala University. During
1996-2003 the material is considered historical, while informed consents are required from
women who had their diagnoses from 2004-01-01 and onwards in accordance to the
biobanking law. Results will only be given at group levels.
Complete project plan
Project plan breast 110218
PhD
Dr Gyula Pekar was accepted PhD student at Department of Genetics and Pathology 2010-1214. Main superviser is associate professor Tibor Tot. Additional supervisers are professor Dan
Hellberg, Center for Clinical Reasearch, Falun and professor Jan Dumansky, Department of
Genetics and Pathology, Uppsala University, Uppsala.
References
1. Parkin DM, Fernandez LMG. Use of statistics to assess the global burden of breast cancer. Breast J 2006;12
(suppl 1):S70-S80.
2. National Board of Health and Welfare. Cancer Incidence in Sweden 2004. Health and diseases 2005:9.
3. Tot T. The diffuse type of invasive lobular carcinoma of the breast: morphology and prognosis. Virchows
Arch 2003;443:718-24.
4. Tot T. Clinical relevance of the distribution of the lesions in 500 consecutive breast cancer cases documented
in large-format histologic sections. Cancer 2007;10:2551-2560.
5. Lerwill MF. Current practical applications of diagnostic immunohistochemistry in breast pathology. Am J
Surg Pathol 2004;28:1076-91.
Complete reference list:
Entire project plan 110218