HISTOPATHOLOGICAL ANALYSIS, GRADING AND VALUE OF PROLIFERATION INDEX, Ki-67 IN
BREAST CARCINOMA
DISSERTATION
SUBMITTED FOR M.D. BRANCH III [PATHOLOGY]
APRIL – 2012
THE TAMILNADU DR. M.G.R. MEDICAL UNIVERSITY
CHENNAI - TAMILNADU
CERTIFICATE
This is to certify that this dissertation entitled “HISTOPATHOLOGICAL ANALYSIS, GRADING AND VALUE OF PROLIFERATION INDEX, Ki-67 IN BREAST CARCINOMA” is a bonafide record work done by Dr. S. LATHA submitted as partial fulfillment for the requirements of M.D Degree Examination -Pathology to be held in APRIL 2012.
DR. N. ARUMUGAM, M.D., DR.T.B.UMA DEVI, M.D., Professor and Head of the Department, The Dean,
Department of Pathology, Thanjavur Medical College, Thanjavur Medical College, Thanjavur.
Thanjavur.
ACKNOWLEDGEMENT
I wish to express my sincere and profound gratitude to DR.N.ARUMUGAM,M.D., Professor and Head of the Department of Pathology, Thanjavur Medical College, Thanjavur, for his valuable guidance at every stage, constant encouragement and words of advice which have been the motivating forces in bringing forth this piece of work.
I take immense pleasure in thanking Dr.A.Vasahar,M.D., Associate Professor of pathology who has offered many valuable suggestions and encouragement during this period, whose help has been invaluable to me.
My heartfelt thanks to Dr.M.Saraswathy,M.D.,DGO, Dr.AL.Shanthi,M.D.,DGO, Dr.M.Senthil Kumar,M.D., Associate Professors for their valuable guidance and encouragement throughout my study.
I am also thankful to all my Assistant Professors Dr.K.G.Padmanaban M.D., Dr.S.Jenita Christiana Ranjana M.D. for their constant support and help at every stage in this study.
I would also like to express my sincere thanks to my fellow-postgraduates, Lab technicians, Librarians, statisticians Dr.Jeevithan and Dr.Kalaiselvi, for their generous help throughout my study.
Finally I would like to express my heartful thanks to my parents, husband, brothers, well wishers and almighty for their blessings and immense support in completing this study.
Above all, I thank our DEAN, for granting me the permission to carry out this work.
CONTENTS
S.NO. TOPICS PAGE NO.
1 INTRODUCTION 1
2 AIM OF THE STUDY 4
3 MATERIALS AND METHODS 5 4 REVIEW OF LITERATURE 7
MASTERCHART
5 OBSERVATION AND RESULTS 42
6 DISCUSSION 51
7 CONCLUSION 65
APPENDIX
BIBLIOGRAPHY
INTRODUCTION
Carcinoma of the breast is the most common non-skin malignancy in women accounting for 22.9 % of all cancers in women worldwide.102 A woman who lives to age 90 has a one in eight chance of developing breast cancer. In 2008, an estimated 13,84,155 women were diagnosed with breast cancer and about 4,58,503 women died of the disease.
As the demographic bulge of the baby boomers continues to grow older, the number of women with breast cancer is expected to increase by about a third over the next twenty years. It is both ironic and tragic that malignant neoplasm arising from this organ, readily accessible to self-examination and clinical diagnosis, continue to exert such a heavy toll among women.
The incidence of breast cancer is higher in North America and Northern Europe, intermediate in South European and Latin American countries and low in most Asian and African countries.63
The incidence is rising in every country of the world especially in developing countries such as India. This is because more and more women in India are beginning to work outside their homes which allow the various risk factors of breast cancer to come into play. These include late age at first childbirth, fewer children and shorter duration of breast-feeding. Thankfully, the incidence of breast cancer is much lower in India compared to western countries. The incidence varies between urban and rural women; the incidence in urban cities of India is about 27 new cases per 100,000 women per year while in rural areas it is only 8 per 100,000. The chances of cure in women who develop the disease are related to early diagnosis.
Triple assessment which includes clinical examination, imaging and tissue sampling been traditionally used for the diagnosis of breast lesions.
The prognosis of women with breast cancer is currently based on the established prognostic markers particularly nodal status, tumor size, histological grade, histological subtype and rate of proliferation.49 Improvements in survival can be expected from early detection and adjuvant treatment. Adjuvant chemotherapy and hormonal treatment improve survival but have potential serious side effects and are costly.100
Adjuvant treatment should be given only to high risk patients which requires good prognostic factors for patient risk stratification.
A standard histologic grading system is a powerful prognostic factor. Histological grading has often been criticized for poor reproducibility and lack of agreement among different observers. However when performed at single institutions by experienced pathologists does correlated well with clinical outcome. Elston and Ellis modification of Scarff Bloom Richardson system (also called the Nottingham combined histologic grade) is the widely used grading system and is based on assessment of three morphological features namely, degree of tubule formation, nuclear pleomorphism and mitotic counts. This system has been shown to be both predictive and reproducible.31,68
As per the literature, high tumor grade and increased proliferation correlate strongly with poor prognosis.100 Tumor proliferative activity / Growth fraction as defined by the proportion of cycling cells in the total population has been shown to be of prognostic value in various solid neoplasms, including breast carcinoma.8
Mitotic index, S phase fraction and immunohistochemistry using Ki-67 and MIB-1 are the best characterized proliferation markers.49 A high growth fraction identifies a subgroup of node negative patients who can benefit from systemic adjuvant therapy.71
Ki-67 is a monoclonal antibody specific for a nuclear antigen expressed in proliferating cells (late G1, S, M and G2 phases of the cell cycle).
Mitoses counting provide the most reproducible and independent prognostic value among the proliferative indices but Ki-67/ MIB 1 labeling index is a promising alternative.
Recently molecular analysis of gene and gene products have shown that gene expression analysis, microarray technology can accurately divide breast cancers into those with high and low risk of recurrence.49
This study is undertaken in view of evaluating the actual incidence of breast carcinoma in semi urban area like Thanjavur with particular attention to prognostic factors like grading and expression of Ki-67 proliferation marker. In addition, the recent literatures, journals and research publications regarding breast carcinoma are also immensely reviewed.
AIM OF THE STUDY
1. To evaluate the incidence of breast carcinomas in our institution.
2. To evaluate the histomorphological features of breast carcinoma with light microscopy.
3. To determine the grade of breast carcinomas by Elston and Ellis modification of Scarff Bloom Richardson system.
4. To evaluate the expression of Ki-67 proliferative index in breast carcinoma by immunohistochemical method.
5. To evaluate the association between histological Grade and other clinicopathological parameters like age, tumor size, lymph node status and Ki-67 immunoexpression.
METHODS AND MATERIALS
Among the 139 breast malignancies reported in the two year study period from January 2009 to December 2010, in the Department of Pathology, Thanjavur Medical College, Thanjavur, 117 breast carcinomas treated by lumpectomy or mastectomy (simple / modified radical) were alone included in the study and thoroughly analysed.
Preliminary slicing was done within 2 hours of surgery to allow adequate penetration of the fixative and specimens were fixed in 10% neutral buffered formalin. Mastectomy specimens were sliced at 1 cm interval like opening a book. A detailed study of the morphological features was done with the specimen. Emphasis was given to the size of tumor. As many lymph nodes as possible was dissected from all the specimens. Adequate tissue samples were taken from the tumor periphery, base, both lateral margins, nipple areola and ulcerated skin. Nodes less than 5mm in diameter were embedded intact, those greater than 5 mm were cut into slices not exceeding 5mm. The tissues were processed, embedded in paraffin blocks and 3-5 µm thick sections were cut in a rotary microtome and the slides were stained with conventional Haematoxylin and Eosin stain (Appendix I).
In all the cases histomorphological evaluation was done. The histologic type was recorded according to the criteria of WHO and grading done by Elston and Ellis modification of Scarff Bloom Richardson system assessing the three morphological parameters, the relative degree of tubule formation, nuclear pleomorphism and mitotic counts. In mucinous / colloid carcinoma of the breast, special histochemical stains were also done to identify mucin (Appendix II).
IMMUNOHISTOCHEMISTRY
In randomly selected 12 cases comprising 4 cases of grade I, 5 cases of grade II and 3 cases of grade III carcinoma including one each of mucinous carcinoma, metaplastic carcinoma and papillary carcinoma, Ki-67 proliferation marker was also assessed by immunohistochemical method.
Formalin fixed paraffin embedded 3 - 5µm thick tissue sections were stained with an antibody directed against the Ki-67 antigen using advanced polymer staining system (Dako,
Netherland). The staining was performed according to the immunohistochemical staining protocol (Appendix III)
A tumor cell was designated positive for Ki-67 when there was any discernible nuclear staining. The percentage of positive cells was determined by counting the number of positively stained and unstained nuclei of tumor cells in the most immunoreactive areas. At least 10 High power fields were evaluated. Benign breast epithelium or stromal nuclei were not included in the count. Tumor samples were interpreted as positive when more than 1% of the tumor cells expressed immunoreactivity. If few tumor cells and benign ductal epithelial cells showed weak cytoplasmic staining; this finding was interpreted as negative.
Ki–67 LABELING INDEX ( Ki–67 LI ) was expressed as the percentage of Ki–67 positive cells.
Also immunohistochemistry with p63 antibody was done in intracystic papillary carcinoma to identify the presence of myoepithelial cells within the lesion and hence to assess invasiveness.
REVIEW OF LITERATURE
ANATOMY
Breast is a modified sweat gland. It lies in the superficial fascia of the pectoral region. It extends from 2nd to 6th rib and from sternal edge to the mid axillary line. A small extension called the axillary tail of Spence pierces the deep fascia and lies in the axilla. Approximately three quarters of the breast is on the pectoralis major muscle normally.
HISTOLOGY
Human breast consists of 15 to 20 lobes, each of which is an individual compound tubuloalveolar type of gland. A lactiferous duct independently emerges from each lobe at surface of the nipple being expanded by lactiferous sinuses just below the nipple.
Successive branching of the large ducts eventually leads to the terminal duct lobular unit.
In adult women, the terminal duct branches into a grape like cluster of small acini to form a lobule. A glandular lobule consists of small tubules or intralobular ducts lined by inner secretory cuboidal or low columnar epithelium and an outer contractile myoepithelial or basal cell layer.75
The acini and intralobular ducts are lined by loose connective tissue stroma which contrasts with the dense interlobular stroma surrounding the lobules.
At puberty, the female breast develops its characteristic adult structure. Minor variations occur during menstrual cycle, but major physiological changes occur during pregnancy and lactation. After menopause, there is atrophy of the breast tissue.
EPIDEMIOLOGY
Invasive breast cancer is the most common carcinoma in women accounting for 22% of all female cancers, 26% in affluent countries which is more than twice the occurrence of cancer in women at any other site.37
In the affluent countries of North America, Europe and Australia, 6% of women develop invasive breast cancer before age 75, whereas in less developed regions of sub-Saharan Africa
and southern and eastern Asia including Japan, the probability of breast cancer by age 75 is one third that of rich countries.
Significant improvements in survival have been recorded in western countries since late 1970’s but advancements have been dramatic in the 1990’s due to the combined effect of population screening and adjuvant hormonal treatment. The risk of the disease had been increasing until the early 1980s in both developed and developing countries and continues to increase in particular in the developing countries.
RISK FACTORS
1. Age – incidence rises throughout a woman’s lifetime peaking at the age of 75 to 80 years.
2. Reproductive lifestyle – breast cancer occurs more frequently among women who have an early menarche, remain nulliparous, or have few children with late age at first delivery, late age at menopause and shorter breast feeding time.
3. Nutrition – fruits and vegetables reduce the risk whereas total fat as well as saturated animal fat increases the risk. Rapid growth and greater adult height and a high body mass index linked to high calorie intake are associated with increased risk.
4. Alcohol – consumption of alcohol causes mild increase in risk.
5. Body weight – the incidence rate increases among postmenopausal women with BMI more than 24 kg/m2. But among premenopausal women, the association between BMI and breast cancer risk is inverse.
6. Physical activity – there is 20 to 40% decrease in risk among most physically active women independent of the menopausal status.
7. Race / ethnicity – non Hispanic women have higher rates of breast cancer. However women of African or Hispanic ancestry tend to develop cancer at younger age, more likely to be poorly differentiated, ER negative, present at a more advanced stage and have increased mortality rate.
8. Estrogen exposure – postmenopausal hormone replacement therapy increases the risk 1.2 to 1.7 fold and adding progesterone increases the risk further. Oral contraceptives have not been shown convincingly to affect the breast cancer risk. Reducing endogenous estrogens by oophorectomy decreases the risk by upto 75%.
9. First degree relatives with breast cancer – risk increases with number of affected first degree relative (mother, sister or daughter) especially if occurred at a young age, due to interaction of low risk susceptibility genes and non genetic factors.
10. Atypical ductal hyperplasia and carcinoma of contralateral breast – increases the risk.
11. Radiation exposure – radiation to chest due to cancer therapy, atomic bomb exposure or nuclear accidents results in a higher rate of breast cancer. The risk being greater with exposure at young ages and with high radiation doses.
ETIOPATHOGENESIS
The major risk factors for the development of breast cancer are hormonal and genetic.
Breast carcinomas can therefore be divided into sporadic cases, probably related to hormonal exposure and hereditary cases associated with germline mutations.102
HEREDITARY BREAST CANCER
` Hereditary breast cancer accounts for about 12% of all breast cancers and are due to inheritance of a susceptibility gene or genes. Single mutations in the highly penetrant breast cancer genes BRCA1 (17q21) and BRCA2 (13q12-13) account for about 3% of all breast cancers and are associated with familial breast carcinoma and ovarian carcinoma. BRCA1 associated breast cancers are commonly poorly differentiated, have medullary features with triple negative phenotype (ER,PR,HER2/neu negative) and gene profiling signature very similar to basal like subtype of breast cancers.
Other less commonly implicated genes include mutated p53 (Li Fraumeni syndrome) CHEK2, PTEN (Cowden syndrome), LKB1/STK11 (Puetz Jeghers syndrome) and ATM (Ataxia Telangiectasia)
LOCALIZATION
Breast carcinoma arises from the epithelial cells of terminal duct lobular unit. There is slight higher frequency of invasive breast cancer in left breast. 40 to 50% of tumors occur in upper outer quadrant with decreasing frequency in central, upper inner, lower outer to lower inner quadrant.37
CLINICAL FEATURES
Most common findings in symptomatic women are breast lumps which may or may not be associated with pain. Nipple abnormalities (discharge, retraction, distortion or eczema) are less common.
Breast abnormalities should be evaluated by triple assessment including clinical examination, imaging (mammography and ultrasound) and tissue sampling by either aspiration cytology or needle core biopsy.37
CLASSIFICATION OF BREAST CARCINOMA DUCTAL CARCINOMA IN SITU (DCIS)
It is a non invasive breast lesion that predominantly affects segmental and larger ducts.
Common patterns – comedo, solid, cribriform, papillary, micropapillary, cystic hypersecretory and mixed.77 Divided into three grades based on cytological features and intraductal gland formation or polarization.19
High grade DCIS – composed of large pleomorphic cells with high nuclear cytoplasmic ratio, coarse chromatin, frequent mitoses and necrosis. Coarse or dystrophic calcifications are often present.
Intermediate DCIS – nuclei less pleomorphic but lack uniformity, nucleoli may be present but not large, necrosis may be present but not extensive, there may be some cell polarization.
Low grade DCIS – evenly spaced cells with small regular nuclei, indistinct nucleoli and with regular round smooth cookie cutter like punched out spaces (in cribriform pattern). Cells well polarized.
Rare variants – spindled, apocrine, signet ring, neuroendocrine, squamous, flat or clinging type, small cell solid and clear cell types.31,77
By the criteria of Page and colleagues, the diagnosis of DCIS requires atleast two duct spaces entirely filled with round or cuboidal proliferation of ductal cells that are evenly spaced with distinct cell borders.
Van nuys grading system uses a combination of nuclear grade and necrosis for grading DCIS.
Anthony S Y Leong et al showed that the biological markers varied between different grades of DCIS and suggested the progression of Low grade DCIS to Low grade IDC and High grade DCIS to High grade IDC.5
IHC – Cytokeratin (CK) 5/6 and 34βE12 negative, E – cadherin positive.63,77
LOBULAR CARCINOMA IN SITU
It predominantly grows within lobules or Terminal duct lobular units. Usually incidental, can be multicentric and bilateral.37 There is 25% increased risk of subsequent invasive breast cancer which can be either lobular or ductal carcinoma of no special type.31Anderson has suggested the criteria for lobular carcinoma in situ which include
uniform or monotonous small dyscohesive cells with regular nuclei, dispersed chromatin and inconspicuous nucleoli comprise the entire cells in a lobular unit;
Filling of all acini (no interspersed intercellular lumens);
Filling or distortion of atleast one half of the acini in the lobular unit.
Patterns – type A of usual morphology, type B with larger and more atypical cells.
Variants – large cell, apocrine, signet ring cell, pleomorphic.77
IHC – almost always E – cadherin negative and positive for CK 5/6.77,45 INTRACYSTIC PAPILLARY CARCINOMA
It is a solitary and central type of intraductal papillary carcinoma that requires >90% of papillary processes devoid of myoepithelial cell layer.37
Gross – similar to papilloma presents as a well circumscribed cauliflower like mass attached to a dilated duct containing serous or serosanguinous fluid, but tends to be larger and occurs centrally.77
Microscopy – characterized by thin fibrovascular stalks devoid of myoepithelial cell layer and neoplastic cells arranged in solid, cribriform, micropapillary or stratified spindle cell patterns of DCIS. A myoepithelial cell layer is usually present in the lining of the duct wall. Variants include solid and transitional cell types.
INVASIVE BREAST CARCINOMA
Invasive ductal carcinoma of no special type is the most common invasive carcinoma accounting for 40 to 75%. Invasive breast carcinoma of special type applies to tumors composed entirely or largely ≥ 90% of the special pattern and accounts for 35%. Mixed ductal and special type carcinoma includes invasive carcinoma containing > 10% but < 90% of the special type patterns.77
INVASIVE DUCTAL CARCINOMA-NOT OTHERWISE SPECIFIED (IDC-NOS)
Gross – lacks circumscription and has irregular stellate outline or nodular configuration. May have gritty feel when cut.37
Microscopy – grows in diffuse sheets, cords, clusters or as individual cells. Glandular differentiation may be well developed, barely detectable or absent. Tumor cells are larger, more pleomorphic with prominent nucleoli. May have areas of necrosis and numerous mitotic figures.
Distinct lymphoplasmacytic infiltrate may be seen in minority of cases. Foci of squamous metaplasia, apocrine metaplasia or clear cell change may be seen. Associated DCIS present in 80% cases.77
Variants
Pleomorphic carcinoma - > 50% pleomorphic and bizarre tumor giant cells.
Carcinoma with osteoclast type giant cells - osteoclastic giant cells are present in an inflammatory, fibroblastic and hypervascular stroma.
Carcinoma with choriocarcinomatous features.
Carcinoma with melanotic features.37
IHC – positive for Low Molecular Weight Cytokeratin (LMWK) 8, 18 & 19 and EMA (Epithelial Membrane Antigen), Human milk fat globule protein, Lactalbumin, CEA & B72.3.
S-100 +ve in 60%, GCDFP (Gross Cystic Disease Fluid Protien )-15 in 50% and rarely HMB45+ve. 63,77
INVASIVE LOBULAR CARCINOMA
It is more common in women older than 75 years. Multicentricity and bilaterality are present in as high as 25-50% of cases.37,63,77
Gross – irregular, poorly delimited, mean diameter slightly larger than IDC
Microscopy – diffuse or multifocal infiltration of small round, regular dyscohesive tumor cells arranged in single file linear cords between collagen bundles, which sometimes encircles ducts in a targetoid or onion skin fashion. Occasional intracytoplasmic lumen with central mucoid material is present. Mitoses infrequent. Metastases in GIT, gynecologic system, meninges, peritoneum – retroperitoneum are more prevalent.
Variants – pleomorphic, solid, alveolar, mixed, apocrine, signet ring, tubulolobular and histiocytoid (myoblastomatoid or granular cell).
IHC – absence of E–cadherin in 80 to 100% cases, almost always positive for hormone receptors. HMWK+. Low proliferation rate.37,45,26
TUBULAR CARCINOMA
Occurs in older patients, are smaller in size with less nodal involvement and are well differentiated grade 1 carcinomas.37
Gross – Pure type-stellate shape radiating arms and central yellow flecks.
Sclerosing type-more diffuse, ill defined.37
Microscopy – well defined tubules, oval or rounded often angulated lined by single layer of epithelial cells which are small, regular with little nuclear pleomorphism and only scanty mitotic figures. > 90% of tumor must exhibit classic tubular pattern. Apical snouts present in a third of the cases. Myoepithelial cells absent. Secondary important feature is cellular desmoplastic stroma. Majority associated with low grade cribriform and micropapillary DCIS and also flat epithelial atypia.63 Have excellent long term prognosis.
IHC – nearly always ER, PR positive, low growth fraction, HER 2 negative.
MEDULLARY CARCINOMA Occurs under 50yrs of age.63
Gross – well circumscribed, rounded and soft in consistency.37 Microscopic criteria for diagnosis
1. >75% syncytial growth pattern (broad anastamosing sheets of tumor cells with indistinct cell borders).
2. Absence of glandular structures
3. Diffuse lymphoplasmacytic infiltrate (moderate to marked) 4. Complete histological circumscription with pushing margins.
5. nuclear pleomorphism moderate to marked
Atypical medullary – tumors showing predominantly syncytial architecture with only two or three of the other criteria. Typical medullary carcinoma observed in 7.8 to 13% of BRCA associated carcinomas. Better prognosis than usual IDC.
IHC – hormone receptors and HER 2 negative, p53 mutation very common, high proliferation rate.
MUCIN PRODUCING CARCINOMAS
Characterized by production of abundant extracellular and or intracellular mucin. WHO classifies them into mucinous (colloid) carcinoma, mucinous cystadenocarcinoma, columnar cell mucinous carcinoma and signet ring cell carcinoma.37
Mucinous carcinoma
>60 years of median age, higher than usual IDC. Mostly grade 1 tumor.
Gross – typical glistening gelatinous appearance with bosselated pushing borders and soft consistency.37,63
Microscopy – clusters of uniform round cells with minimal eosinophilic cytoplasm floating in lakes of mucin. Delicate fibrous septa divides the mucous lake into compartments. Mucin lakes may be acid or of neutral type.63 DCIS component seen in 30 to 75% of cases. Notable proportion (25 – 50%) have neuroendocrine differentiation with argyrophilic granules.
IHC – hormone receptors and Bcl – 2 positive, HER 2 usually absent.
NEUROENDOCRINE TUMORS
Occurs in 6th or 7th decade and express neuroendocrine markers in >50% of the cell population. WHO classifies them into solid, small cell or oat cell and large cell neuroendocrine carcinoma.37
Microscopy – solid carcinoma consists of densely cellular solid sheets and trabeculae of cells that are spindle to plasmacytoid to large clear cells separated by delicate fibrovascular stroma.
Small cell tumors are composed of densely packed hyperchromatic cells with scant cytoplasm.
Large cell are poorly differentiated tumors composed of large clusters of cells with moderate to abundant cytoplasm, vesicular nuclei, finely granular chromatin and high mitoses.
Argyrophilia demonstrated by Gremelius silver precipitation technique.
IHC – expresses chromogranin, synaptophysin, and NSE. CK 7+ve, CK20 -ve. ER, PR +ve mostly.
INVASIVE PAPILLARY CARCINOMA
Common in postmenopausal patients.37,63 Has relatively good prognosis.
Gross – circumscribed in two- thirds of cases. Can be large due to bulky cystic component.
Microscopy – shows delicate or blunt papillae and focal solid areas of tumor growth. Cells have amphophilic cytoplasm, typically intermediate grade nuclei and may show apocrine features.
Most tumors are histologic grade 2. DCIS present in > 75% cases.
INVASIVE MICROPAPILLARY CARCINOMA Occurs at same age as IDC – NOS.
Gross – lobulated outline due to expansive growth.37
Microscopy – consists of hollow aggregates of malignant cells without fibrovascular cores (micropapillae) which lie within artifactual stromal spaces (caused by shrinkage of the surrounding tissue). Nuclear pleomorphism is moderate, mitotic activity low. Tumor cells display reverse polarity with an `inside out’ morphology. Prognosis generally poor.19
IHC – EMA demonstrates reverse polarity of the tumor cells.
METAPLASTIC CARCINOMA
It is a heterologous group of neoplasm characterized by an intimate admixture of adenocarcinoma with areas of spindle cell, squamous and or mesenchymal differentiation.37
Azzopardi grouped them into 4 categories; adenocarcinoma with varying degrees of squamous metaplasia, squamous cell carcinoma with extensive spindle cell metaplasia or marked desmoplasia, SCC arising in cystosarcoma phyllodes, pure SCC without spindle cell metaplasia and not arising in preexisting tumor.9 But use of this scheme was limited.
WHO classifies metaplastic carcinoma into purely epithelial and mixed epithelial and mesenchymal. Purely epithelial carcinoma can be Squamous with Large cell keratinizing, Spindle cell or Acantholytic variants or Adenocarcinoma with spindle cell differentiation or Adenosquamous including muco-epidermoid. Mixed epithelial and mesenchymal can be Carcinoma with chondroid metaplasia, Carcinoma with osseous metaplasia or Carcinosarcoma with malignant heterologous elements.
Gross – well circumscribed, often solid. Squamous or chondroid differentiation is reflected as pearly white to firm glistening areas on cut section.
Microscopy - The metaplastic spindle cell and squamous cell carcinomas may present in a pure form without any admixture with a recognizable adenocarcinoma. Extensive sampling of metaplastic tumours should be done to identify carcinomatous foci and distinguish them from true sarcomas because of differences in biologic behaviour and response to therapy. Matrix producing carcinomas are those which show an apparently abrupt transition from carcinoma to an osseous or cartilaginous matrix without an intervening transitional phase.
Spindle cell carcinoma is composed of predominantly bipolar spindle cells of relatively bland appearance with only mild or moderate atypia arranged in interweaving bundles. Such tumours frequently also contain areas of squamous differentiation. Matrix producing carcinomas and spindle cell carcinomas have generally favourable prognosis. When the mesenchymal component is malignant the designation of carcinosarcoma is used, which has an aggressive behavior. Among squamous cell carcinomas, acantholytic variant is a very aggressive tumor.37,63
IHC – basal cytokeratins CK5 and CK 34βE12 positive, p63 positive. ER, PR& HER2 negative.26,37
PAGET’S DISEASE
It is a weeping crusted eczematous lesion of nipple caused by insitu breast carcinoma cells permeating the squamous epithelium. In more than 95% cases underlying DCIS present with or without invasion.77
Microscopy – large atypical adenocarcinoma cells are scattered within the epidermis, along dermo – epidermal junctions and also as single cells or as clusters in the upper layers of epidermis.
IHC – CK 7 is the single best stain positive in almost all cases. HER 2/ neu positive in 80 to 90%
cases.26 Other less helpful markers are GCDFP 15, pCEA, ER & PR.
OTHER RARE TYPES
Various entities such as Infiltrating cribriform carcinoma, Lipid rich carcinoma, Secretory carcinoma, Oncocytic carcinoma, Adenoid cystic carcinoma, Acinic cell carcinoma, Glycogen rich clear cell carcinoma, Apocrine carcinoma, Sebaceous carcinoma, Inflammatory carcinoma have been reported in the literature.
PROGNOSTIC FACTORS IN BREAST CANCER
PROGNOSTIC MARKERS – are factors that determine or correlate with the natural history of the disease in the absence of intervening adjuvant systemic therapy and therefore are reflective of the inherent aggressiveness of the tumor.
PREDICTIVE MARKERS – are those associated with response or lack of response to a particular therapy. Under the auspices of the College of American Pathologists, a multidisciplinary group of clinicians, pathologists, and statisticians considered prognostic and predictive factors in breast cancer and stratified them into categories reflecting the strength of published evidence.80
Category I – are factors proven to be of prognostic importance and useful in clinical patient management; it includes tumor size, nodal status, micrometastasis, sentinel lymphadenectomy, histologic grade, histologic type, mitotic figure count and hormone receptor status.
Category II - are factors that had been extensively studied biologically and clinically, but whose importance remains to be validated in statistically robust studies; it includes HER 2 /neu (c–erb B2), p53, lymphatic or vascular channel invasion, additional proliferative indices like Ki 67/MIB -1, DNA analysis:phase fraction.
Category III - are all other factors not sufficiently studied to demonstrate their prognostic value;
it includes DNA ploidy analysis, tumor angiogenesis, Epidermal growth factor receptor, Transforming growth factor - , Bcl-2, pS2 and cathepsin D.
1) AXILLARY LYMPHNODE STATUS
Presence or absence of axillary lymphnode metastasis is the single most powerful prognostic factor for primary breast cancer.49 The five year disease free survival for node negative patients is approximately 80%. There is direct relationship between the number of lymphnodes and clinical outcome. Patients with four or more involved nodes have a worse prognosis.80
Regional lymphnodes may be divided into macrometastasis (>2mm), micrometastasis (0.2 – 2mm) and isolated tumor cells (< 0.2mm).49 Several studies have found that prognosis of patients with micrometastasis in sentinel lymphnodes with other axillary lymphnodes being negative is same as that for patients with negative nodes while others have suggested that such patients have worse prognosis.21,24,90 Some investigators have found that immunocytochemical detection of micrometastatic cells in the bonemarrow is an independent prognostic risk factor in patients with node negative breast cancer.14,15
Bernard Fisher et al studied the relation of number of positive axillary nodes to the prognosis of patients with primary breast cancer.11 The hazard rate for those with negative, or 1 to 3 positive nodes, was relatively low and constant, whereas in those with ≥4 positive nodes the risk in the early years was much greater, but by the fifth year it was similar to that occurring when 1-3 nodes were involved, and not much different from negative node patients.
The same pattern existed whether 4 to 6 or ≥13 nodes were positive.
Tatjana Ivkovic-Kapicl studied 74 patients with pT1 ductal invasive breast carcinoma with known nodal status and found that tumor size was related directly to nodal positivity. Also nodal positivity was significantly related to negative PR status, p53 protein overexpression and high Ki-67 index. No significant association was found between nodal positivity and patient age, tumor grade, ER status, and c-erbB2 expression.97
2) TUMOR SIZE
After nodal status, tumor size is the most consistent and powerful prognostic factor for distant relapse especially in node negative patients.49 Disease recurrence generally increases as tumor size increases.
Adair et al, in a longterm follow up of systemically untreated breast cancer patients over 30 years of age found that tumor sizes under 2 cm have a distant recurrence risk of approximately 25%, risk increases to 35% with tumors 2 to 2.9 cm, 45% with tumors 3 to 3.9 cm and to greater than 50% in tumors 4 to 4.9 cm.1 Literature says very large tumors (i.e >5cm) actually have a better prognosis than those between 3 and 4.9 cm, as the tumors that grow to very large size without nodal metastases may have lower inherent ability to metastasize.
Data from Memorial Sloan-Kettering Cancer Center indicate that tumors less than 1 cm have a 20 year relapse rate of 12%.87
Jeffrey D. Seidman et al in their study of 37 ductal carcinomas found that size of the invasive component is a more important factor in predicting axillary lymph node status and hence the prognosis than is the size of the entire tumor in patients having an intraductal component at the periphery.58
Similarly Anthony L Abner et al studied 118 patients with clinically negative axillary lymph nodes and found that microscopic invasive tumor size is a better predictor of 10 year relapse free survival than macroscopic tumor size and the prognosis for patients with tumors that microscopically measured ≤ 1 cm was excellent.4
3) HISTOLOGIC SUBTYPE
Most 80 to 90% primary breast cancers are infiltrating ductal carcinoma of no special type. Inflammatory breast cancer, a distinct form of invasive breast cancer is a clinical entity with extremely poor prognosis. Another distinct subtype is invasive lobular carcinoma. A large retrospective study of more than 4000 patients with invasive lobular carcinoma identified that these tumors were more likely to occur in older, to be larger in size, to be ER or PR positive, to be associated with lower S phase fraction and good prognosis.6
Special histologic types (pure Papillary, Tubular and Mucinous) identify a small subset of patients who have a better prognosis compared with Infiltrating Ductal Carcinoma of no special type.83 Node negative tumors less than 3 cm that are pure papillary, Mucinous or Tubular have a long term risk of recurrence of under 10%.
Medullary carcinomas have a better prognosis than other Infiltrating Ductal carcinoma but may not have same excellent prognosis as the other special type. In the series of Ridolfi et al, the 10 year survival rate was 84% as opposed to 63% for ordinary ductal carcinoma.31,86 The prognosis was good particularly for tumors smaller than 3cm and it remained better than that for ductal carcinoma even when nodal metastases were present.
In summary, patient with the common histological types of breast carcinoma may be stratified into four broad prognostic groups as shown in the table 1.
Table 1:
PROGNOSIS 10–YR SURVIVAL
TYPE OF CARCINOMA
Excellent > 80% Tubular, Cribriform, Mucinous
Good 60 to 80% Mixed ductal NOS
with special type
Moderate 50 – 60% Medullary, Invasive
Papillary, Lobular
Poor < 50% Ductal NOS
4) HISTOLOGIC GRADE
Although inspired by Broders, histologic grading of breast carcinoma was first introduced in 1925 at Massachusetts General Hospital in Boston by Greenough. He formulated three prognostically significant categories of invasive carcinoma based on two features:
1. The degree of arrangement of cells around an open gland lumen, which he referred to as
‘adenomatous’ or Adenocarcinoma.
2. Uniformity or lack thereof, of the size of cells and nuclei as well as hyperchromasia and mitotic activity of the latter. He assessed eight morphological parameters, namely the amount of gland formation, the presence of secretory vacuoles, cell size, nuclear size, variation in the size of both cells and nuclei, degree of nuclear pleomorphism and number of mitoses. On this basis he recognized three ‘classes’ of breast cancer in ascending order of malignancy, that is dedifferentiation.
The next landmark is provided by the studies of Patey and Scarff and his colleagues in 1927, who followed Greenough method, but placed emphasis on the amount of tubule formation, inequality in size of nuclei and hyperchromatism. Mitoses were considered to be of less
importance. They correlated histological grade with survival outcome and axillary lymphnode status.
In 1957, Bloom, a radiotherapist and Richardson, a surgical research fellow introduced a numerical scoring system to grade breast cancers.12 They divided breast carcinomas into three categories based on tubular arrangement of cells, variation in the size, shape and staining of the nuclei and frequency of mitotic figures. They allotted points 1,2 or 3 to each of the three histological factors and added together making a possible score of 3 to 9, the smallest number referring to the lowest grade of malignancy. They followed Scarff in dividing this malignant scale into approximately three equal parts. Thus 3 to 5 points – low (grade 1), 6 or 7 points – intermediate (grade 2), and 8 or 9 – high (grade 3). This grading scheme has become the most popular worldwide, adopted by the WHO and referred to as the Bloom – Richardson or Scarff – Bloom – Richardson (SBR) technique. They also concluded that grading reflects the potential malignancy of the tumor and indicates which cases are more likely to have distant metastases at the time of treatment and provide a guide to the speed with which such metastases become active, produce symptoms and cause death.12,32,78,81
Black and colleagues in 1975, reevaluated the multifactorial method which Bloom advocated, concluding that tubular differentiation made no contribution to prognosis and devised four grades of malignancy based on the regularity of the nuclear outline, delicacy of chromatin strands, presence or absence of nucleoli and mitotic figures. They reversed the numerical order of their grades so that grades 0 to 1 apply to poorly differentiated carcinomas and grades 3 to 4 to well differentiated tumors. Black’s method has been used exclusively in the United States where it is referred to as nuclear grading.
In Fisher’s modification of Black’s grading method, three grades are used with grade 1 representing the highest level of differentiation and grade 3 equivalent to anaplasia as shown in table 2. Grade 1 and 2 may be clumped together as a ‘favourable nuclear grade’, for they are prognostically similar and favourable while grade 3 is considered an ‘unfavourable nuclear grade’ and has a poorer prognosis. These grading schemes have been used for ductal carcinomas of all types.
Table 2: Fisher’s modification of Black’s Nuclear grading Fisher’s
modification
Black Anisonucleosis Size Mitosis Nucleoli Chromatin
3 0 ++ ++ ++ ++ ++
1 + ++ + + +
2 2 ± + + + +
1 3 _ _ ± , + ± , + _
4 _ _ - , ± - , ± _
Histological grading of breast carcinoma has been severely criticized for its apparent lack of reproducibility among pathologists yet, when carefully defined criteria are used, the same histological grade is assigned by highly trained observers in upto 90% of cases.68 A second criticism is that the various grading schemes divide patients unequally into significantly different prognostic categories. It appears that SBR scheme places too many patients in grades 2 and 3 where there is significant prognostic separation (particularly in patients with node negative disease).
Le Doussal and colleagues, in 1989 noted that mitotic figure counts and nuclear grade were more predictive of outcome than was tubular differentiation which could be dropped from consideration in grading and they created modified Scarff – Bloom – Richardson scheme (MSBR) composed of five categories based on sum of 1 to 3 points assigned to nuclear grade and mitotic figure count.66 This showed statistically significant risk ratios for metastases free survival, hence they concluded that MSBR scheme is more accurate and predictive than standard SBR system particularly useful when nodal status of the patient is negative or unknown.
Schumacher and colleagues in 1993 also found that a combination of nuclear pleomorphism and mitotic count gave most significant results.
Parham et al assessed stromal fibrosis and tumor necrosis in addition to the three conventional components of histologic grade. They concluded that mitotic count and tumor necrosis correlated best with survival proposed and that these two factors be combined to form a
new simplified method for grading breast cancer. Unfortunately methodology supplied in the paper was so imprecise that the proposed method must be virtually impossible to reproduce.
Elston and Ellis in 1991, introduced greater objectivity to the assessment of the component factors and formulated the Elston – Ellis – Modified SBR system also referred to as the Nottingham combined histologic grade (NCHG).31
Technique
Grade is based on assessment of three morphological features, relative degree of tubule formation, nuclear pleomorphism and mitotic counts and each assigned a score of 1 to 3 as shown in the following table 3 and 4.
Table 3: Elston and Ellis’ Modification of the Bloom-Richardson System
Feature Score 1 Score 2 Score 3
Tubule formation Majority of tumor (>75%)
Moderate degree (10- 75%)
Little or none (< 10%
or none)
Nuclear
pleomorphism
Small, regular and uniform size and shape, Dispersed chromatin, No prominent nucleoli
Intermediate size, somewhat pleomorphic, Presence of nucleoli.
Large, varying size and shape, Coarse chromatin, Prominent, multiple nucleoli.
Mitotic count (per 10 high power fields)
As per the microscopic field area
Tubule formation- Modifications include more objective criteria like, tubular structures must exhibit central clear lumina, all parts of each tumor block are scored and proportion occupied by such tubular structures assessed semi quantitatively assigning a score of 1 when more than 75%
of the area is composed of definite tubules.
Nuclear pleomorphism- They suggested that size and shape of normal epithelial cells in the breast tissue adjacent to the tumor or inflammatory cells such as lymphocytes be used as reference point in order to introduce a degree of objectivity.
Mitotic counts- Significant modifications have been made in this area. Recommendations included are
mitotic counts are determined at the most cellular invasive periphery of the tumor.
Start with a mitotic figure, and then count a minimum of 10 contiguous high power fields of invasive tumor at the periphery.
Ignore carcinoma in situ.
Count only clear cut mitotic figures (metaphase, anaphase and telophase). Criteria include absence of a nuclear membrane, hairy extensions of the nuclear material, either clotted (beginning metaphase), in a plane (metaphase/ anaphase), or in separate clors (telophase).67
Ignore hyperchromatic, apoptotic and lymphoid cell nuclei.
Table 4: Calibrated field area for assignment of points.
Field diameter in mm
Number of mitoses corresponding to
Score 1 Score 2 Score 3
0.40 Up to 1 5 to 8 9 or more
0.41 Up to 4 5 to 9 10 or more
0.42 Up to 4 5 to 9 10 or more
0.43 Up to 4 5 to 10 11 or more
0.44 Up to 5 6 to 10 11 or more
0.45 Up to 5 6 to 11 12 or more
0.46 Up to 5 6 to 11 12 or more
0.47 Up to 5 6 to 12 13 or more
0.48 Up to 6 7 to 12 13 or more
0.49 Up to 6 7 to 13 14 or more
0.50 Up to 6 7 to 13 14 or more
0.51 Up to 6 7 to 14 15 or more
0.52 Up to 7 8 to 14 15 or more
0.53 Up to 7 8 to 15 16 or more
0.54 Up to 7 8 to 16 17 or more
0.55 Up to 8 9 to 16 17 or more
0.56 Up to 8 9 to 17 18 or more
0.57 Up to 8 9 to 17 18 or more
0.58 Up to 9 10to 18 19 or more
0.59 Up to 9 10 to 19 20 or more
0.60 Up to 9 10 to 19 20 or more
0.61 Up to 9 10 to 20 21 or more
0.62 Up to 10 11 to 21 22 or more
0.63 Up to 10 11 to 21 22 or more
0.64 Up to 11 12 to 22 23 or more
0.65 Up to 11 12 to 23 24 or more
0.66 Up to 11 12 to 24 25 or more
0.67 Up to 12 13 to 25 26 or more
0.68 Up to 12 13 to 25 26 or more
0.69 Up to 12 13 to 26 27 or more
0.70 Up to 13 14 to 27 28 or more
Allocation of grade
To obtain overall tumor grade, the scores for each factor are added together, giving a possible total of 3 to 9 points.
Grade I = 3 – 5 points (well differentiated/low grade)
Grade II = 6 – 7 points (Moderately differentiated/intermediate grade) Grade III = 8 – 9 points (Poorly differentiated/high grade)
Elston and Ellis continue to grade all histological types of invasive carcinoma. Most special types fall into SBR grade 2 suggesting that the grade explains the better prognosis. An exception is Medullary carcinoma, which almost always is grade 3 but has controversial prognosis.
Significance
Dalton et al did a study in which a single slide from 10 invasive breast carcinomas was submitted to 25 pathologists. Their study indicated that reproducibility of grading breast cancers can be achieved when a histologic grading scheme with specific guidelines is used.
Another reproducibility study was done by John S. Meyer and colleagues in 9000 breast carcinoma specimens, grading been evaluated by 7 pathologists. They concluded that Nottingham histologic grade is only modestly reproducible. Prognostically useful components of grade are mitotic index and tubularity, nuclear grade being the weakest. They stated that
Nottingham system can be improved by lowering cutoffs of mitotic index and by counting 20-30 rather than 10 high power fields and that measurement of proliferation index by immunohistochemically detected markers will probably give superior prognostic results in comparison to grade.
Emad A. Rakha et al have stated that as the prognostic value of NGS has been proven in operable breast cancer (stages I and II), in which decisions about systemic therapy usage and its regimen need to be made, histological grade could be incorporated in the TNM system to improve its ability to stratify cases into risk-associated subcategories.34 However grading has limited value in advanced or metastatic breast cancer (stages III and VI) and grading is not expected to change treatment decisions and therefore need not be considered in these cases.
Clayton and Hopkins showed the prognostic significance of histologic grading within the category of IDC no special type. Pereira and colleagues found that histologic type and grade when used together, more accurately predict prognosis.21
Evidence that histological grade can be used to predict outcome is that it correlates with other features of breast carcinoma associated with prognosis like rapid tumor cell replication (shown by flow S phase, high H- thymidine uptake or bromodeoxyuridine labeling) or frequent immunostaining of tumor cells by Ki-67 proliferation marker. Flow or static aneuploidy, HER 2 expression and absence of ER or PR also correlate with poor outcome and higher histologic grade.
Bloom and Richardson in their study of 1409 cases of breast cancer divided them into three grades of malignancy depending upon simple histological criteria and found a good correlation with prognosis based upon 5, 10- and 15-year survival rates.12 The 5-year survival rate varied from 86 per cent for low grade cases with the axilla free to 19 per cent for high grade cases with the axilla involved. The corresponding figures at 10 years were 61 and 9 per cent, and at 15 years 49 and 7 per cent. They stated that grading reflects the potential malignancy of the tumor and indicates which cases are more likely to have occult distant metastases at the time of treatment. However, as metastases appear to be common in all three grades of tumor when the patient first seeks advice, the real value of histological grading is in providing a guide to the speed with which such metastases become active, produce symptoms and cause death.
Davis and colleagues studied the prognostic significance of histologic tumor grade in 1537 women with node-positive breast cancer.27 The 5-year overall survival rates were: Grade 1, 86%; Grade 2, 70%; and Grade 3, 57% (P < 0.0001). They showed that tumor grade is a statistically significant prognostic factor for disease free survival and overall survival in multivariate analyses controlling for nodal status, tumor size, estrogen receptor status, menopausal status, age, peritumoral vessel invasion, and treatment assigned.
Donald Earl Henson et al studied the survival rates for 22,616 cases of breast cancer listed in the SEER program.29 They found that patients who were assigned Stage II, Grade 1 had the same survival as those assigned Stage I, Grade 3. Their survival was better than patients assigned Stage I, Grade 4. The 5-year relative survival rate for patients listed as Stage I, Grade 1 was 99% and for patients listed as Stage I, Grade 2, it was 98%. At 10 years, the survival rate of patients assigned Stage I, Grade 1 was 95%. They concluded that histologic grade when used in conjunction with stage of disease can improve the prediction of outcome.
Baak et al in their study of 271 breast cancer patients showed that the 5 year survival rate for grade I breast carcinoma to be 97%, grade II to be 78% and for grade III to be 55%.55
Arthur K. C. Lee et al studied the outcomes of 88 T1a,bN0M0 carcinomas in 87 patients and found that they have a low recurrence rate, especially those tumors ≤ 0.5 cm, or of low histologic or nuclear grade, or of favorable histologic type.7 Also the disease free survival rate of grade I carcinomas was found to be 100% and that of grade II & III to be 89%.
Clayton et al studied the long term prognosis of 378 lymph node-negative Infiltrating ductal breast carcinomas and found that the 5 year survival rate of grade I carcinomas to be 93%, grade II to be 91% and that of grade III to be 75 %.40
Nottingham prognostic index (NPI)
Analysis of a range of prognostic factors using multiple regression technique of cox, showed that only tumor size, histological grade and lymphnode stage gave a significant correlation with overall survival and a simple composite prognostic index has been devised as follows: 31
NPI = 0.2 x tumor size + lymphnode stage (1 – 3) + histological grade (1 -3)
Thus the prognostic value of grading can be improved by this NPI, which has also been shown to be a reproducible method of prognostication. Prognosis worsens as the numerical value of NPI increases and by using cut off points of 3.4 and 5.4, patients may be stratified into good, moderate and poor prognostic groups having an annual mortality of 3, 7 and 30% respectively.
5) Estrogen and Progesterone Receptors (ER &PR)
Current assays use immunohistochemistry to detect the nuclear hormone receptor status.
Women with hormone receptor-positive cancers have a slightly better prognosis than do women with hormone receptor-negative carcinomas. The evaluation of hormone receptors is most valuable to predict response to hormonal therapy.63,102
Jean F. Simpson et al studied 560 axillary node positive breast cancer patients and found that estrogen receptor status was significantly associated with histologic grade and also with time to recurrence and disease free survival.57
6) HER-2/neu (Human Epidermal growth factor Receptor 2)
HER-2/ neu or c-erb B2 is a transmembrane glycoprotein involved in cell growth control encoded by a gene on chromosome 17. HER2/neu is overexpressed in 20% to 30% of breast carcinomas and is associated with a poor prognosis. Overexpression of this oncogene as determined by immunohistochemistry or Fluorescent in situ hybridization (FISH) is an excellent predictor of response to Herceptin/Trastuzumab but a weak predictor of response to chemotherapy. Trastuzumab is a humanized monoclonal antibody to HER-2/neu developed to specifically target tumor cells and it is hoped to spare normal cells.63,102
Gloria peira et al studied the expression of HER2 in 162 lymphnode negative breast carcinoma through IHC, Chromogenic in situ hybridization (CISH) and Fluorescent in situ hybridization and stated that HER2 overexpression was present in tumors of high grade, with necrosis and lympho vascular invasion.46,91 They concluded that IHC is a reliable method that can be complemented by CISH in nondefinite cases and a promising practical alternative to FISH for the assessment of HER2 gene status.
7) Lymphovascular invasion
Tumor cells may be seen within vascular spaces (either in lymphatic or in small capillaries surrounding tumors) in half of all invasive carcinomas. This finding is strongly associated with the presence of lymph node metastasis. It is a poor prognostic factor and a risk factor for local recurrence.
Donald Earl Henson et al through their study stated that the presence of vessel invasion by primary tumor cells was a stronger predictor of early recurrence than was increasing tumor grade in postmenopausal patients who received no adjuvant therapy.29
Chisako Yamauchi et al studied the lymph vessel tumor emboli by H & E staining and D2-40 staining in the intratumor area or nontumor area and stated that they significantly increased the risk for tumor recurrence or death of patients with IDC, independent of hormone receptor status or nodal status.17
8) p53
Accumulation of p53 protein as a result of gene mutation have been said to correlate with reduced patient survival. It has been shown that loss of heterozygosity for p53 is strongly associated with high histologic and nuclear grade.63
Philippe bertheau et al studied p53 gene products in 112 primary breast carcinomas and demonstrated that p53 positivity was associated with high histological grade and shorter overall survival.82
9) DNA content
The amount of DNA per tumor cell can be determined by flow cytometric analysis or by image analysis of tissue sections. Aneuploid tumors are those with abnormal DNA indices and have a slightly worse prognosis.
Henry F. Frierson et al in their study of ploidy in 118 infiltrating ductal carcinomas found that 40% of grade 1, 71% of grade 2 and 83% of grade 3 neoplasms were DNA aneuploid.51
Jean F. Simpson et al studied 560 axillary node positive breast cancer patients and found that 19.6% of grade 1, 43.6% of grade 2 and 36.7% of grade 3 neoplasms were DNA aneuploid.59
10) PROLIFERATION INDICES
The ability of the cancer cells to divide and replicate is the basis of the continued expansion of tumors. There are many different techniques for evaluating the rate of cell proliferation : mitotic index, Thymidine labeling index (TLI), S- phase fraction by flow cytometry (SPF), Bromodeoxyuridine index (BrdU) and immunohistochemistry using antibodies against specific cell nuclear antigens (Ki-67/MIB-1, Ki-S5, Ki-S1), various cyclins and mitosin.
Cell proliferation takes place through a defined process in which several phases can be recognized. From the resting G0 phase, they join the active cycling population after appropriate stimuli and enter the first gap G1 phase. Both phases have a highly variable duration. In G1, the cell prepares for the synthesis S phase, in which DNA synthesis and doubling of the genome takes place. The S phase is followed by a period of apparent inactivity known as the second gap phase G2, in which the cell prepares for further separation of chromatids during the mitotic M phase. After the M phase, each daughter cell may enter G0 phase or move on to the G1phase to repeat the cell cycle.
Mitotic index
It is the oldest method for quantitating proliferation by counting the number of mitotic bodies by light microscopy on hematoxylin and eosin stained paraffin embedded tumor specimens.
The interface which comprises the G1, S and G2 phases, forms the largest part of the cell cycle. But cells in these phases cannot be morphologically recognized. However, cells in the mitotic phase can be easily identified because of the typical appearance of the chromosome cells during the different subphases of the M phase. This has been the basis for light microscopical counting of mitotic figures.100
Mitotic index is the total number of sharply defined mitosis in ten high power fields. It is the most important constituent of histological grade.55
Few studies have validated this approach in assessing clinical outcome and these indicate that higher mitotic rates are associated with increased mortality.
Clayton et al, reported a study of 378 node negative breast carcinomas and found that on multivariate analysis, mitotic count was a stronger predictor of survival than tumor size, lymphatic invasion or skin invasion and patients with more than 4.5 mitotic figures per 10 high power field had a 2.8 fold increase in the risk of death.21
Baak et al did a study to validate the independent strong prognostic value of mitotic activity index (MAI) in lymphnode (LN) –negative invasive breast cancer patients younger than 55 years in a nationwide multicenter prospective study comprising 516 patients.56 The absolute difference in 10-year Kaplan-Meier estimates of time to distant recurrence as well as survival was 22% between MAI less than 10 versus ≥ 10. This effect was independent of age, estrogen receptor (ER) status, and tumor diameter. They concluded that MAI is the strongest, most widely available, easily assessable, inexpensive, well reproducible prognosticator and is well suited to routinely differentiate between high- and low-risk LN-negative breast cancer patients younger than 55 years.
Baak et al in another study of 271 breast cancer patients evaluated the value of morphometry to classic prognosticators of breast cancer and showed the significance of several features such as lymphnode status, tumor size, nuclear and histological grade, and morphological variables like mitotic activity index.55 They concluded that mitotic activity index is the best single predictor of the prognosis.
Jean F. Simpson et al studied 560 axillary node positive breast cancer patients and stated that a subset of axillary lymph node–positive patients with improved prognosis may be identified by using a lower (< 3 mitoses/10 HPF) mitotic count than is usually performed.57
Anne Marie Mandard et al studied the prognostic value of DNA cytometry in 281 premenopausal patients with lymph node negative breast carcinoma randomized in a control trial Metastasis free survival was 97% and 73% when MI was less than 10 and MI was greater than or equal to 10, respectively.3
Although the methods of calculating mitotic index and the cut off to define high and low mitotic index varies, mitotic index appears to be a simple and cost effective method for measuring proliferation rate. Limitations include variability in counting, poor reproducibility, confusion with apoptosis or nuclear pyknosis and correlation with proliferation rate not necessarily linear due to variability in the duration of the mitotic phase of the cell cycle.
Thymidine labeling index
Thymidine labeling index measures the number of cells synthesizing DNA during the incubation and therefore provides an estimate of the proportion of the cells in S and G2/M phases of the cell cycle.
It is assessed by counting the number of labeled nuclei on auto-radiographed micro sections after incubation of the fresh tumor specimen with titrated thymidine.
Several studies have shown improved survival in node negative breast carcinoma patients with slow proliferating tumors and the relative risk of relapse on multivariate analysis in fast proliferating tumors was approximately two fold.23,98
Advantage - Accurate even in slowly proliferating tumors and reproducible.
Disadvantage - Requires handling of radioisotope. Requires time-consuming autoradiography. Needs fresh tissue.
S – phase fraction by flow cytometry
The S phase fraction is a measure of cells in the S phase of the cell cycle undergoing DNA synthesis. It is based on estimating the proportion of cells with partially replicated DNA (i.e., cells with more DNA than the normal amount in a cell in G1 and less than double the normal amount as is seen in G2/M phases of the cell cycle).
Cells are mechanically dispersed, stained with propidium iodide and passed through a flow cytometer, which produces a DNA histogram with distributions corresponding to phases in the cell cycle.69 The major peak corresponds to G1/G0 phase. DNA content (ploidy) and proliferation can be assessed. If there is a second major peak, the tumor is considered aneuploid, otherwise it is considered diploid.
