Immunohistochemistry of breast lesions(IHC)

IHC of Breast lesions.

1. DR. NOOPUR S. PATIL IMMUNOHISTOCHEMISTRY IN BREAST PATHOLOGY

2. Immunohistochemistry (IHC) BASICS: Technique used to detect & localize antigens by means of labeled antibodies through Ag – Ab interaction that are visualized by enzyme - substrate – chromogen reaction It takes its name from the roots " immuno ," in reference to antibodies used in the procedure, " histo ," meaning tissue & “chemistry” meaning enzymatic reaction. Visualizing an antibody-antigen interaction can be accomplished in a number of ways. In the most common instance, an antibody is conjugated to an enzyme, such as peroxidase , that can catalyse a colour -producing reaction. PRINCIPLES: Direct labeling method - enzymes are directly labeled with the primary antibody, less sensitive •Indirect labeling method – enzymes are labeled with the secondary Ab which is produced against the primary Ab , more sensitive. •ABC method – the sec. Ab & the avidin are labeled with biotin & enzyme respectively. The avidin has high affinity to biotin . IHC makes use of this high affinity by using biotinylated Abs to amplify tissue Ags

3. INTRODUCTION: Immunohistochemistry (IHC) is used to characterize intracellular proteins or various cell surfaces in all tissues. Individual markers or more often panels of various marker proteins can be used to characterize various tumour subtypes, confirm tissue of origin, distinguish metastatic from primary tumour and provide additional information which may be important for prognosis, predicting response to therapy or evaluating residual tumour post-treatment. There is a growing list of available products (antibodies) or antigen retrieval techniques, which all contribute to the broader utility of immunohistochemistry for solving diagnostic problems or for determining prognosis and response to therapy in breast pathology.

4. It is well known that normal glandular breast tissue is composed of three cell types which express different subsets of proteins: The most important diagnostic problems that occur in mammary gland tumor pathology are: the differential diagnosis of various types of benign lesions and carcinoma; differentiating between carcinoma in situ and invasive carcinoma, diagnosis and differentiation of microinvasion and its imitating lesions and confirming the breast as the primary site in metastatic carcinoma. In the absence of advanced molecular biological techniques, IHC can be use to identify histologic subtype or molecular phenotype.

5. basal cell types express different cytokeratins (5/6, 14, 17 )

6. Analysis of prognostic markers e.g. Prognostic or Predictive Factors in Breast Carcinoma: Hormone Receptors Solving common diagnostic dilemmas- Differential Diagnosis of Epithelial Lesions: Myoepithelial Cells and papillary lesions of breast Tumour typing and confirming diagnoses- e.g. Lobular v Ductal; Luminal v Basal/Myoepithelial; Distinction DIN (DCIS) and LIN (LCIS) Cell Population in Intraductal Proliferative Lesions: Homogeneous Versus Heterogeneous Cell Population (Neoplasia Versus Hyperplasia ) INDICATIONS OF IHC IN BREAST LESIONS :

7. Subtle foci of invasion/Microinvasive Carcinoma and status of margins Lymph node metastases- Micrometastatic Disease in Axillary Lymph Nodes (Including Sentinel Nodes ) Demonstrating epithelial cells in necrotic material Paget’s Disease Systemic Metastasis of Breast Carcinoma Metastases to breast Spindle cell lesions

8. Endocrine treatment for breast cancer- late 1890s by Beatson , when he observed what would now be called responses in a few young women with apparent locally advanced breast cancer after he performed surgical oophorectomy . Beatson presumed that he was interrupting neural connections between the ovaries and the breast, although he was actually removing the ligand estrogen from the patient’s circulation, which diminished estrogen availability to its protein receptor ( ER ) in the breast HISTORICAL IMPORTANCE :

9. MARKERS OF PROGNOSIS AND RESPONSE TO THERAPY The most common immunohistochemical breast cancer prognostic and therapeutic markers used include: ER, HER2 , Ki-67, PR, and p53. In addition markers of angiogenesis and apoptosis are used . Nowadays, immunohistochemical detection of ER and PR is part of the routine work-up of breast cancer, and in some cases of DCIS the presence of ERs is an indication for tamoxifen therapy.

10. ER and PR in Carcinoma breast Normal breast epithelial cells- ER and PR receptors , proliferate under their influence. 75% to 85% Ca breast stimulated to grow when these hormones are present . ER Clinical rational e : identify pat i ents who may benefit from hormonal therapy. – St r ong predictive factor It is also a minor prognostic factor . PR PR status is determined to identify a small number of carcinomas (in most series <5%) that are PgR positive and ER negative but which may respond to hormonal therapy. – Predictive factor Progesterone receptor is also a minor prognostic factor

11. Prognostic factor - any measurement available at the time of diagnosis or surgery that is associated with clinical outcome in the absence of systemic adjuvant therapy. Predictive factor - associated with response or lack of response to a particular therapy. ER- Strong predictive factor for response to endocrine therapy. Weak prognostic factor PR expression is induced by ER - surrogate marker for ER activity ( Additional predictive factor for hormonal therapy in breast cancer ) One of the effects of estrogen is to induce the PR (ER and PR + tumors form 99% of receptor positive tumors) and ER-negative, PR-positive tumors account for fewer than 1% of all breast cancers Nuclear immunostaining for both receptor proteins can be demonstrated in normal breast acini (15-20%), which serve as internal controls for the testing procedure

12. Estrogen receptors (ER) ER-alpha: “classic” functions of ER; susceptible to proliferative stimulation of estrogen; breast and endometrium; ER- beta:“ housekeeping” functions; normal ovary and granulosa cells, carcinoma of breast, colon , prostate ;

13. ER status is strongly influenced by tumor grade and histology. Virtually all grade I tumors are ER positive, bcl2+ tumors, as are pure tubular, colloid, and classic lobular carcinoma. PR is an estrogen-regulated gene , and its synthesis in normal and cancer cells requires estrogen and ER . Some studies have shown that ER and PR status can change over the natural history of the disease or during treatment

14. Response rate ER(+) PR(+) 78% ER(+) PR(-) 34% ER(-) PR(+) 45% ER(-) PR(-) 10%

15. Assay methods Cytosol ligand binding assay Enzyme immunoassay Immunohistochemical assay Antibody used : * H 222 * 1D5 ER is detected in the nucleus by immunohistochemical studies.

16. Tissue fixation ER are thermolabile and short half life Incised immediately after resection to ensure rapid penetration of fixative Most tissue fixatives can be used to preserve receptor reactivity if microwave predigest is used Control : To avoid risk of false negative classification

17. FALSE NEGATIVE Exposure of the carcinoma to heat ( cautery during surgery). Prolonged cold ischemic time (the time between tissue removal at surgery and initiation of fixation). Type of fixative: ER is degraded in acidic fixatives such as Bouin’s and B-5. Prolonged fixation in formalin: Optimally at least 8 hours in buffered formalin. Fixation for more than 3 weeks can diminish immunoreactivity Decalcification: results in loss of immunoreactivity. Incorrect antigen retrieval method. Non-optimized antigen retrieval.

18. Quantification systems Quantification systems may use only the proportion of positive cells or may include the intensity of immunoreactivity . Number of positive cells : As a percentage or within discrete categories . Intensity : Refers to degree of nuclear positivity (ie, pale to dark). The intensity can be affected by the amount of protein present, as well as the antibody used and the antigen retrieval system . Most cancers - Heterogeneous immunoreactivity with pale to darkly positive cells present

19. Scoring for ER Immunostains Conventional scoring : Semi quantitative fashion incorporating both the intensity and the distribution of specific staining as described by Mc Carthy, Jr et al . Allred scoring : Semi quantitative system that takes into consideration the proportion of positive cells(scored on a scale of 0-5) and staining intensity (scored on a scale of 0-3 ). The proportion and intensity were then summed to produce total scores of 0 or 2 through 8.

20. Scoring system for ER/PR Score for propo r tion Score for intensity 0= No staining 0= No staining 1<1% staining 1=Weak staining 2=(1-10)% staining 2= Moderate staining 3=(11-33)% staining 3=Strong staining 4=(34-66)% staining 5=(67-100)% staining Total score ranges from to 8. Tumors scoring ≤2 are regarded as ER negative and have a negligible chance of response.

21. IHC for ER/PR

22. Preanalytical optimum formalin exposure time for ER determination is 8 hours antigen can be retrieved with increasing retrieval times for over-exposed tissue, but an under-fixed tissue is completely useless for biomarker study. Analytical All commercially available antibodies for ER assessment in breast carcinoma target only ER-alpha isoform. Post analytical NIH Consensus Conference of December 2000 states, “Any nuclear expression of HRs should be regarded as a positive result and render a patient eligible for hormonal therapy

23. HER 2/ neu (c- erb B2) Proto oncogene Homologous to human epidermal growth factors Encodes 185 KD transmembrane protein having intra and extra cellular domain Intra cellular domain with tyrosine kinase activity and extracellular domain with binding site for growth factor.

24. Over expression of c- erb B2 in 1/3 rd of breast cancer Over expression is associated with High histologic grade Reduced survival Lower response to methotrexate , tamoxifen Higher responsiveness to doxorubicin Monoclonal antibody trantuzumab ( Herceptin ) have role in treatment of tumor cell lines over expressing the HER – 2 protein

25. HER2 Testing by Im m unohistochemistry It is a marker for sensitivity to Herceptin ( trastuzumab ), and resistance to tamoxifen . Although Her-2/ neu can be detected using many methods, only two are currently approved and recommended for its detection: IHC and fluorescence in situ hybridization (FISH). Scientific rationale : A subset of breast carcinomas (approximately 15% to 25%) overexpress the epidermal growth factor receptor HER2. The mechanism of overexpression - amplification of the gene resulting in increased amounts of protein Clinical rationale: To determine if a carcinoma will respond to treatment directed against the protein (eg, treatment with trastuzumab or lapatinib

26. IH C - according to ASCO/ CAP

27. Clinical Implication The availability of trastuzumab, a humanized monoclonal antibody against HER2 has improved progression-free survival and overall survival, as part of adjuvant treatment for both early high-risk disease and metastatic disease

28. BRCA 1 mutation (17q21) IHC - new and powerful predictor of BRCA1 mutation status ER-negative (usually triple negative) will roughly double the probability that the individual is a BRCA1 carrier . Er positive - reduce the probability by approximately fivefold . ( a/w BRCA2-13q12)

29. Markers of apoptosis and cell proliferation: Ki-67 proliferation index, BCl-2, p53  Ki-67 ( MIB-1 ) : Ki-67, a non- histone protein, involved in the early steps of polymerase I-dependent ribosomal RNA synthesis is a predictive and prognostic marker in cancers and has been extensively studied. When Ki-67 level is above 10%-14%, breast cancer patients are defined as high-risk Proliferation marker, stains cells in all cell cycle phases except the resting phase (G0); the percentage of stained nuclei is the proliferation index. A low proliferation index is associated with slower tumor growth,better prognosis, whereas the converse is true for a high proliferation index.

30. According to the St. Gallen Consensus (2009), the Ki-67 index is useful for selecting patients with hormone receptor-positive breast cancers for the addition of chemotherapy to endocrine therapy. Thus, breast tumours are classified as low, intermediate, and highly proliferating according to a Ki-67 labelling index of under 15%, 16%-30%, and over 30%, respectively. Data from the Clinical Cancer Registry Regensburg showed that Ki-67 expression was associated with common histopathological parameters, especially grading and survival, but is an additional independent prognostic parameter for diseasefree survival and overall survival in breast cancer patients. Ki-67 expression has been used to determine the effects of different doses of tamoxifen on breast cancer proliferation[42,46]. The change in Ki-67 expression induced by lower doses of tamoxifen was comparable to that achieved with the standard dose, indicating that tamoxifen retains antiproliferative activity at low doses

31. With regard to the molecular breast cancers, high Ki-67 proliferation index can be used to classify triple negative breast cancer into subtypes with different prognosis or responses to treatment. For this purpose, the number of Ki-67 positive cells among the total number of counted tumour cells was determined and the high expression of Ki-67 was defined as ≥ 10%. It is known that patients with triple negative breast cancer have a poor survival, despite their high response rate to neoadjuvant chemotherapy, and those with high Ki-67 have more aggressive clinical features.

32. P53 The other marker, p53 is well studied in cancers, but its value in predicting clinical outcome in breast cancer is debatable. The p53 gene is located on the short arm of chromosome 17 and encodes a 375 amino acid nuclear phosphoprotein that prevents propagation of genetically modified cells. Wild-type p53 is a tumour suppressor protein and plays an essential role in regulating genomic stability by controlling the cell cycle and inducing apoptosis when cell damage cannot be repaired. In normal cells, p53 has a very short half-life due to ubiquitylation and proteasome degradation. IHC can be used, as wild-type p53 protein is rapidly degraded, while TP53 mutations (18%-25% of primary breast carcinomas) are often associated with the production of a stable protein. In addition, sequencing of the p53 gene in all breast cancers would be expensive and time-consuming for routine practice. A higher tumour grade, negative ER and PR status, and the more aggressive basal subtype were associated with abnormal p53 immunohistochemical expression or p53-positive status. With regard to early breast cancers, some scientists have reported that a p53 mutation has no influence on the outcome and therefore, the value of p53 status is too weak to be recommended as a routine marker in clinical practice.

33. Bcl 2 : BCL2 belongs to a group of protein key regulators of apoptosis or programmed cell death Zhang et al. - bcl-2 expression - better response to hormone therapy, and the expression of bcl-2 is a favorable prognostic factor regardless of nodal status. Berardo et al .- high bcl-2 expression was associated with a significantly improved disease-free survival and overall survival, Gee et al. found that patients with estrogen-receptor and bcl-2-positive tumors were particular responsive to endocrine therapies that included an anti-estrogen. van Slooten et al. found no association between bcl- 2 expression and response to perioperative chemotherapy in node-negative patients. Bonetti et al. reported a higher response rate to chemotherapy among tumors classified as bcl-2 positive with immunostaining in ≥40% of tumor cells.

34. Angiogenesis markers: Tumor growth and metastasis are dependent on tumour angiogenesis and this complex process involves a delicate balance between angiogenic and antiangiogenic factors. Numerous studies have investigated the relationship between tumour angiogenesis, prognosis and response to antiangiogenic drugs. Analysis of these factors in tumour or serum of breast cancer patients by IHC or multiplex protein assay ( FASTQuant ® Microspot Assays) can improve diagnosis and prognosis of the disease. There is a large list regarding angiogenesis markers: Angiogenin , Ang2, keratinocyte growth factor (KGF), fibroblast growth factor basic, intercellular adhesion molecule (ICAM)-1, platelet-derived growth factor-BB and the vascular endothelial growth factor family.

35. The commonly used method to determine angiogenesis is counting intratumoral blood vessels (MVD) stained with factor Ⅷ related antigen or anti CD31 or CD34 using light microscopy. Counting newly formed stained microvessels is a useful tool in the early detection of metastatic potential and in the selection of patients for whom anti angiogenesis drugs might be beneficial. The reactivity level of CD34 antigen was assessed by IHC in all types of invasive ductal breast cancer and its level seems to be a useful predictor for the development of local lymph node metastasis and can indicate the benefit of antiangiogenic treatment

36. Blood vessel invasion stained for CD31 positive endothelium CD31 positive endothelium D2-40 positive endothelium. D2-40 negative endothelium

37. D2-40 CD 31

38. VEGF: Most of the biological and clinical activity of the antiangiogenic drugs currently approved for cancer therapy is against the VEGF-related pathways. The VEGF system is part of the platelet-derived growth factor gene family, and interacts with its specific receptors; VEGFR-1 (flt-1) and VEGFR-2 (flt-2) for VEGF-A, a very potent angiogenic growth factor. VEGF-B, interacting with VEGFR-1, seems to have an important role in the maintenance of existing vessels, but this protein is not well studied. VEGF A and B, their receptors VEGFR-1 and 2 are expressed in a variety of normal cells, and overexpression has been described in malignant tumors. The prognostic importance of VEGF in invasive breast cancer is associated with tumour stage and ER status, and inversely correlated with tumour grade and measurement of tumour VEGF, as an indicator of angiogenesis, which is more reliable prognostically than measurement of microvessel density or serum VEGF

39. DIAGNOSTIC MARKERS: Myoepithelial markers: (SMA,P63,CALPONIN,SMMHC) Myoepithelial markers are useful in helping to distinguish invasive carcinoma from benign proliferations with a similar morphological appearance, benign proliferative lesions and most preinvasive lesions with an intact myoepithelium . Invasive carcinomas lack the myoepithelial cell layer that normally surrounds benign breast glands. There is an exception, microglandular adenosis , a benign proliferative lesion which lacks the myoepithelial cell layer. Using IHC, positive myoepithelial staining is seen in the benign area with attenuated or absent staining in areas of atypia or in situ carcinoma.

40. p63 The nuclear protein p63 - homologue of p53 that is expressed in the basal epithelia of multiple organs. In the breast, p63 is positive in nearly 100% of normal MECs and those associated with benign proliferations Advantages Nuclear staining pattern, which removes the interpretation difficulties that may be associated with the cross-reactivity for myofibroblasts seen with many of the other markers High sensitivity.

41. Discontinuous staining pattern (Focal gaps) - morphologic impression that MECs are absent

42. SMA : Smooth muscle actin (SMA) has long been used as a myoepithelial marker in breast pathology diagnosis as a sensitive marker of myoepithelial differentiation, even if it is not specific, because any cell with substantial expression of actin is positive for SMA ( myofibroblasts and blood vessels are positive for SMA). This becomes problematic in lesions where there are either myofibroblasts or blood vessels in close proximity to the epithelial lesion. One pitfall is the presence myofibroblasts within desmoplastic stroma adjacent to nests/ glands of invasive carcinoma being misinterpreted as myoepithelial cells, resulting in a false-negative diagnosis. This is why the use of a panel of markers (p63, calponin , smooth muscle myosin, CD10, S100) or a more specific marker such as p63 are recommended.

43. Smooth Muscle Myosin Heavy Chain SMMHC is a structural component of myosin - specific for smooth muscle cells,terminal smooth muscle differentiation . SMMHC is associated with contractile elements and is present in all cells with such properties. It is expressed primarily in myoepithelial cells, but is also expressed in blood vessels. An advantage of SMMHC is that it demonstrates less crossreactivity in myofibroblasts than calponin and SMA. Overall, the studies so far suggest that among smooth muscle markers, SMMHC provides the best results, in terms of both sensitivity and specificity.

44. Ideal panel Combination of SMMHC and p63

45. Detecting Absence Detecting the absence – more problematic than detecting its presence. When IHC fail to reveal myoepithelial cells around tumor- the diagnosis of stromal invasion is supported. Truly absent or whether they are merely markedly attenuated and out of the plane of section. Reassuring features - Medium to large tumor nests without detectable myoepithelial cells, multiple tumor nests without detectable myoepithelial cells, and lack of reactivity with two different myoepithelial markers.

46. Avoidance of Pitfalls p63 and SMM-HC complement each other well. P63 - sensitive and specific nuclear myoepithelial marker but staining can be discontinuous Cytoplasmic myoepithelial marker, such as SMMHC or calponin, as well will aid interpretation. If these two stains yield unclear results, the slightly more sensitive but less specific markers calponin and SMA can be used. . Combination of the sensitive marker smooth muscle actin, and two more specific markers such as smooth muscle myosin heavy chain and p63.

47. THANK YOU

48. DR. NOOPUR S. PATIL IMMUNOHISTOCHEMISTRY IN BREAST PATHOLOGY -II

49. MEP MARKER LOCATION MEP CELLS MYOFIBROBLAST VESSELS P63 Nuclear +++ _ _ CALPONIN Cytoplasmic +++ + ++ SMMHC Cytoplasmic +++ + ++ SMA Cytoplasmic +++ +++ +++ BASAL TYPE CK Cytoplasmic +Membranous +++ _ _

50. Recommendations for a Diagnostic Panel The optimal antibody depends upon the type of lesion being evaluated. Reactive stroma - p63 is an excellent choice because it does not stain myofibroblasts or blood vessels. p63 is less adroit at highlighting architecture in small glandular proliferations such as sclerosing adenosis, and in these cases a cytoplasmic marker such as SMA may be easier to interpret .

51. MYOEPITHELIAL CELL MARKERS IN THE EVALUATION OF BENIGN SCLEROSING LESIONS : To assess intraductal proliferative lesions, high-molecular-weight cytokeratins ( cytokeratin 14 and cytokeratin 5/6) can be helpful. Radial scar and complex sclerosing lesion - P roliferation of benign glands and tubules within a fibrous/fibroelastotic stroma. MECs associated with sclerosing lesions may have different immunophenotypic characteristics from the MEC layer . Reduced expression of CK5/6 in 32% of cases, SMMHC in 20%, CD10 in 15%, p63 in 10%, and calponin in 6%, compared to that of normal MECs

52. Central sclerosis with fibroelastotic stroma and entrapped glands radiating outward.

53. Benign lesions without MEP cells include microglandular adenosis , which is an infiltrative, benign breast lesion consisting of small, round, open glands formed by a single layer of flat to cuboidal epithelial cells with fibrous or fatty stroma . Microglandular adenosis , which is surrounded by an often-thickened basement membrane, lacks MEP cells and contains periodic acid–Schiff diastase–positive secretions. Microglandular adenosis is a mimic of tubular carcinoma. Microglandular adenosis is positive for collagen IV; negative for ER, PR, and p63; and often strongly positive for S100, whereas tubular carcinoma is ER+/PR+ and p63-ve and S100-ve.

54. MEP markers serve as important adjuncts for distinguishing between radial scars, invasive ductal carcinoma, and DCIS involving sclerosing adenosis . Basal-type cytokeratins , smooth muscle myosin heavy chain, smooth muscle actin , calponin , and p63 that are the frequently used MEP markers with varying specificities and sensitivities that are used to make the distinction. Clinical practices often use more than one MEP marker. Collagen IV and laminin have been used for distinguishing invasive carcinoma from in situ carcinoma. Benign or in situ lesions should demonstrate positive staining for MEP markers, whereas invasive carcinomas should show a loss of MEP staining at the periphery of each glandular structure . EXCEPTION: There are invasive carcinomas that express MEP markers; this group of cancers consist of tumor cells that stain positive for MEP markers. They include Adenoid cystic carcinoma and Metaplastic carcinoma, In such cases location of MEP marker( Non peripheral and liner) must be taken into consideration Invasi ve Carcinoma Versus In Situ Carcinoma

55. Detection of microinvasion Stromal invasion occurs when malignant epithelial cells extend beyond the myoepithelial cell layer and Earlier investigators- antibodies to basement membrane components - collagen IV and laminin to differentiate between in situ and invasive carcinomas. Cytokeratin markers may be helpful in highlighting the presence of malignant cells of a microinvasive carcinoma (1 mm) arising in a background of DCIS.

56. Irregularly shaped nests of neoplastic cells in a sclerotic background SMMHC highlights the myoepithelial cells surrounding the DCIS within the complex sclerosing lesion. Small foci of invasive ductal carcinoma - apparent in the immunostained section

57. A papillary lesion of the breast is characterized by an epithelial proliferation arising within the ductal /lobular system, supported by fibrovascular cores, with or without an intervening MEP cell layer. Papillary lesions consist of a range of benign, atypical, in situ, and even invasive lesions. Papillary lesions are among the most challenging in diagnostic breast pathology, and IHC analysis is often used in making the diagnosis. The most commonly used markers are CK5 (or CK5/6) for the presence or absence of UDH and MEP cells, and p63 for the presence or absence of MEP cells

58. p63 - highest sensitivity and lowest cross reactivity , and the nuclear staining is easy to interpret. CK5/6 appears to have a better sensitivity and specificity than other markers. Neuroendocrine markers are useful in differentiating solid papillary carcinoma (spindle cell type, neuroendocrine type) from papilloma with extensive florid epithelial hyperplasia.

59. Intraductal Papilloma .—Intraductal papilloma (IP) is a benign papillary lesion characterized by fibrovascular cores lined by MEP and epithelial layers. Immunohistochemistry analysis for MEP markers highlights the presence of MEP cells along the fibrovascular cores and at the periphery of the lesions. Intraductal papilloma with atypia is characterized by the presence of a population of monotonous cells with cytologic and architectural features of LG-DCIS. The MEP cells are typically scant or absent in atypical areas. Lack of CK5 or ADH-5 (the breast marker cocktail-CK8/18, CK5/14,P63) staining and uniform ER expression are helpful clues for the diagnosis of IP with ADH/LG-DCIS. An IP with atypia can be classified as IP with ADH or IP with LG-DCIS, depending on the size of the atypical proliferation. If the size of the atypical area within an IP is smaller than 3 mm, the lesion is classified as IP with ADH; if the atypical area is 3 mm or larger, it is called IP with LG-DCIS.

60. P apilloma with ADH vs P apilloma with DCIS Both show absent MEC markers within Both show present MEC around the ducts Positive 34βE12, CK5/6 is within proliferative component is indicative of ADH

61. Intraductal Papillary Carcinoma .—Intraductal papillary carcinoma lesions are considered a de novo, in situ papillary malignant process without a recognizable benign IP in the background. Intraductal papillary carcinoma consists of slender fibrovascular cores covered by a single layer of monotonous neoplastic cells without the presence of MEP cells ; however, the MEP cells are retained at the periphery of the lesions but, often, in a more-attenuated form . The neoplastic cells are usually low to intermediate nuclear grade and are often strongly positive for ER, PR, and luminal cytokeratins. Solid Papillary Carcinoma .—Solid papillary carcinoma (SPC ) is a distinctive variant of papillary carcinoma with a solid growth pattern and inconspicuous fibrovascular cores. It typically has a single, large, expansile mass or multiple solid , closely opposed nodules, and it may show spindle cell morphology and/or mucin production. The neoplastic cells are strongly positive for ER and PR,few neuroendocrine markers and are negative for CK5 , CK5/6, and MEP markers . Solid papillary carcinoma should be differentiated from UDH, which is positive for CK5 and CK5/6. Regardless of the presence or absence of peripheral MEP cells, SPC is staged as an in situ tumor ( Tis ) if it has a smooth, nodular tumor border . A lesion is considered invasive SPC only with the presence of a geographic ‘‘jigsaw’’ pattern with ragged, irregular borders in the absence of MEP cells.

62. Encapsulated papillary carcinoma (EPC ) is a variant of intraductal papillary carcinoma and is characterized by fine fibrovascular cores covered by neoplastic cells surrounded by a fibrous capsule . Encapsulated papillary carcinoma lacks MEP cells, both at the periphery of the lesion and within the fibrovascular cores. Most of the EPCs are low- to intermediate-grade , usually have a favorable prognosis, and are currently staged and managed as in situ carcinoma ( pTis ). Some authors consider EPCs to be slow-growing, invasive lesions or lesions in transition from in situ to invasion. High-grade EPCs are rare and account for approximately 3% of the EPCs. A high-grade EPC is defined by marked nuclear pleomorphism and increased mitotic activities . It is also often negative for hormone receptor, tends to be larger, and is more frequently associated with stromal invasion as compared with low- to intermediate grade EPCs. A recent study reported that 1 of 10 patients with pure, high-grade EPC developed recurrence and died of the disease. Because of its aggressive clinical behavior, those authors suggest managing high-grade EPC in a similar fashion to invasive breast carcinoma.

63. Residual benign intraductal papilloma with s olid and cribriform pattern, indicative of DCIS . p63 – MECs are present within the papillae of the residual papilloma are greatly reduced in number in the portion of the papilloma occupied by DCIS/atypia

64. Intrinsic Breast Cancer Subtypes : Breast cancers are classified as follows: Luminal-like Breast Cancer Types Luminal A Luminal B HER2 enriched breast cancer subtype Basal-like breast cancer subtype Claudin-low breast cancer subtype.

65. Luminal A Derives its name from its similarity to the expression profile of normal luminal breast epithelium. Overexpression of ER-regulated genes Underexpression of an HER2 gene cluster Underexpression of proliferation-related genes. Sensitive to endocrine manipulation( hormonal therapy). Less sensitive to cytotoxic agents in both the neoadjuvant and metastatic settings. Approximately 40% of all breast cancers are classified as luminal A. They have favorable prognosis

66. Luminal B Have lower expression of ER-related genes Variable expression of an HER2 cluster of genes, Relatively higher expression of proliferation- related genes. They represent about 20% of breast cancers. They also been shown to have genomic instability, and to harbor mutations in TP53 . less sensitive to cytotoxic chemotherapy, sensitive to hormonal therapy Associated with a relatively higher risk of relapse.

67. HER2 enriched breast cancer subtype It is characterized by high expression of HER2 Proliferation genes and low expression of luminal clusters. Constitute 20% to 30% of all breast tumors. Clinically, they are associated with a poorer prognosis

68. Basal-like breast cancer subtype Constitute about 15% of invasive ductal breast cancers. Its name is derived from shared gene expression patterns with normal basal epithelial cells. They are considered ER/PR and HER2 negative (“triple negative ”) - TNBCs This subtype is also characterized by relatively high frequency of BRCA1 mutations, increased genomic instability, high expression of the proliferation cluster of genes, and a high histologic grade

69. Claudin-low breast cancer subtype Is characterized by overexpression of genes associated with epithelial-to-mesenchymal (EMT) transition. Have no expression of luminal differentiation markers, are HER2 and hormone-receptor- negative by IHC Frequently exhibit metaplastic and medullary differentiation, and are often part of the basal intrinsic subgroup.

70. Luminal A Luminal B Her-2/neu Basal-like Gene e x p r es s ion pattern Expression(LMW) c y t o k e r a ti n s , and h i g h expression of HR’s and associated genes Expression (LMW) cytokeratins, and moderate to weak expression of HR’s and associated genes. High expression of Her-2/neu . L o w e x p r ession of E R and ass o ci a t ed genes. High expression of B asal epithelial genes, basal cytokeratins . Low expression of ER and Her-2/neu associated genes. Clinical ~ 50% of invasive breast cancer ~ 20% of invasive breast cancers ~15% of invasive breast cancers ~15% of invasive breast cancers ER/PR status ER/PR positive ER/PR positive ER/PR negative Most ER/PR negative Her-2/neu status Her-2/neu negative Her-2/neu E x p r ession v aria b le (+/-) Her-2/neu positive (by definition) Her-2/neu N e g at i v e (“tri p l e negative”) Biological features High proliferation than luminal A High proliferation High proliferation

71. Luminal A Luminal B Her-2/neu Basal-like Luminal B tends to be TP53 mutati on TP 53 mutation H igher histological common common; BRCA-1 grade than luminal A More likely to be high dysfunction (germline grade and node sporadic) positive. Histological correlation Tubular carcinoma Cribriform carcinoma Low grade IDC (NOS) IDC (NOS) Mic r o papi l l a r y carcinoma . High grade IDC (NOS) High grade IDC (NOS) Metaplastic carcinoma Medullary carcinoma lobular carcinoma Treatment Respond to endocrine therapy Respond to endocrine the r a p y( t a m o xi f e n & aromatase inhibitors) Respond to trastuzumab No response to Endocrine therapy and trastuzumab Response to chemotherapy variable variable (> in luminal A) Good ( a n th r ac y cl i ne b a s ed chemotherapy) Good ( p l a tinum b a sed chemotherapy ) Prognosis Good prognosis Prognosis not as good as for luminal A Generally poor prognosis Generally poor prognosis

72. Prognostic and Predictive Values of Androgen Receptor in TNBCs: Androgen receptor (AR) expression has been shown to have prognostic implications in breast cancers, and higher AR expression levels have been associated with higher expression of ER or PR, lower nuclear grade, and smaller tumors , with lower risk of recurrence and death. Significant differences in AR protein expression have been detected in the various molecular subtypes of breast cancers . Triple-negative breast cancers, which are by nature ER- ve , characteristically have much lower frequency of AR expression compared with ER+ breast cancers. The AR signaling pathway may represent a molecular driver that can be therapeutically targeted in Ar expressing TNBCs by using an AR inhibitor as a novel therapeutic agent( Bicalutamide ) Therefore , knowing the AR expression status of a TNBC is necessary for selecting patients who could benefit from antiandrogen therapy. Antiandrogen therapy may be of particular interest to those who do not respond to conventional chemotherapy, or it could even be used as an additional first line drug.

73. Lobular or ductal carcinoma: E- cadherin , CK8 Determining whether an in situ lesion is lobular carcinoma or ductal carcinoma has clinical management implications and is another situation in which IHC proves its worth . Generally, ductal and lobular carcinomas, either invasive or in situ can be distinguished in hematoxylin -eosin stained sections . In cases with non-specific morphologic characteristics , categorization can be performed through IHC , and E- cadherin is currently used to differentiate between the two. The majority of ductal carcinomas express cytoplasmic E- cadherin , whereas most lobular carcinomas lack expression of E- cadherin . In addition, the differences in CKs expression may be used: high molecular- weight CK (clone 34βE12) is usually expressed by lobular carcinomas, but is absent or expressed at low levels in most cases of DCIS. In the same context, CK 8 is stained in ductal carcinoma cells in the peripheral cytoplasm, while perinuclear staining is characteristic of lobular carcinoma.

74. E cadherin and p120ctn

75. E - cadherin Strong E-cadherin (ECAD) membranous staining has been long used to define ductal carcinoma Ductal carcinomas, both in situ and invasive, retain membranous ECAD Lobular carcinomas studied at the genetic level have often shown mutation that accounts for the loss of cohesiveness This correlates with either a complete absence of the ECAD protein or abnormal localization (apical or perinuclear)

76. p120ctn When ECAD is absent, the cytoplasmic pool of p120 increases. Therefore in normal ducts and in ductal carcinomas, p120 shows a membranous pattern of staining In contrast, lobular carcinomas, with absent or non functional E- cadherin, show strong cytoplasmic p120 immunoreactivity

77. ECAD – BROWN P120 - PINK

78. METASTATIC ADE N OCAR C INOMAS The breast itself is an uncommon site of metastatic disease. Cutaneous melanoma is the most common extramammary solid malignancy to metastasize to the breast . Pulmonary, ovarian, gastric, and renal carcinomas are also common sources of metastases to the breast, as is prostatic carcinoma in males In 24% to 40% of cases the breast lesion is the first presentation of an occult malignancy. Absence of an in situ component in a tumor that is negative for ER, PR, and HER2/neu is a ‘‘red- flag ’

79. Ovary- WT-1 antibody together with a negative reaction for GCDFP-15 and/or mammaglobin, provides strong evidence for metastases . Negativity for pan-CK, coupled with immunoreactivity to HMB-45 and Melan-A , is consistent with metastatic melanoma . Positivity for S100 protein alone is of limited value. Metastasis from a gastric signet ring cell carcinoma vs invasive lobular carcinoma - IHC panel of ER, PR, and CDX-2 (caudalrelated homeobox gene 2) antibodies is helpful.

80. UNKNOWN PRIMARY ORIGIN

81. Breast Versus Lung Solitary lung lesions in patients with a history of breast cancer and in the workup of metastases of unknown primary. GCDFP-15 and TTF-1 Breast Versus Ovary Same patient population, particularly in those women who harbor BRCA mutations. GCDFP-15 and sometimes WT-1 WT-1, being expressed in 96% of ovarian serous ca and 2% in all breast carcinoma

82. Breast Versus Stomach GCDFP-15, ER, and CK20, + for GCDFP-15 is consistent with a breast primary An ER+ signet-ring cell carcinoma is more likely to be of breast origin, and a CK20+ tumor is more likely to be of gastric origin. A CK20+/ ER+ signet ring cell carcinoma is more likely to be of breast origin Breast Versus Melanoma Positive reactions for HMB-45 and MART-1, and a negative reaction for cytokeratin S-100 is of limited value

83. Sentinel lymph node Rationale : Historically, complete axillary lymph node dissection was performed with lumpectomy or mastectomy specimen, primarily for staging purposes, providing information that was used to determine adjuvant chemotherapy The morbidity associated with this procedure is substantial in terms of arm pain, limitation of arm motion, and chronic lymphedema The SLN is identified by injecting a radioisotope and blue dye before planned surgical excision, Identified by a combination of visual inspection for blue dye and intraoperative scanning for radioactivity The rationale for this approach is that for patients who are SLN negative, a further morbid procedure of axillary cleanout is unnecessary

84. SLN invasion SLNs are histologically examined on multiple levels and by cytokeratin stains (AE1/AE3) on at least two levels If the primary breast carcinoma is of the ductal type, most pathologists would agree that they would be able to identify micro metastases (0.2- 2mm) on HE. Therefore cytokeratin stains on SLN do not add any significant information in a primary ductal cancer However, when the primary breast tumor shows a lobular morphology; Because of single-cell infiltration, small metastases (micro metastases) of lobular carcinoma require IHC

85. Paget’s disease The presence of malignant epithelium with breast cancer immunophenotype (CK-7+) within the nipple epidermis The majority (>90%) of the underlying breast carcinomas are high grade ductal carcinoma in situ with apocrine differentiation and comedo-necrosis, they are her 2 neu positive and ER PR negative Accordingly the Paget cells are also her 2 neu positive and ER, PR negative The differential for Paget disease includes melanoma and squamous cell carcinoma in situ aka (Bowen disease)

86. THANK YOU