IHC in breast pathology

The utilisation of Immunohistochemistry in breast pathology

1. NAMRATHA RAVISHANKAR IMMUNOHISTOCHEMISTRY IN BREAST PATHOLOGY

2. 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)

3. 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 Other markers

4. 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).

5. 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 rationale : identify patients who may benefit from hormonal therapy. – Stong 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

6. Hormone receptors breast cancer (ER) as a prognostic and predictive factor 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.

7. 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 ; Values differ from ER-alpha in BRCA1 associated breast carcinoma 

8. ER 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. Nadji M, Gomez-Fernandez C, Ganjei-Azar P, et al. Immunohistochemistry of estrogen and progesterone receptors reconsidered: experience with 5,993 breast cancers. Am J Clin Pathol 2005;123:21–27. Oestrogen receptor-negative breast carcinoma with a high concentration of microvessels stained with anti-CD34

9. 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 Nuclear staining for PR more heterogeneous than ER - cause of false-negative results PR cytoplasmic and membranous staining is considered negative

10. ER-positive/PR-negative breast tumors Nonfunctional and unable to stimulate PR production and that the tumor is, therefore, no longer dependent on estrogen for growth and survival. Low circulating levels of endogenous estrogens in postmenopausal women Hypermethylation of PR promoter Growth factor down regulator of PR Brief treatment of patients with ER-positive/PR-negative tumors with estrogen can restore PR levels in some of the patients .

11. Recommendations ER, PgR , and HER2 determined on all invasive carcinomas. The largest Invasive carcinoma should always be tested and the results reported in the case summary. If invasive carcinomas - different histologic type or of higher grade , performing the studies on these cancers as well is recommended If immunohistochemistry for ER and PR is negative in a core biopsy , it should be repeated on the excisional breast specimen.

12. 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.

13. 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

14. 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 systemthat 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.

15. Sensitivity of Allred method = 99.4% Specificity of Allred method = 99.5% Sensitivity of conventional score= 88 % Specificity of conventional score= 84%. Allred scoring for ER reporting and it's impact in clearly distinguishing ER negative from ER positive breast cancers Asim Qureshi , Shahid Pervez Department of Pathology, Shaukat Khanum Cancer Hospital, Lahore,1 Department of Histopathology, Aga Khan University Hospital, Karachi.2

16. Shousha  S Oestrogen receptor status of breast carcinoma: Allred/H score conversion table . Histopathology  2008 ; 53 ( 3 ): 346 - 347

17. HER2 Testing by Immunohistochemistry 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

18. IHC Score Criteria % of Cases % of Cases With Amplification by FISH or CISH 0 (Negative) No immunoreactivity or immunoreactivity in ≤10% of tumor cells. ~ 60% 0% – 3% 1+ (Negative) Faint weak immunoreactivity in >10% of tumor cells but only a portion of the membrane is positive. ~ 10% 0% – 7% 2+ (Equivocal)# Weak to moderate complete membrane immunoreactivity in >10% of tumor cells or circumferential intense membrane staining in ≤30% of cells. ~ 5% – 10% 25% – 35% 3+ (Positive) More than 30% of the tumor cells must show circumferential intense and uniform membrane staining. A homogeneous (chicken wire) pattern should be present. ~15% – 20% 95%##

19. BRCA 1 mutation IHC - new and powerful predictor of BRCA1 mutation status ER-negative will roughly double the probability that the individual is a BRCA1 carrier , Er positive - reduce the probability by approximately fivefold. The Pathology of Familial Breast Cancer: Predictive Value of Immunohistochemical Markers Estrogen Receptor, Progesterone Receptor, HER-2, and p53 in Patients With Mutations in BRCA1 and BRCA2 By Sunil R. Lakhani , Marc J. van de Vijver , Jocelyne Jacquemier , Thomas J. Anderson, Peter P. Osin , Lesley McGuffog , and Douglas F. Easton for the Breast Cancer Linkage Consortium Journal of Clinical Oncology, Vol 20, No 9 (May 1), 2002: pp 2310-2318

20. Standardization for HR testing Prompt fixation of breast tissue- 8 to 72 hours of 10% neutral buffered formalin fixation Processing by conventional (not microwave enhanced) tissue processors. Formalin newly replenished in the processor Processor fluids should not exceed 37°C. In vitro diagnostic kits should be used that utilize one of these ER clones: 6F11, 1D5 or SP1. Positive and negative controls, internal and external, should be used on each run. A positive cut-off of 1% - nuclear expression, and results should be semiquantitated with the percentage of cells staining and their intensity.

21. 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

22. Other Prognostic Assays Ki-67 (MIB-1) 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.

23. Myoepithelial cells - easily identified in normal breast ductules and acini Distinguished from luminal epithelial cells by the presence of smooth muscle fibres. Structures dilate and fill with intraluminal proliferating cells or are compressed- difficult to recognize the attenuated myoepithelial cells.

24. 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 (1 ) 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 (2) High sensitivity.

25. Discontinuous staining pattern, - morphologic impression that MECs are absent Positivity in tumor cells, - 15.7% to 23% of invasive ductal carcinomas,85% to 100% of adenoid cystic carcinomas, and the majority of metaplastic carcinomas Potential for a temporal reduction in its expression within archival material

26. SMA Normal MEC in 88% to 100% of cases, normal luminal/epithelial cells in 37% of cases Myofibroblasts , and vascular smooth muscle and pericytes , 10%–16.1% of invasive ductal carcinomas, scattered epithelial cells in UDH Muscle-Specific Actin also known as HHF-35 is comparable to SMA in its sensitivity for significantly lower specificity. In addition to the SMA-like patterns of cross-reactivity with stromal myofibroblasts , luminal/epithelial cells, and vessels

27. Smooth Muscle Myosin Heavy Chain SMMHC is a structural component of myosin - specific for smooth muscle cells, terminal smooth muscle differentiation. 100% of the MECs associated with normal breast ductules and benign breast proliferations, as well as vascular smooth muscle. As compared with SMA and MSA, SMMHC is significantly easier to interpret 8% of cases displayed significant crossreactivity with myofibroblasts

28. CD 10 and Calponin CD10 Stains stromal myofibroblasts - lesser intensity than is seen with MSA or SMA, and does not stain vessels., Values change post-chemotherapy Calponin - Contractile element expressed in differentiated smooth muscle cells. Highly sensitive for MECs. normal breast MECs, vascular smooth muscle Collagenous spherulosis (CD10+, HHF35+) from adenoid cystic carcinoma (CD10-, HHF35-)

29. Basal-Type and High-Molecular-Weight Cytokeratins - CK 1, CK5, CK5/6, CK10, CK14, and CK17, react variably with breast MECs. The sensitivities of CK5/6, CK14, and CK17 for MECs- comparable to those of SMA, MSA, p63, and SMMHC , whereas 34bE12 (which recognizes CK1, CK5, CK10, and CK14) exhibits considerably lower sensitivity. 32% of high-grade DCIS and 38% of invasive carcinomas - positive for at least one basal-type and high- molecularweight cytokeratin .

30. 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.

31. 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. 

32. Recommendations for a Diagnostic Panel The optimal antibody also 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 .

33. MYOEPITHELIAL CELL MARKERS IN THE EVALUATION OF BENIGN SCLEROSING LESIONS 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

34. Adenosis - benign proliferative lesion that usually occurs as part of the spectrum of proliferative fibrocystic change Myoepithelial cells Collagen Other markers Tubular Carcinoma Invasive tubules Apical snouts Desmoplasia Absent Absent EMA + ER/PR + Microglandular adenosis Round glands in fat Flat to cuboidal cells Secretions Absent Present S100+ ER/PR negative EMA negative EGFR + Tubular adenosis Lobulocentric Tubules Present Present S100 negative Sclerosing adenosis Lobulocentric Lobular fibrosis Present Present S100 negative

35. Invasive Carcinoma Versus In Situ Carcinoma DCIS mimicking invasion Epihelial tributaries adjacent to DCIS Inflammation and sclerosis DCIS with sclerosing adenosis and radial scar, cancerisation of lobules - Invasive appearance Invasive carcinoma mimicking CIS Cribriform carcinoma and Adenoid cystic carcinoma Myoepithelial cells can become diminished in number in both in-situ carcinomas(5%)

36. Detection of microinvasion Stromal invasion occurs when malignant epithelial cells extend beyond the myoepithelial cell layer and break through the basement membrane. Earlier investigators- antibodies to basement membrane components - collagen IV and laminin to differentiate between in situ and invasive carcinomas. Invasive tumor cells are also capable of synthesizing basement membrane.

37. 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

38. MARKER COLLAGENOUS SPHERULOSIS ACC CRIBRIFORM DCIS INVASIVE CRIBRIFORM CARCINOMA p63 + at the periphery and surrounding lumens + at the periphery and the basaloid cells + at periphery only Negative SMMHC + at the periphery and surrounding lumens Negative + at periphery only Negative CALPONIN + at the periphery and surrounding lumens Negative + at periphery only Negative C- KIT Negative Positive tumour cells Negative Negative MYOEPITHELIAL CELL MARKERS IN THE EVALUATION OF CRIBRIFORM LESIONS OF THE BREAST

39. Special types of carcinomas - myoepithelial markers Adenoid cystic carcinoma, low-grade adenosquamous carcinoma, malignant adenomyoepithelioma , and malignant myoepithelioma Metaplastic carcinomas, including spindle cell carcinomas , may also stain for myoepithelial markers. Myoepithelial markers can stain the periphery of the invasive adenosquamous tumor nests , simulating an intact myoepithelial cell layer Current Practical Applications of Diagnostic Immunohistochemistry in Breast Pathology Melinda F. Lerwill , MD Am J Surg Pathol 2004;28:1076–1091)

40. Signet ring cell carcinoma - (GCDFP-15 or BRST2), Estrogen receptor and MUC1 ,CK7 +,and negative for CK20 Ca with neuroendocrine differentiation : specific but quite heterogeneous positive reaction chromogranin or synaptophysin and NSE Secretory carcinoma - alpha- lactalbumin , beta-casein, and HMFG-2 (all milk proteins), S100 protein and CEA. ER - negative Sebacceous Ca- Pan CK + .PR+, ER- Medullary Ca- ER and PR- . HER2/ neu – Tumor cells - + for HMW-CK and CK19, basal/ myoepithelial markers such as CK5/6, CK14, p63, and CD10 Lipid-rich ca - lactoferrin and alpha- lactalbumin .

41. + for both LMW-CK (such as CK8/18) and HMW-CK or basal-type cytokeratins (such as CK34BE12, CK5/6, CK14). . SMA,Calponin , and p63+ CD117 + .ER,PR, and HER2/ neu are very often negative in adenoid cystic carcinoma Adenoid  cystic   carcinoma  showing positive staining with SMA KIT expression: cribriform pattern and solid  pattern  , more than 90% of cells  expressed  KIT, with membranous and/or cytoplasmic +  Benign   ductal   epithelial cells  expressed KIT whereas myoepithelial cells did not express KIT

42. HER2 overexpression (95%) and p53 (70%).  MUC1 - unusual immunoreactivity limited to the basal surface of the cells Conventional- MUC1- intracytoplasmic or apical.  Reversal  of cell orientation - important factor in the morphogenesis and pathogenesis of invasive micropapillary carcinoma

43. Papillary lesions of breast

44. 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.

45. 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

46. Encapsulated papillary carcinoma Encapsulated papillary carcinoma- CD10. Myoepithelial cells are not present either within the papillae or at the periphery of the nodule (note myoepithelial cell reactivity of adjacent normal duct).

47. Solid papillary carcinoma Endocrine features, immunoreactivity for chromogranin and synaptophysin .

48. Basal like carcinomas  Expression signature similar to that of the basal/ myoepithelial cells of the breast Transcriptomic characteristics similar to those of tumors arising in  BRCA1   germline mutation carriers   High nuclear grade, high mitotic activity, coexpression of high-molecular-weight cytokeratins , EGFR and vimentin , and a lack of expression of ER and PR

49. Poorer survival if express CK 17 or CK 5/6 CK 5/6 or EGFR , HER2+ Not all triple negative tumors express basal cytokeratins , and a subset of basal-like carcinomas are not triple negative. The combination of ER(-) Her2/ neu (-) CK5/6(+) and EGFR(+) has a 76% sensitivity and 100% specificity

50. TUMOUR TYPING- Definition of Lobular Differentiation Morphology ( the traditional method); Immunohistochemical (loss of E- cadherin immunoexpression and mislocalization of p120); or molecular (mutation in E- cadherin gene 16q22.1) . Occasional cases with morphologic and molecular criteria for lobular differentiation - variable degrees/patterns of Ecadherin expression May be valid to equate “loss of E- cadherin staining” with lobular differentiation, Not valid to exclude lobular differentiation in morphologically “lobular” tumors just because of positive E- cadherin staining.

51. Molecular defects in the E- cadherin gene - may still express E- cadherin , protein may not be functional in terms of achieving cell to cell adhesion ( Da Silva et al. Am J Surg Pathol 2008; 32: 773), Granular cytoplasmic pattern, dot-like or patchy discontinuous membranous pattern or even continuous membranous pattern “aberrant Ecadherin reactivity” should not automatically exclude a diagnosis of lobular differentiation Not automatically equated with ductal differentiation Complete lack of E- cadherin : Supports a diagnosis of lobular differentiation. Presence of E- cadherin : Does not exclude lobular differentiation. Revert to morphology and/or p120

52. E-CD is a calcium-dependent, epithelial-specific cell-cell adhesion molecule whose reduced or lost expression is associated with tumor dedifferentiation and increased metastatic potential in human carcinomas . Membrane staining in luminal cells and granular in myoepithelial cells extended spread of lobular carcinoma in situ and the peculiar diffuse invasion mode of invasive lobular carcinoma.

53. p120 catenin (a.k.a. p120): Inner membrane bound protein associated with E- cadherin . In ductal epithelium (benign or malignant), p120 - membranous expression. In lobular cancer, loss of E- cadherin is associated with loss of the anchoring of p120 to the membrane Cytoplasmic expression Helpful adjunct to E- cadherin - in the setting of aberrant E- cadherin reactivity Membranous p120: Ductal differentiation. Cytoplasmic p120: Lobular differentiation.

54. Mammary intraepithelial neoplasia , NOS type: Both E- cadherin and CK34BE12 negative: negative hybrid lesion. Both E- cadherin and CK34BE12 positive: positive hybrid lesion.

55. UDH VERSUS ADH/DUCTAL CARCINOMA IN SITU Normal breast glands and ducts are composed of 3 cell types : luminal, basal, and myoepithelial . The luminal cell types express LMW cytokeratins (CKs) Myoepithelial cells express basal cell–type CKs- HMW CKs Thee HMW-CKs are CK34BE12(CKs 1, 5, 10, and 14), and monoclonal HMW-CKs such as CK5/6 or CK14.

56. Luminal epithelial cells and myoepithelial (or basal) cells UDH - H eterogeneous cell population - epithelial and modified myoepithelial cells (divergent cell population). Both low (CK8, CK18, CK19) and high molecular weight cytokeratins (HMW-CK), such as CK5/6, CK14, and CK34betaE12 ADH/LG-DCIS- clonal derivation- Markers of only 1 cell type, usually luminal (CK8, CK18, CK19) Small percentage show basal cell differentiation , a phenotype more typical of high-grade DCIS Note that CK5/6 is more specific than CK34BE12

57. Current Practical Applications of Diagnostic Immunohistochemistry in Breast Pathology Melinda F. Lerwill , MD Am J Surg Pathol 2004;28:1076–1091)

58. UDH, keratin 903, note strong cytoplasmic staining of cells , ADH, keratin 903, note minimal to no staining of cells

59. The loss of TGF-β2 expression in UDH has been suggested as a predictive factor for the development of invasive breast cancer in the future TGF-α which showed an increasing staining intensity in malignant tumors

60. CHARACTERIZATON OF METASTATIC ADENOCARCINOMAS 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 ’

61. 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 - Benign and malignant breast epithelium. 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.

62. METASTATIC CARCINOMA, UNKNOWN PRIMARY ORIGIN

63. 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

64. 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

65. SENTINEL LYMPH NODE EVALUATION Multiple step levels, cytokeratin immunostains , and/or molecular diagnostics are all variously used. Increased detection of micrometastases and isolated tumor cells.  Although routine IHC staining with cytokeratin is not - Invasive lobular carcinoma Suspicious for, but not diagnostic of, lymph node metastases on H&E rather than as a routine method of evaluating nodes in cases of invasive lobular cancer.

66. The keratin cocktail AE1/AE3, which stains predominantly high-molecular-weight keratins, or a wide-spectrum pan-CK are most commonly used. CAM 5.2 and AE1 , which stain only low-molecular-weight keratins. Not all epithelial cells in lymph nodes represent metastatic carcinoma. A biopsy may displace benign breast epithelia, which then drain to the axillary nodes, so-called benign transport.

67. Reticulum cells -+ Cam5.2 or pan- cytokeratin is used,but much less so when AE1/AE3 or AE1 alone is used. Plasma cells

68. Current AJCC staging criteria include a special identifier “ i +” to indicate when metastatic deposits >0.2 cm are detected only on immunohistochemical stains. Metastases larger than 0.2 cm are considered N1 regardless of the method of detection.

69. Paget’s Disease Positive LMW-CK (CK8, CK18, CK19) CK7,EMA GCDF-15 , CEA, CD138, p53 Overexpress HER2/ neu Negative CK20, ER and PR CK7

70. Toker cells CK 7 +/- ER+, PR+, CD138-, p53-, HER2-, Low Mib-1 Melanoma S100, HMB 45 and Vimentin Melanin, cytoplasmic mucin vacuoles.  Extramammary Paget’s disease HMWCK, p63+ CK 20 and CK 7 positive MUC1 - only MUC in both MPD and EMPD. Paget cells in MPD- about 75%) express MUC3 > 40% of EMPD were positive for MUC5AC

71. Spindle cell lesions Pure spindle cell lesion or only subtle cohesive foci- IHC for cytokeratins Bland spindle cell lesions as spindle cell carcinoma can resemble fibromatosis. Spindle cell carcinomas typically express basal cytokeratins or myoepithelial markers. No marker is expressed by all spindle cell carcinomas -important to use a panel of antibodies to both basal and luminal cytokeratins . Cytokeratin 14, cytokeratin 5/6, 34 β E12 (basal), CAM5.2, cytokeratin 7 (luminal), MNF116 and AE1/AE3 (broad spectrum). Cytokeratin expression per se in a spindle cell lesion does not exclude other diagnoses.

72. Panel of IHC often needed to demonstrate tumor cell reactivity Antibodies to high molecular weight cytokeratins most sensitive – Pankeratin MNF116 – 34β E12 – CK 5/6 – CK 14 ER/PR/Her2neu Myoepithelial markers- SMA, S100, P63, CD10 and Actin Laminin

73. Spindle cell carcinoma that is positive with CAM5.2 - more cohesive areas (B). There is nuclear expression of β- catenin by the carcinoma cells. Note that the endothelial cells show cytoplasmic staining, but the nuclei are negative

74. p63 was consistently negative in the spindle and epithelial cell components of all benign and malignant Phyllodes tumors The sensitivity and specificity of p63 as a marker for metaplastic carcinoma is 86.7% and 99.4%, respectively, with a 100% specificity for MCB with spindle cell and/or squamous areas.

75. Bcl 2 Zhang et al. - a) 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.

76. Antibodies to D2-40, LYVE-1, and other endothelial markers - uncertainty about the presence of lymphovascular invasion at a particular site in the tissue VEGF, CD31 and CD34 Angiogenesis in breast carcinoma - relevance of tumor vascularity to known prognostic markers and to prognosis UPA1 and PAI 1

77. Prognostic or Predictive Factors in Breast Carcinoma: Hormone Receptors Differential Diagnosis of Epithelial Lesions: Myoepithelial Cells (Panel of p63, SMMHC and SMA and basal cytokeratins )- Sclerosing lesions, Adenosis and Cribriform lesions Papillary lesions of breast- Myoepithelial markers Tumour typing and confirming diagnoses- Lobular v Ductal ; (DCIS) vs LIN (LCIS)- E cadherin,p120 catenin , CK8, CK34 betaE12 Homogeneous Versus Heterogeneous Cell Population ( Neoplasia Versus Hyperplasia)- CK34 betaE12 and CK5/6 ER

78. Basal like carcinomas- ER, PR(-) Her2/ neu (-) CK5/6(+) and EGFR(+) Special types of invasive carcinoma- ACC- CD117 Micropapillary-MUC1, ER/PR Metastatic carcinoma- Unknown primary- GCDF15, ER,PR, CK7, CK20 Metastasis to breast- Melanoma markers, Ovary, Sentinel lymph node evaluation- Keratins in lobular carcinoma

79. Spindle cell lesions of breast- Metaplastic carcinoma- – Pankeratin MNF116 – 34β E12, CK 5/6, CK 14, ER/PR/Her2neu Myoepithelial markers- SMA, S100, P63, CD10 and Actin Laminin Pagets disease of breast- LMW-CK (CK8, CK18, CK19)CK7,EMA GCDF-15 , CEA, CD138, p53 Overexpress HER2/ neu

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