The Story of the Magee Equations: The Ultimate in Applied Immunohistochemistry.

Magee equations (MEs) are a set of multivariable models that were developed to estimate the actual Onco type DX (ODX) recurrence score in invasive breast cancer. The equations were derived from standard histopathologic factors and semiquantitative immunohistochemical scores of routinely used biomarkers. The 3 equations use slightly different parameters but provide similar results. ME1 uses Nottingham score, tumor size, and semiquantitative results for estrogen receptor (ER), progesterone receptor, HER2, and Ki-67. ME2 is similar to ME1 but does not require Ki-67. ME3 includes only semiquantitative immunohistochemical expression levels for ER, progesterone receptor, HER2, and Ki-67. Several studies have validated the clinical usefulness of MEs in routine clinical practice. The new cut-off for ODX recurrence score, as reported in the Trial Assigning IndividuaLized Options for Treatment trial, necessitated the development of Magee Decision Algorithm (MDA). MEs, along with mitotic activity score can now be used algorithmically to safely forgo ODX testing. MDA can be used to triage cases for molecular testing and has the potential to save an estimated $300,000 per 100 clinical requests. Another potential use of MEs is in the neoadjuvant setting to appropriately select patients for chemotherapy. Both single and multi-institutional studies have shown that the rate of pathologic complete response (pCR) to neoadjuvant chemotherapy in ER+/HER2-negative patients can be predicted by ME3 scores. The estimated pCR rates are 0%, <5%, 14%, and 35 to 40% for ME3 score <18, 18 to 25, >25 to <31, and 31 or higher, respectively. This information is similar to or better than currently available molecular tests. MEs and MDA provide valuable information in a time-efficient manner and are available free of cost for anyone to use. The latter is certainly important for institutions in resource-poor settings but is also valuable for large institutions and integrated health systems.

Best Practice (Efficient) Immunohistologic Panel for Diagnosing Metaplastic Breast Carcinoma.

Immunohistochemistry (IHC) plays a key role in the diagnosis of metaplastic breast carcinomas (MBCs), particularly the spindle cell variant. The most efficient immunopanel has yet to be developed. We studied the immunoprofile of 45 MBCs including 23 matrix-producing MBCs, 11 squamous cell carcinomas, 6 spindle cell carcinomas, and 5 mixed-subtypes (2 cases including spindle cell components). Representative sections from mastectomy or core biopsy specimens were subject to IHC using a list of antibodies including OSCAR, a recently developed antibody against pooled cytokeratins. The staining was interpreted as positive when >1% of tumor cells demonstrated unequivocal staining. As a result, OSCAR showed similar sensitivity to AE1/AE3 and CAM 5.2 (89.1% vs. 89.4% vs. 89.4%) for MBCs, but the former showed more diffuse pattern of staining, particularly in spindle cell carcinomas. High molecular weight cytokeratin CK14, CK5, and CK17 were positive in 91.3%, 87.2%, and 73.3% of MBCs, respectively. CK7 was much less likely to be positive in spindle cell carcinomas (37.5%) than in other variants of MBCs (97.4%). P63 and CK14 were the most useful markers for spindle cell carcinomas, positive in 87.5% and 85.7% of cases, respectively. GATA 3 was positive in 63% MBCs, and nonspecific staining for vimentin and smooth muscle actin were common. Random combination of up to 3 antibodies against keratins including p63 showed sensitivities ranging from 80.9% to 97.9%. Our results suggested the combination of OSCAR, CK14 and p63 is the most efficient panel (sensitivity 97.9%) for diagnosing MBCs.

Isolated Flat Epithelial Atypia on Core Biopsy Specimens Is Associated With a Low Risk of Upgrade at Excision.

OBJECTIVES: We present a retrospective review of 111 breast core biopsy specimens with flat epithelial atypia (FEA) and corresponding excision to better understand rates of upgrade following this diagnosis. METHODS: In total, 252 breast core biopsy specimens were identified. Cases were excluded if biopsy slides or excision results were unavailable, for ipsilateral carcinoma or other findings warranting excision, or biopsy performed for indications other than mammographically detected calcifications. Coincident atypical lobular hyperplasia was not excluded. Diagnoses were confirmed by breast pathologists, and mammographic images were reviewed. RESULTS: Ultimately, 111 biopsy specimens with FEA were included. In subsequent excisions, one (1%) of 111 showed invasive carcinoma, 20 (18%) atypical ductal hyperplasia, 20 (18%) lobular neoplasia, 31 (28%) FEA, and 39 (35%) no atypical findings. CONCLUSIONS: FEA on core biopsy specimens is associated with a low rate of upgrade to invasive carcinoma. Close follow-up may be a reasonable alternative to excision for isolated FEA on core needle biopsy specimens.

Effects of Hydrochloric Acid and Formic Acid Decalcification on Breast Tumor Biomarkers and HER2 Fluorescence In Situ Hybridization.

Biomarker analysis of metastatic breast carcinoma (MBC) is routinely recommended by ASCO/CAP guidelines, and establishing a diagnosis of MBC often requires immunohistochemistry (IHC). The reliability of breast tumor biomarkers and breast-specific markers on decalcified tissues has not been extensively studied. We performed IHC studies on breast tumors exposed to hydrochloric acid (HCl) and formic acid (FA) decalcification solutions, and HER2 fluorescence in situ hybridization on a subset of these tumors to establish a protocol for handling bone specimens with suspicion for MBC. Fifteen fresh cases of primary breast carcinoma and 8 HER2+ paraffin-embedded core biopsy cases were studied. Fresh tissue was divided into 5 fragments to approximate a bone core biopsy. One fragment (control) was fixed in 10% neutral buffered formalin. The remaining fragments were also exposed to FA or HCl decalcification for 1 or 5 hours. All fragments were embedded in 1 block and tested with an IHC panel. The known HER2+ cases were exposed to either 1 or 5 hours of FA, and HER2 fluorescence in situ hybridization was also performed. Results were interpreted as follows: H-scores for estrogen receptor, progesterone receptor, and GATA-3 were assigned from 0 to 300; HER2, cytokeratin 7, gross cystic disease fluid protein-15, Pax-8, TTF-1, cytokeratin 20, and mammaglobin were scored from 0 to 3+; and Ki67 from 0% to 100%. Mean scores were compared using the t test or Wilcoxon test for paired samples. No significant differences in mean score were seen between NF and 1 hour FA for any IHC immunoreactivity. After 5 hours of FA, only Ki67 average score was significantly less than NF. Mean scores for estrogen receptor, progesterone receptor, HER2, Ki67, and GATA-3 were significantly lower than NF in the tissue after either 1 or 5 hours of HCl. Mean scores for gross cystic disease fluid protein-15, mammaglobin, and cytokeratin 7 staining were not significantly lower than NF after 1 or 5 hours of HCl.

Breast Cancers With Magee Equation Score of Less Than 18, or 18-25 and Mitosis Score of 1, Do Not Require Oncotype DX Testing: A Value Study.

Objectives: To investigate use of Magee equations (MEs) to determine which breast cancer cases can be excluded from Oncotype DX testing. Methods: A prospective value study was carried out using data from pathology reports. Results: If all three MEs scores were less than 18 or 31 or higher, the cases were labeled do not send for testing. If any or all scores were 18 to 25, cases were labeled do not send if mitosis score was 1. Of the total 205 cases, 146 (71%) were labeled do not send; of these, the correct call was made in 143 (98%) cases. Two of the three discordant cases had associated nontumor factors, likely resulting in higher scores. Conclusions: Cases with ME scores less than 18, or 18 to 25 and mitosis score 1, do not require Oncotype DX testing, an estimated saving of US$280,000 per 100 clinical requests.

High Fidelity of Breast Biomarker Metrics: A 10-Year Experience in a Single, Large Academic Institution.

PURPOSE: Recommendations for standardization of breast biomarkers including estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 (HER2) led to the creation of American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines to provide continuous guidance. Included in these recommendations is the "ongoing assay assessment procedures." We report these biomarker metrics as there is a dearth of published information on this topic. MATERIALS AND METHODS: ER, PR, and HER2 positivity rates of all newly diagnosed, recurrent, and metastatic invasive breast cancers on core biopsies, and repeated testing on resection specimen by immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH) were collected from April 1, 2008 to December 31, 2017. RESULTS: The positivity rates of ER, PR, and HER2 over almost 10 years of monitoring showed high fidelity. Total ER-positive rate was 83.6% (81.4% to 86.8%), ER+/PR+ was 71.7% (68.6% to 75.5%), ER+/PR- was 17.6% (11.0% to 15.0%), ER-/PR- was 16.0% (13.5% to 18.2%), and ER-/PR+ was 0.6% (0.2% to 1.0%). The HER2-positive rate was 13.7% (10.2% to 17.4%) including 9.9% (7.3% to 11.9%) by IHC and 3.8% (1.9% to 5.9%) by FISH reflexed from IHC 2+ results. FISH amplification rate of HER2 IHC 2+ cases was 11.0% (5.8% to 19.2%). Annual quality-assurance check for HER2 IHC/FISH percent positive and percent negative agreement (as defined by Food and Drug Administration) was 96% to 100%. CONCLUSIONS: This longitudinal active assessment of 9564 breast biomarker cases shows the achievement of high fidelity of breast biomarker results when following the ASCO/CAP guidelines. Continuous monitoring of breast biomarkers may minimize assay analytical drift and assure quality clinically relevant results.

Low Estrogen Receptor (ER)-Positive Breast Cancer and Neoadjuvant Systemic Chemotherapy: Is Response Similar to Typical ER-Positive or ER-Negative Disease?

OBJECTIVES: Pathologic complete response (pCR) rate after neoadjuvant chemotherapy was compared between 141 estrogen receptor (ER)-negative (43%), 41 low ER+ (13%), 47 moderate ER+ (14%), and 98 high ER+ (30%) tumors. METHODS: Human epidermal growth factor receptor 2-positive cases, cases without semiquantitative ER score, and patients treated with neoadjuvant endocrine therapy alone were excluded. RESULTS: The pCR rate of low ER+ tumors was similar to the pCR rate of ER- tumors (37% and 26% for low ER and ER- respectively, P = .1722) but significantly different from the pCR rate of moderately ER+ (11%, P = .0049) and high ER+ tumors (4%, P < .0001). Patients with pCR had an excellent prognosis regardless of the ER status. In patients with residual disease (no pCR), the recurrence and death rate were higher in ER- and low ER+ cases compared with moderate and high ER+ cases. CONCLUSIONS: Low ER+ breast cancers are biologically similar to ER- tumors. Semiquantitative ER H-score is an important determinant of response to neoadjuvant chemotherapy.

Prognostic Significance of Modified Residual Disease in Breast and Nodes (mRDBN) Algorithm After Neoadjuvant Chemotherapy for Breast Cancer.

OBJECTIVES: We hypothesized that prognostic accuracy of the residual disease in breast and lymph nodes (RDBN) method, which is calculated using residual tumor size, nodal involvement, and tumor grade, may be improved by incorporating residual tumor cellularity. METHODS: Cases included 614 patients who underwent neoadjuvant therapy for breast cancer. Tumor size was adjusted for residual cellularity of invasive carcinoma and used to calculate modified RDBN (mRDBN) and compared with unmodified gross tumor size (gRDBN). RESULTS: RDBN could be calculated in 428 cases. Relative risks of recurrence and death were significantly higher for RDBN-3 and RDBN-4 compared with RDBN-1. Kaplan-Meier analysis showed significant differences in disease-free survival and overall survival for estrogen receptor (ER)-negative/human epidermal growth factor receptor 2 (HER2)-negative and ER-positive/HER2-negative subgroups (P < .0001). CONCLUSIONS: Both mRDBN and gRDBN provide prognostic information, particularly in HER2-negative carcinoma; however, mRDBN showed better stratification of RDBN-3 and RDBN-4 patients.

Male Breast Cancer.

Objectives: A clinicopathologic study with an emphasis on tumor immunohistochemical profile is presented. Methods: Sixty-one cases of male invasive breast cancers were studied. Median age of the cohort was 65 years. Results: Ninety-seven percent were estrogen receptor positive+ and 10% human epidermal growth factor receptor 2 positive. The individual diagnostic marker positivity was 98% for GATA-binding protein 3, 95% for androgen receptor, 90% for progesterone receptor, 88% for deleted in pancreatic cancer 4, 75% for gross cystic disease fluid protein 15, 72% for cytokeratin 7, 55% for mammaglobin, and 15% for vimentin and Wilms tumor protein 1. Caudal type homeobox 2 protein, cytokeratin 20, Napsin A, paired box gene 8, prostate-specific antigen, thyroid transcription factor 1, and uroplakin II were negative in all cases. Survival analyses showed tumor stage, receptor status, and Nottingham prognostic index to be prognostic. The overall survival was 70%, but the breast cancer­specific survival was 92% (mean follow-up, 59 months); 33% developed second malignancy. The immunohistochemistry profile was similar to female breast cancers. Conclusions: The second malignancies in this cohort affected overall survival and suggest the possibility of other germline mutations in addition to BRCA2 in male patients with breast cancer.

Characterizing Molecular Variants and Clinical Utilization of Next-generation Sequencing in Advanced Breast Cancer.

Women with advanced breast carcinomas have few therapeutic options. Recent advances in genomic profiling represent a new paradigm of cancer classification and treatment, but experience with genomic testing in a clinical setting remains limited. We retrospectively determined the genomic variants and correlate these with histology [histomorphologic subtype, nuclear grade, standard immunohistochemistry (IHC)] and clinical utilization (ordering, turnaround time, report review, and targeted therapy). Among 48 patients, 2 showed no genetic alterations, 11 (23%) showed variants of unclear significance only and 35 (73%) showed variant(s) affecting function (VaF) and/or variants of unclear significance. Overall, 119 variants were observed in 20 of 50 tested genes. Each patient had a unique molecular profile, with numerous (n=58) variants not previously reported in breast cancer. VaF detected in more than 2 patients included: TP53 (n=21), PIK3CA (n=20), and FGFR1 (n=3). VaF comprised 46 single nucleotide variants (79%), 7 amplifications (12%), 3 frameshifts (5%), 1 insertion (2%), and 1 deletion (2%). The tested samples had very high Ki67 index (average 57%±23%) and approximately half were hormone receptor and HER2 negative (25/46, 54%). Metastatic breast carcinomas showed a higher average VaF versus breast-localized tumors (1.3±0.99 vs. 0.18±0.60, P<0.05). Next-generation sequencing reports were promptly reported and reviewed (average 1 to 2 d) and 7 (∼25%) of potentially eligible patients received targeted therapy. Advanced breast cancers show unique landscapes of genetic variants. Most testing was done in late disease, often in metastatic and receptor-negative carcinomas. Next-generation sequencing results were promptly reported and reviewed, but the utilization of targeted therapies was limited.

Immunohistochemical profile of breast cancer with respect to estrogen receptor and HER2 status.

There are a few studies that have evaluated a panel of stains on a single large data set of breast cancers, which is required for direct comparison between antibodies. The immunohistochemical panel in this study was chosen to include breast-specific markers and markers that are expressed in tumors resembling breast cancer. The individual marker positivity in decreasing order was 95% (177/186) for GATA-3, 92% (172/186) for cytokeratin (CK)7, 80% (151/189) for AR, 80% for estrogen receptor (158/198), 69% for progesterone receptor (137/198), 55% (105/190) for NY-BR-1, 52% (99/189) for mammaglobin, 31% (59/191) for vimentin, 26% (51/195) for GCDFP-15, 0.5% (1/186) for CK20, and 0% (0/188) for PAX-8. When tumors were categorized based on estrogen receptor and HER2 status; a total of 45 profiles were identified. In addition, some tumors showed an unconventional profile-although the majority of breast carcinomas were CK7-positive/CK20-negative, a CK7-negative/CK20-negative profile was seen in ∼8% of the cases. Such a profile can create confusion in investigation of a carcinoma of unknown origin. The results define the individual sensitivity of each marker and establish a baseline diagnostic profile of breast cancer in a large data set. In addition, the results support the use of immunohistochemical panel for confirming or determining breast as the source of metastasis.

Semiquantitative GATA-3 immunoreactivity in breast, bladder, gynecologic tract, and other cytokeratin 7-positive carcinomas.

OBJECTIVES: To evaluate GATA-3 immunohistochemical expression semiquantitatively in breast, gynecologic, gastric, pancreatic-biliary tract, urothelial, and vulvar/cervical squamous cell carcinomas. METHODS: GATA-3 expression was evaluated by immunohistochemistry in 198 invasive breast carcinomas on tissue microarrays. Tissue microarrays of other tissues included 144 gynecologic tumors, 28 bladder carcinomas, 63 cholangiocarcinomas, 20 pancreatic carcinomas, and 62 gastric carcinomas. Full tissue sections of 10 invasive squamous cell carcinomas were also stained. GATA-3 expression was semiquantitatively scored using an H-score method. H-score greater than 10 was considered a positive result. RESULTS: Of 186 breast carcinomas, 95% were positive (mean H-score of 217). GATA-3 expression was uncommon in 139 nonsquamous gynecologic tumors, with often weak reactivity (mean H-score <50) seen in 18% of endocervical, 7% of endometrial, and 10% of ovarian tumors. Six (60%) of 10 squamous cell carcinomas expressed GATA-3 (mean H-score of 102). Of 22 urothelial carcinomas, 95% expressed GATA-3 (mean H-score of 170). A few cholangiocarcinomas (3%), pancreatic adenocarcinomas (10%), and gastric carcinomas (2%) weakly expressed GATA-3 (mean H-score <50). CONCLUSIONS: Strong GATA-3 expression is a reliable marker of primary breast carcinoma in the appropriate clinical context. GATA-3 reactivity in around 70% of triple-negative breast carcinomas is also clinically useful. Significant reactivity in gynecologic squamous cell carcinomas suggests that GATA-3 alone cannot reliably distinguish these tumors from urothelial carcinoma.

Morphologic features do not influence response to trastuzumab-containing neoadjuvant chemotherapy in HER2-positive breast cancer.

Trastuzumab-containing neoadjuvant chemotherapy in patients with HER2-positive breast cancer is highly effective in reducing tumor volume and enables more patients to have breast-conserving surgery. The tumor hormone receptor level has been shown to significantly influence response to trastuzumab-containing neoadjuvant chemotherapy. In this study, we comprehensively evaluated various morphologic features and proliferative activity in 50 HER2-positive invasive breast carcinomas treated with trastuzumab-containing neoadjuvant chemotherapy to determine whether any of these features have the same predictive value as tumor hormone receptor content. The tumor proliferation activity as measured by Ki-67 and various morphologic parameters were compared between tumors that achieved >50% tumor volume reduction including pathologic complete response (pCR) and tumors that showed ≤50% tumor volume reduction. Thirty-seven cases (74%) showed >50% tumor volume reduction including 18 cases with pCR. Thirteen cases (26%) showed ≤50% tumor volume reduction. Neither morphologic variables nor Ki-67 labeling index were predictive of >50% tumor volume reduction. In contrast, hormone receptor status and semiquantitative H scores for hormone receptors were predictive of>50% tumor volume reduction. The mean estrogen and progesterone receptor H scores for tumors that showed >50% tumor volume reduction (including pCR) were 77 (median 10) and 31 (median 0), respectively, compared with 168 (median 200) and 79 (median 75) for cases that showed ≤50% tumor volume reduction. Semiquantitative scoring for hormone receptors provides useful information in terms of therapeutic response in HER2-positive tumors.

Diagnostic Value of Calretinin in Mast Cell Lesions of the Skin.

The diagnosis of mast cell lesions of the skin can occasionally be challenging. Calretinin, a 29 kD neuron-specific calcium-binding protein found mostly in the CNS and retina, has been shown to be a positive marker for mesotheliomas, and is also expressed in mast cells. We studied the diagnostic value of calretinin and compared our results to other established ancillary studies used to identify mast cells, such as Toluidine blue and the Leder stain. Sixty-three cases were studied, including 45 mast cell lesions (22 urticaria pigmentosum, 17 mastocytomas, and six telangiectasia macularis eruptiva perstans [TMEP]), seven nevi, three melanomas, four granular cell minors of the skin, three cutaneous lymphomas, and one granulocytic sarcoma. Patients ranged in age from less than 1 to 85 years with a median age of 29 years. The group consisted of 36 females and 27 males. Calretinin was expressed in all 45 mast cell lesions. Negative staining for calretinin was seen in all skin lesions that potentially could be considered in the differential diagnosis of mast cell lesions such as nevi, melanomas, lymphomas, and the granulocytic sarcoma. However, calretinin expression was noted in four/four granular cell tumors. Leder and Toluidine blue stains were positive in all 45 mast cell lesions, and all nonmast cell lesions were negative with these stains. In conclusion, our study demonstrated that calretinin is a sensitive and specific marker of mast cells and can be an aid in distinguishing mast cell lesions from other skin lesions considered in the differential diagnosis. Calretinin may be more sensitive than the currently used special stains utilized to diagnose mast cell lesions having few diagnostic mast cells such as TMEP. However, this immunoperoxidase stain does not add significant diagnostic information in most cases, when compared with the currently used less expensive special stains and, therefore, is not cost-effective. Int J Surg Pathol 8(2):119-122, 2000