Fit-for-Purpose Ki-67 Immunohistochemistry Assays for Breast Cancer.

New therapies are being developed for breast cancer, and in this process, some "old" biomarkers are reutilized and given a new purpose. It is not always recognized that by changing a biomarker's intended use, a new biomarker assay is created. The Ki-67 biomarker is typically assessed by immunohistochemistry (IHC) to provide a proliferative index in breast cancer. Canadian laboratories assessed the analytical performance and diagnostic accuracy of their Ki-67 IHC laboratory-developed tests (LDTs) of relevance for the LDTs' clinical utility. Canadian clinical IHC laboratories enrolled in the Canadian Biomarker Quality Assurance Pilot Run for Ki-67 in breast cancer by invitation. The Dako Ki-67 IHC pharmDx assay was employed as a study reference assay. The Dako central laboratory was the reference laboratory. Participants received unstained slides of breast cancer tissue microarrays with 32 cases and performed their in-house Ki-67 assays. The results were assessed using QuPath, an open-source software application for bioimage analysis. Positive percent agreement (PPA, sensitivity) and negative percent agreement (NPA, specificity) were calculated against the Dako Ki-67 IHC pharmDx assay for 5%, 10%, 20%, and 30% cutoffs. Overall, PPA and NPA varied depending on the selected cutoff; participants were more successful with 5% and 10%, than with 20% and 30% cutoffs. Only 4 of 16 laboratories had robust IHC protocols with acceptable PPA for all cutoffs. The lowest PPA for the 5% cutoff was 85%, for 10% was 63%, for 20% was 14%, and for 30% was 13%. The lowest NPA for the 5% cutoff was 50%, for 10% was 33%, for 20% was 50%, and for 30% was 57%. Despite many years of international efforts to standardize IHC testing for Ki-67 in breast cancer, our results indicate that Canadian clinical LDTs have a wide analytical sensitivity range and poor agreement for 20% and 30% cutoffs. The poor agreement was not due to the readout but rather due to IHC protocol conditions. International Ki-67 in Breast Cancer Working Group (IKWG) recommendations related to Ki-67 IHC standardization cannot take full effect without reliable fit-for-purpose reference materials that are required for the initial assay calibration, assay performance monitoring, and proficiency testing.

Ki67 in Breast Cancer Assay: An Ad Hoc Testing Recommendation from the Canadian Association of Pathologists Task Force.

Ki67, a marker of cellular proliferation, is commonly assessed in surgical pathology laboratories. In breast cancer, Ki67 is an established prognostic factor with higher levels associated with worse long-term survival. However, Ki67 IHC is considered of limited clinical use in breast cancer management largely due to issues related to standardization and reproducibility of scoring across laboratories. Recently, both the American Food and Drug Administration (FDA) and Health Canada have approved the use of abemaciclib (CDK4/6 inhibitor) for patients with HR+/HER2: high-risk early breast cancers in the adjuvant setting. Health Canada and the FDA have included a Ki67 proliferation index of ≥20% in the drug monograph. The approval was based on the results from monarchE, a phase III clinical trial in early-stage chemotherapy-naïve, HR+, HER2 negative patients at high risk of early recurrence. The study has shown significant improvement in invasive disease-free survival (IDFS) with abemaciclib when combined with adjuvant endocrine therapy at two years. Therefore, there is an urgent need by the breast pathology and medical oncology community in Canada to establish national guideline recommendations for Ki67 testing as a predictive marker in the context of abemaciclib therapy consideration. The following recommendations are based on previous IKWG publications, available guidance from the monarchE trial and expert opinions. The current recommendations are by no means final or comprehensive, and their goal is to focus on its role in the selection of patients for abemaciclib therapy. The aim of this document is to guide Canadian pathologists on how to test and report Ki67 in invasive breast cancer. Testing should be performed upon a medical oncologist's request only. Testing must be performed on treatment-naïve tumor tissue. Testing on the core biopsy is preferred; however, a well-fixed resection specimen is an acceptable alternative. Adhering to ASCO/CAP fixation guidelines for breast biomarkers is advised. Readout training is strongly recommended. Visual counting methods, other than eyeballing, should be used, with global rather than hot spot assessment preferred. Counting 100 cells in at least four areas of the tumor is recommended. The Ki67 scoring app developed to assist pathologists with scoring Ki67 proposed by the IKWG, available for free download, may be used. Automated image analysis is very promising, and laboratories with such technology are encouraged to use it as an adjunct to visual counting. A score of <5 or >30 is more robust. The task force recommends that the results are best expressed as a continuous variable. The appropriate antibody clone and staining protocols to be used may take time to address. For the time being, the task force recommends having tonsils/+pancreas on-slide control and enrollment in at least one national/international EQA program. Analytical validation remains a pending goal. Until the data become available, using local ki67 protocols is acceptable. The task force recommends participation in upcoming calibration and technical validation initiatives.

Metastatic Mammary Carcinoma Presenting as a Large Cystic Axillary Mass: A Report of an Unusual Case.

The differential diagnosis of cystic axillary masses is broad and includes intranodal lesions. Cystic metastatic tumor deposits are rare, and have been reported in a few tumor types, most commonly in the head and neck region, but rarely described with metastatic mammary carcinoma. We report a case of a 61-year-old female who presented with a large right axillary mass. Imaging studies revealed a cystic axillary mass and ipsilateral breast mass. She was managed with breast conservation surgery and axillary dissection for invasive ductal carcinoma, no special type, Nottingham grade 2 (21 mm). One of nine lymph nodes contained a cystic nodal deposit (52 mm), which resembled a benign inclusion cyst. Oncotype DX recurrence score for the primary tumor was low (8), conferring a low risk of disease recurrence despite the large size of the nodal metastatic deposit. A cystic pattern of metastatic mammary carcinoma is rare and important to recognize for accurate staging and management decisions.

Using Deep Learning to Predict Final HER2 Status in Invasive Breast Cancers That are Equivocal (2+) by Immunohistochemistry.

Invasive breast carcinomas are routinely tested for HER2 using immunohistochemistry (IHC), with reflex in situ hybridization (ISH) for those scored as equivocal (2+). ISH testing is expensive, time-consuming, and not universally available. In this study, we trained a deep learning algorithm to directly predict HER2 gene amplification status from HER2 2+ IHC slides. Data included 115 consecutive cases of invasive breast carcinoma scored as 2+ by IHC that had follow-up HER2 ISH testing. An external validation data set was created from 36 HER2 IHC slides prepared at an outside institution. All internal IHC slides were digitized and divided into training (80%), and test (20%) sets with 5-fold cross-validation. Small patches (256×256 pixels) were randomly extracted and used to train convolutional neural networks with EfficientNet B0 architecture using a transfer learning approach. Predictions for slides in the test set were made on individual patches, and these predictions were aggregated to generate an overall prediction for each slide. This resulted in a receiver operating characteristic area under the curve of 0.83 with an overall accuracy of 79% (sensitivity=0.70, specificity=0.82). Analysis of external validation slides resulted in a receiver operating characteristic area under the curve of 0.79 with an overall accuracy of 81% (sensitivity=0.50, specificity=0.82). Although the sensitivity and specificity are not high enough to negate the need for reflexive ISH testing entirely, this approach may be useful for triaging cases more likely to be HER2 positive and initiating treatment planning in centers where HER2 ISH testing is not readily available.

HER2 testing in metastatic breast cancer - Is reflex ISH testing necessary on HER2 IHC-equivocal (2+) cases?

Current guidelines recommend HER2 testing on all primary invasive breast cancers and re-biopsy at disease relapse. The discordance rate between HER2-negative primaries and HER2 IHC2+ metastases that are ISH-amplified is unknown. We hypothesize that the majority of such cases are non-amplified. ISH testing is time-consuming and resource-intensive, and there may be situations where it is unnecessary. A retrospective review of IHC2+ metastatic lesions assessed with ISH at our center from 2013 to 2021 was undertaken. 105 cases were identified after exclusion of cases missing HER2 results, with primaries of unconfirmed origin, and cases of synchronous primary and metastatic disease. IHC and ISH results were recorded with detailed slide review of discordant cases. 91/105 metastases had HER2-negative primaries (87%). A metastasis was significantly more likely to be HER2-negative when the primary was HER2-negative (93%) versus positive (43%) (p < 0.0001). 54/91 primaries were IHC2+/ISH-non-amplified, and 50/54 (93%) corresponding metastases had identical results. Of the 37 HER2-negative primaries that were IHC0/1+, 35 (95%) corresponding metastases were ISH-non-amplified. Six metastases in cases with HER2-negative primaries were discordant. Characteristics of metastases suggesting ISH testing was warranted to assess for discordance included IHC heterogeneity, morphological discordance, increased staining of moderate intensity, and ER/PR discordance. One or more of these factors were present in all discordant metastases. Our results suggest selective ISH testing on HER2 IHC2+ breast cancer metastases in the context of HER2-negative primary disease may be appropriate when there is careful review of the IHC. Validation of our findings awaits further studies with larger sample sizes.

Assessment of HER2 using the 2018 ASCO/CAP guideline update for invasive breast cancer: a critical look at cases classified as HER2 2+ by immunohistochemistry.

In 2018, the American Society of Clinical Oncology/College of American Pathologists revised the criteria for HER2 immunohistochemistry (IHC) equivocal (2+) classification in their updated guideline. We reviewed invasive breast cancer specimens originally classified as equivocal (2+) under the 2018 guideline that underwent HER2 fluorescence in situ hybridization (FISH) testing from August 2018 to August 2019 at our Canadian reference hospital to investigate cases with ambiguous staining patterns between the 1+ and 2+ definitions. Demographics, pathologic features, and pre-analytic conditions were recorded. The H&E and corresponding HER2 IHC slides were reviewed to confirm tumor type and grade, and classify as HER2 indeterminate, 0, 1+, 2+, or "Intermediate" (staining features between the 1+ and 2+ classifications). FISH testing was performed on 289 cases and 273 met inclusion criteria. The FISH-amplified rate was 12.1%. Upon IHC review, 44.7% (122/273) of cases were reclassified as Intermediate. These cases had incomplete staining with moderate intensity (43/122, 35.3%) and/or <10% complete weak or moderate staining (102/122, 83.6%). Intermediate cases had a significantly lower frequency of amplified FISH results than 2+ cases (p < 0.0001), with only four (3.3%) FISH positive and two (1.6%) FISH heterogeneous. Our study highlights the ambiguity in the current guideline for classifying some HER2 IHC patterns. As the rate of gene amplification in these cases was low (4.9%), we recommend adhering to the 2018 HER2 2+ criteria for reflex FISH testing. However, cases with <10% moderate complete staining and certain heterogeneous patterns warrant special consideration. Further descriptive clarification of 1+ criteria is needed.

Cystic neutrophilic granulomatous mastitis - a review of 12 consecutive cases.

AIMS: Cystic neutrophilic granulomatous mastitis (CNGM) is an uncommon but increasingly recognised cause of mastitis, often associated with Corynebacterium ssp. infection. We studied the histopathological and clinical features of CNGM in a Canadian setting, and the work-up required to identify pathogenic microorganisms. METHODS AND RESULTS: A retrospective search for breast specimens with abscess, acute, chronic and/or granulomatous inflammation from 1998 to 2018 was performed. Haematoxylin and eosin slides were reviewed for typical histological features of CNGM. Histochemically stained slides for microorganisms were also reviewed. Repeat Gram stains were performed if initially negative. Electronic medical records were abstracted for microbiology results and relevant clinical data. Twelve cases were identified. All were female, aged 25-57 years, mainly Caucasian, with one Venezuelan and two of Chinese ethnicity. Most were parous (10 of 12); five of 12 had an endocrinopathy. Bacteria were identified in one or more specimens from eight of 12 patients; additional Gram stains revealed organisms in four of 12 cases. Of four bacterial cultures, one grew Corynebacterium kroppenstedtii. 16S polymerase chain reaction for three samples was negative. Two patients had multiple breast biopsies, showing early palisaded granulomas followed by classic features of CNGM. The patients had various management approaches, including surgery and antimicrobials. CONCLUSIONS: CNGM may present as palisaded granulomatous inflammation, without the expected 'cystic' pattern, suggesting that there is an evolution of histomorphology with this infection. Most patients with CNGM are parous, and there may be an association with endocrinopathies. Application of multiple Gram stains increases the yield of microorganism identification. Recognition of CNGM in breast biopsies and collaborative communications are essential to direct appropriate therapy.

Assessing breast cancer risk within the general screening population: developing a breast cancer risk model to identify higher risk women at mammographic screening.

OBJECTIVES: To develop a breast cancer risk model to identify women at mammographic screening who are at higher risk of breast cancer within the general screening population. METHODS: This retrospective nested case-control study used data from a population-based breast screening program (2009-2015). All women aged 40-75 diagnosed with screen-detected or interval breast cancer (n = 1882) were frequency-matched 3:1 on age and screen-year with women without screen-detected breast cancer (n = 5888). Image-derived risk factors from the screening mammogram (percent mammographic density [PMD], breast volume, age) were combined with core biopsy history, first-degree family history, and other clinical risk factors in risk models. Model performance was assessed using the area under the receiver operating characteristic curve (AUC). Classifiers assigning women to low- versus high-risk deciles were derived from risk models. Agreement between classifiers was assessed using a weighted kappa. RESULTS: The AUC was 0.597 for a risk model including only image-derived risk factors. The successive addition of core biopsy and family history significantly improved performance (AUC = 0.660, p < 0.001 and AUC = 0.664, p = 0.04, respectively). Adding the three remaining risk factors did not further improve performance (AUC = 0.665, p = 0.45). There was almost perfect agreement (kappa = 0.97) between risk assessments based on a classifier derived from image-derived risk factors, core biopsy, and family history compared with those derived from a model including all available risk factors. CONCLUSIONS: Women in the general screening population can be risk-stratified at time of screen using a simple model based on age, PMD, breast volume, and biopsy and family history. KEY POINTS: • A breast cancer risk model based on three image-derived risk factors as well as core biopsy and first-degree family history can provide current risk estimates at time of screen. • Risk estimates generated from a combination of image-derived risk factors, core biopsy history, and first-degree family history may be more valid than risk estimates that rely on extensive self-reported risk factors. • A simple breast cancer risk model can avoid extensive clinical risk factor data collection.

"Interchangeability" of PD-L1 immunohistochemistry assays: a meta-analysis of diagnostic accuracy.

Different clones, protocol conditions, instruments, and scoring/readout methods may pose challenges in introducing different PD-L1 assays for immunotherapy. The diagnostic accuracy of using different PD-L1 assays interchangeably for various purposes is unknown. The primary objective of this meta-analysis was to address PD-L1 assay interchangeability based on assay diagnostic accuracy for established clinical uses/purposes. A systematic search of the MEDLINE database using PubMed platform was conducted using "PD-L1" as a search term for 01/01/2015 to 31/08/2018, with limitations "English" and "human". 2,515 abstracts were reviewed to select for original contributions only. 57 studies on comparison of two or more PD-L1 assays were fully reviewed. 22 publications were selected for meta-analysis. Additional data were requested from authors of 20/22 studies in order to enable the meta-analysis. Modified GRADE and QUADAS-2 criteria were used for grading published evidence and designing data abstraction templates for extraction by reviewers. PRISMA was used to guide reporting of systematic review and meta-analysis and STARD 2015 for reporting diagnostic accuracy study. CLSI EP12-A2 was used to guide test comparisons. Data were pooled using random-effects model. The main outcome measure was diagnostic accuracy of various PD-L1 assays. The 22 included studies provided 376 2×2 contingency tables for analyses. Results of our study suggest that, when the testing laboratory is not able to use an Food and Drug Administration-approved companion diagnostic(s) for PD-L1 assessment for its specific clinical purpose(s), it is better to develop a properly validated laboratory developed test for the same purpose(s) as the original PD-L1 Food and Drug Administration-approved immunohistochemistry companion diagnostic, than to replace the original PD-L1 Food and Drug Administration-approved immunohistochemistry companion diagnostic with a another PD-L1 Food and Drug Administration-approved companion diagnostic that was developed for a different purpose.

Predictors of Outcome in Mammary Adenoid Cystic Carcinoma: A Multi-Institutional Study.

Mammary adenoid cystic carcinoma (ACC) is a rare subtype of breast cancer with a favorable prognosis. Here we report on predictors of outcome based on a detailed morphologic review and analysis of 108 mammary ACC. Sixty-four tumors (59.2%) were pure conventional ACC, 23 (21.3%) were pure basaloid ACC. Follow-up was available for 87 patients (median: 51 mo). Eighteen patients (20.7%) developed recurrence: 7 (8%) had local recurrence and 14 (16%) had distant metastasis. Two patients died of disease, 1 died of an unrelated cause, 14 were alive with disease (including 8 in palliative care), and 70 (80.5%) were alive with no evidence of disease. Of 90 patients with known lymph node (LN) status 9 (10%) had nodal involvement (all with basaloid ACC). Distant metastases in patients with predominantly basaloid ACC compared with pure conventional ACC were more common (40% vs. 7.7%) and occurred earlier (22 vs. 84 mo). The following factors were found to be predictive of recurrence-free survival: positive margin, Nottingham grade, neovascularization, basaloid component, perineural invasion, lymphovascular invasion, >30% solid growth, necrosis and LN involvement; the first 3 remained statistically significant on multivariate analysis. Factors predictive of distant disease-free survival were neovascularization, Nottingham grade, lymphovascular invasion, solid component >50%, LN involvement, basaloid component >50%, tumor necrosis, perineural invasion, and final margin. Only neovascularization remained statistically significant on multivariate analysis. Basaloid ACC is an aggressive variant of mammary ACC with more frequent nodal involvement and higher incidence of distant spread. LN staging should be performed for all mammary basaloid ACC.

Fit-For-Purpose PD-L1 Biomarker Testing For Patient Selection in Immuno-Oncology: Guidelines For Clinical Laboratories From the Canadian Association of Pathologists-Association Canadienne Des Pathologistes (CAP-ACP).

Since 2014, programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) checkpoint inhibitors have been approved by various regulatory agencies for the treatment of multiple cancers including melanoma, lung cancer, urothelial carcinoma, renal cell carcinoma, head and neck cancer, classical Hodgkin lymphoma, colorectal cancer, gastroesophageal cancer, hepatocellular cancer, and other solid tumors. Of these approved drug/disease combinations, a subset also has regulatory agency-approved, commercially available companion/complementary diagnostic assays that were clinically validated using data from their corresponding clinical trials. The objective of this document is to provide evidence-based guidance to assist clinical laboratories in establishing fit-for-purpose PD-L1 biomarker assays that can accurately identify patients with specific tumor types who may respond to specific approved immuno-oncology therapies targeting the PD-1/PD-L1 checkpoint. These recommendations are issued as 38 Guideline Statements that address (i) assay development for surgical pathology and cytopathology specimens, (ii) reporting elements, and (iii) quality assurance (including validation/verification, internal quality assurance, and external quality assurance). The intent of this work is to provide recommendations that are relevant to any tumor type, are universally applicable and can be implemented by any clinical immunohistochemistry laboratory performing predictive PD-L1 immunohistochemistry testing.

Isolated Tumor Cells in Sentinel Lymph Nodes of Primary Invasive Breast Carcinoma: A Cohort Analysis.

BACKGROUND: The practice of performing routine cytokeratin immunohistochemistry (CK-IHC) on sentinel lymph nodes in early stage invasive breast cancer leads to frequent identification of isolated tumor cells (ITCs), the clinical significance of which remains unclear. After emergence of guidelines that suggested limited clinical utility of ITC detection, routine CK-IHC (rCK-IHC) staining was discontinued at our institution. We studied the rate and clinical utility of ITC detection before and after the discontinuation of rCK-IHC. PATIENTS AND METHODS: We retrospectively reviewed 2 cohorts of 250 consecutive early stage invasive breast cancer (IBC) patients with sentinel lymph node biopsies (SLNBs) in 2010 to 2011 (rCK-IHC) and 2015 to 2016 (selective CK-IHC [sCK-IHC]). Variables abstracted included: tumor histology, tumor size, grade, lymphatic-vascular invasion, hormone receptor expression, HER2 status, and nodal status including ITCs. All cases from the 2015 to 2016 cohort for which sCK-IHC was performed underwent pathology review. A clinical review of treatment decision effect and cost analysis was undertaken. Data were analyzed using descriptive statistics and Fisher exact test. RESULTS: In the rCK-IHC cohort, all 250 cases underwent CK-IHC staining versus 57 cases in the sCK-IHC cohort. There were 23 ITC cases observed in the rCK-IHC cohort compared with 11 in the sCK-IHC cohort (P = .049). Excluding lobular carcinomas, 19 ITC cases were observed with rCK-IHC versus 7 with sCK-IHC (P = .02). ITC detection did not affect adjuvant treatment decision-making and resulted in savings of at least Can$8000. CONCLUSION: Selective rather than routine use of CK-IHC staining for SLNB evaluation in early-stage IBC results in decreased ITC detection without affecting treatment decisions and leads to cost savings.

Impact of Detection Method and Age on Survival Outcomes in Triple-Negative Breast Cancer: A Population-Based Cohort Analysis.

BACKGROUND: Most investigations have compared triple-negative breast cancer (TNBC) to non-TNBC to elucidate clinical or epidemiologic differences between subtypes. We examined a contemporary cohort of patients with primary TNBC by detection and age at diagnosis within a population-based breast screening program to examine survival outcomes. PATIENTS AND METHODS: All women with a diagnosis of primary TNBC between January 1, 2005, and December 31, 2012, in Nova Scotia, Canada, were included. Clinicopathologic and detection variables were abstracted from the Nova Scotia Breast Screening Program. Patient charts were abstracted for adjuvant therapies and survival outcomes, supplemented by provincial vital statistical data. RESULTS: A total of 412 patients comprised the study population, with almost half aged over 60 years (46.3%) and 30.2% having screen-detected disease. There were no significant differences in prognostic variables between age groups. Younger patients were more likely to receive adjuvant chemotherapy (96.3% ≤ 49 years vs. 31.2% ≥ 70 years), but there were no differences in disease-free or breast cancer-specific survival between the age groups. For those with disease recurrence, median time to recurrence and death was shorter for younger patients (17 vs. 26 months, 16 vs. 33 months respectively; age 40-49 vs. 70+). Those with screen-detected disease had better disease-free, breast cancer-specific, and overall survival outcomes. CONCLUSION: Detection method may play a role in TNBC survival outcomes, thus supporting novel screening strategies for TNBC. Shorter time to survival events in the younger patient groups suggests that TNBC is a clinically heterogeneous disease despite similarities in prognostic factors across age.

Performance of residents using digital images versus glass slides on certification examination in anatomical pathology: a mixed methods pilot study.

BACKGROUND: It is anticipated that many licensing examination centres for pathology will begin fully digitizing the certification examinations. The objective of our study was to test the feasibility of a fully digital examination and to assess the needs, concerns and expectations of pathology residents in moving from a glass slide-based examination to a fully digital examination. METHODS: We conducted a mixed methods study that compared, after randomization, the performance of senior residents (postgraduate years 4 and 5) in 7 accredited anatomical pathology training programs across Canada on a pathology examination using either glass slides or digital whole-slide scanned images of the slides. The pilot examination was followed by a post-test survey. In addition, pathology residents from all levels of training were invited to participate in an online survey. RESULTS: A total of 100 residents participated in the pilot examination; 49 were given glass slides instead of digital images. We found no significant difference in examination results between the 2 groups of residents (estimated marginal mean 8.23/12, 95% confidence interval [CI] 7.72-8.87, for glass slides; 7.84/12, 95% CI 7.28-8.41, for digital slides). In the post-test survey, most of the respondents expressed concerns with the digital examination, including slowly functioning software, blurring and poor detail of images, particularly nuclear features. All of the respondents of the general survey (n = 179) agreed that additional training was required if the examination were to become fully digital. INTERPRETATION: Although the performance of residents completing pathology examinations with glass slides was comparable to that of residents using digital images, our study showed that residents were not comfortable with the digital technology, especially given their current level of exposure to it. Additional training may be needed before implementing a fully digital examination, with consideration for a gradual transition.

Postirradiation pseudosclerodermatous panniculitis with involvement of breast parenchyma: a dramatic example of a rare entity and a pitfall in diagnosis.

Postirradiation pseudosclerodermatous panniculitis is a rare complication of external beam radiotherapy. This inflammatory process typically presents as an erythematous indurated plaque in a previously irradiated region of skin. To date, 13 cases have been reported worldwide. We present a case of a 70-year-old female who received breast irradiation following conservation surgery for invasive breast carcinoma. In her third year of follow-up, she developed an enlarging mass, involving the subcutis and underlying breast tissue, associated with mammographically detected coarse calcifications and density, at the surgical site. This was deemed highly suspicious of recurrent malignancy. Following several benign needle core biopsies, she had an excision of the mass. This revealed a lobular panniculitis and irradiation-induced vascular changes affecting subcutaneous fat and underlying breast tissue. This is the 14th reported case of this rare entity. It is unique in the degree of involvement, affecting breast parenchyma as well as subcutaneous fat, and in its corresponding dramatic clinical and radiographic manifestations.

Benign and Malignant Male Breast Diseases: Radiologic and Pathologic Correlation.

Male breast disease comprises a wide spectrum of benign and malignant processes. We present the spectrum of diseases encountered at our institution over the past 7 years (2007-2013) and correlate their radiological and histopathological appearances. Gynaecomastia is the most frequently encountered disease due to its association with a variety of causes. Male breast malignancies, though rare, must be considered. The most frequently encountered pathological characteristic is invasive and the predominant histologic subtypes are infiltrating ductal carcinomas.

Impact of the 2013 American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 (HER2) testing of invasive breast carcinoma: a focus on tumours assessed as 'equivocal' for HER2 gene amplification by fluorescence in-situ hybridization.

AIMS: The updated 2013 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) human epidermal growth factor receptor 2 (HER2) testing guidelines include changes to HER2 in-situ hybridization (ISH) interpretation criteria. We conducted a retrospective review of a consecutive cohort of primary breast carcinomas to assess the impact of updated guidelines on HER2 classification and laboratory resource utilization, and to characterize the pathobiology of HER2 equivocal tumours. METHODS AND RESULTS: A total of 904 dual-probe HER2/chromosome enumeration probe (CEP17) FISH tests on invasive breast carcinomas were studied. Eighty-five (9.4%) cases had a classification change with the updated guidelines; 66 (7.3%) went from HER2-negative to -equivocal, 15 cases (1.7%) were reclassified as HER2-positive and four cases from HER2-equivocal to -negative. A subset of primary breast cancers, reported initially as HER2-negative but -equivocal by 2013 guidelines, was identified. Traditional pathological factors of this subset were compared to HER2-negative and -positive control cases. The three HER2 groups demonstrated statistically significant differences with respect to prognostic factors, including tumour size, grade and nodal involvement. CONCLUSIONS: The updated HER2 testing guidelines will result in the reclassification of approximately 9.4% of primary breast cancers with uncertainty regarding the clinical impact of this reclassification in the majority of cases. Resource utilization will increase as a result of the recommendation for retesting.

CytoLyt® fixation and decalcification pretreatments alter antigenicity in normal tissues compared with standard formalin fixation.

Immunohistochemistry is used on cell blocks constructed from cytopathology samples fixed in methanol-based fixatives, such as CytoLyt (Cytyc Corp), and on surgical pathology tissues exposed to decalcifying agents, often without technical validation. We evaluated a panel of commonly utilized antibodies in normal tissues exposed to differing preanalytic conditions as follows: CytoLyt fixation, formalin fixation followed by exposure to decalcifying agents (Leica Decalcifier I-10% formic acid or Leica Decalcifier II-5% hydrochloric acid), or standard formalin fixation. Altered expression was observed with several antibodies compared with standard formalin fixation. Specifically, there was absent or near absent expression of thyroid transcription factor 1 (TTF-1), D2-40, and CD20 in CytoLyt-fixed tissues, whereas reduced expression was observed for p63, estrogen receptor, S100 protein, CD3, calretinin, chromogranin, and synaptophysin. Absent or near absent expression of TTF-1 was also observed with exposure to hydrochloric acid, whereas reduced expression was observed for CK5/6, CK7, p63, estrogen receptor, leukocyte common antigen, CD3, CD20, and synaptophysin. Exposure to formic acid had less impact with reduced expression observed for only 3 antibodies (CK8/18, CK7, and TTF-1). The results of this study demonstrate the need to validate immunohistochemical protocols on control tissue treated in the same manner as test tissue, including CytoLyt fixation and exposure of tissue to decalcifying agents.

Human epidermal growth factor receptor 2 testing in primary breast cancer in the era of standardized testing: a Canadian prospective study.

PURPOSE: Therapies that target overexpression of human epidermal growth factor receptor 2 (HER2) rely on accurate and timely assessment of all patients with new diagnoses. This study examines HER2 testing of primary breast cancer tissue when performed with immunohistochemistry (IHC) and additional in situ hybridization (ISH) for negative cases (IHC 0/1+). The analysis focuses on the rate of false-negative HER2 tests, defined as IHC 0/1+ with an ISH ratio ≥ 2.0, in eight pathology centers across Canada. PATIENTS AND METHODS: Whole sections of surgical resections or tissue microarrays (TMAs) from invasive breast carcinoma tissue were tested by both IHC and ISH using standardized local methods. Samples were scored by the local breast pathologist, and consecutive HER2-negative IHC results (IHC 0/1+) were compared with the corresponding fluorescence or silver ISH result. RESULTS: Overall, 711 surgical excisions of primary breast cancer were analyzed by IHC and ISH; HER2 and chromosome 17 centromere (CEP17) counts were available in all cases. The overall rate of false-negative samples was 0.84% (six of 711 samples). Interpretable IHC and ISH scores were available in 1,212 cases from TMAs, and the overall rate of false-negative cases was 1.6% (16 of 978 cases). CONCLUSION: Our observation confirms that IHC is an adequate test to predict negative HER2 status in primary breast cancer in surgical excision specimens, even when different antibodies and IHC platforms are used. The study supports the American Society of Clinical Oncology/College of American Pathologists and Canadian testing algorithms of using IHC followed by ISH for equivocal cases.

Canadian Association of Pathologists-Association canadienne des pathologistes National Standards Committee for High Complexity Testing/Immunohistochemistry: guidelines for the preparation, release, and storage of unstained archived diagnostic tissue sections for immunohistochemistry.

OBJECTIVES: Formalin-fixed, paraffin-embedded unstained archived diagnostic tissue sections are frequently exchanged between clinical laboratories for immunohistochemical staining. The manner in which such sections are prepared represents a type of preanalytical variable that must be taken into account given the growing importance of immunohistochemical assays, especially predictive and prognostic tests, in personalized medicine. METHODS: Recommendations were derived from review of the literature and expert consensus of the Canadian Association of Pathologists-Association canadienne des pathologists National Standards Committee for High Complexity Testing/Immunohistochemistry. RESULTS: Relevant considerations include the type of glass slide on which to mount the unstained sections; the thickness of the tissue sections; the time from slide preparation to testing; the environment, particularly the temperature at which the unstained sections will be maintained prior to testing; the inclusion of on-slide positive control tissue where possible; and whether patient identifier(s) should be included on slide labels. CONCLUSIONS: Clear communication between requesting and releasing laboratories will facilitate the proper preparation of unstained sections and also ensure that applicable privacy considerations are addressed.