Is Oncotype DX testing informative for breast cancers with low ER expression? A retrospective review from a biomarker testing referral center.

PURPOSE: It remains unclear whether patients with HER2-negative, low-estrogen receptor (ER-low)-positive early breast cancer (BC) benefit from Oncotype DX® (ODX) testing. METHODS: We conducted a retrospective review of cases referred for ODX testing over a seven-year period from a breast biomarker testing referral center (n = 854). For each case, we recorded the ODX Recurrence Score (RS) along with percentage of ER nuclear positivity and staining intensity on immunohistochemistry. Our criteria for ER-low was defined as ≤10% cells with nuclear positivity and/or weak intensity of staining. Slides from all ER-low cases were reviewed and the reported ODX ER gene scores were recorded. We randomly selected a comparator group of 56 patients with ER > 10% positivity and non-weak staining intensity (ER-high). RESULTS: We identified 27 cases (3.2%) that met our criteria for ER-low. Of these, 92.6% had a high RS (>25), and 7.4% had a RS of 25. All cases with ≤10% ER nuclear positivity had a high RS. Most ER-low cases (85.2%) had ODX quantitative ER gene scores in the negative range, whereas all (100%) ER-high cases had positive ER gene scores. CONCLUSION: ODX does not appear to add significant additional information to inform treatment decisions for most patients with ER-low BC. Incorporating weak ER staining intensity in addition to low percentage of nuclear positivity identifies about twice as many ER-low patients, although with reduced specificity for high RS. Our study supports the contention that most ER-low early BC should be regarded similarly to ER-negative BC.

ALDH1A3 promotes invasion and metastasis in triple-negative breast cancer by regulating the plasminogen activation pathway.

Aldehyde dehydrogenase 1A3 (ALDH1A3) is a cancer stem cell marker that promotes metastasis. Triple-negative breast cancer (TNBC) progression has been linked to ALDH1A3-induced gene expression changes. To investigate the mechanism of ALDH1A3-mediated breast cancer metastasis, we assessed the effect of ALDH1A3 on the expression of proteases and the regulators of proteases that degrade the extracellular matrix, a process that is essential for invasion and metastasis. This revealed that ALDH1A3 regulates the plasminogen activation pathway; it increased the levels and activity of tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA). This resulted in a corresponding increase in the activity of serine protease plasmin, the enzymatic product of tPA and uPA. The ALDH1A3 product all-trans-retinoic acid similarly increased tPA and plasmin activity. The increased invasion of TNBC cells by ALDH1A3 was plasminogen-dependent. In patient tumours, ALDH1A3 and tPA are co-expressed and their combined expression correlated with the TNBC subtype, high tumour grade and recurrent metastatic disease. Knockdown of tPA in TNBC cells inhibited plasmin generation and lymph node metastasis. These results identify the ALDH1A3-tPA-plasmin axis as a key contributor to breast cancer progression.

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.

Tumor levels of the mediators of ErbB2-driven anoikis resistance correlate with breast cancer relapse in patients receiving trastuzumab-based therapies.

PURPOSE: Patients with ErbB2/Her2 oncoprotein-positive breast cancers often receive neoadjuvant therapies (NATs) containing the anti-ErbB2 antibody trastuzumab. Tumors that are still present after NATs are resected, and patients continue receiving trastuzumab. These cancers are associated with high relapse risk. Whether relapse will occur cannot be presently reliably predicted. The ability to make such predictions could improve disease management. We found previously that ErbB2 blocks breast tumor cell anoikis, apoptosis induced by cell detachment from the extracellular matrix, by downregulating the pro-apoptotic protein Irf6 and upregulating the anti-apoptotic protein Epidermal Growth Factor Receptor (EGFR) in the cells and, thus, promotes their three-dimensional growth. We now tested whether tumor levels of these proteins before and after NATs correlate with patients' relapse-free survival (RFS) and overall survival (OS). METHODS: We selected archival breast tumor samples collected from 37 women with ErbB2-positive stages II and III breast cancer before and after NATs. We used immunohistochemistry to test whether levels of the indicated proteins in respective tumors correlate with RFS and OS. RESULTS: We observed that the presence of high Irf6 levels in the tumors following NATs correlated with reduced RFS and OS. Perhaps not by coincidence, we noticed that trastuzumab-sensitive ErbB2-positive breast cancer cells selected for the ability to overproduce exogenous Irf6 in culture acquired trastuzumab resistance. Finally, EGFR presence in patients' tumors before or after NATs was associated with decreased RFS and OS. CONCLUSIONS: This study could help identify patients with ErbB2-positive tumors that are at increased risk of disease relapse following NATs.

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.

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.

ErbB2-driven downregulation of the transcription factor Irf6 in breast epithelial cells is required for their 3D growth.

BACKGROUND: The ability of solid tumor cells to resist anoikis, apoptosis triggered by cell detachment from the extracellular matrix (ECM), is thought to be critical for 3D tumor growth. ErbB2/Her2 oncoprotein is often overproduced by breast tumor cells and blocks their anoikis by partially understood mechanisms. In our effort to understand them better, we observed that detachment of nonmalignant human breast epithelial cells from the ECM upregulates the transcription factor Irf6. Irf6 is thought to play an important role in mammary gland homeostasis and causes apoptosis by unknown mechanisms. We noticed that ErbB2, when overproduced by detached breast epithelial cells, downregulates Irf6. METHODS: To test whether ErbB2 downregulates Irf6 in human ErbB2-positive breast cancer cells, we examined the effect of ErbB2 inhibitors, such as the anti-ErbB2 antibody trastuzumab or the ErbB2/epidermal growth factor receptor small-molecule inhibitor lapatinib, on Irf6 in these cells. Moreover, we performed Irf6 IHC analysis of tumor samples derived from the locally advanced ErbB2-positive breast cancers before and after neoadjuvant trastuzumab-based therapies. To examine the role of Irf6 in anoikis of nonmalignant and ErbB2-overproducing breast epithelial cells, we studied anoikis after knocking down Irf6 in the former cells by RNA interference and after overproducing Irf6 in the latter cells. To examine the mechanisms by which cell detachment and ErbB2 control Irf6 expression in breast epithelial cells, we tested the effects of genetic and pharmacological inhibitors of the known ErbB2-dependent signaling pathways on Irf6 in these cells. RESULTS: We observed that trastuzumab and lapatinib upregulate Irf6 in ErbB2-positive human breast tumor cells and that neoadjuvant trastuzumab-based therapies tend to upregulate Irf6 in human breast tumors. We found that detachment-induced Irf6 upregulation in nonmalignant breast epithelial cells requires the presence of the transcription factor ∆Np63α and that Irf6 mediates their anoikis. We showed that ErbB2 blocks Irf6 upregulation in ErbB2-overproducing cells by activating the mitogen-activated protein kinases that inhibit ∆Np63α-dependent signals required for Irf6 upregulation. Finally, we demonstrated that ErbB2-driven Irf6 downregulation in ErbB2-overproducing breast epithelial cells blocks their anoikis and promotes their anchorage-independent growth. CONCLUSIONS: We have demonstrated that ErbB2 blocks anoikis of breast epithelial cells by downregulating Irf6.

Well-differentiated invasive breast cancers with equivocal HER2 immunohistochemistry: what is the yield of routine reflex in-situ hybridization testing?

AIMS: The 2013 American Society of Clinical Oncology and the College of American Pathologists (ASCO/CAP) HER2 guidelines recommend testing all invasive breast cancers for HER2, typically with immunohistochemistry (IHC) followed by in-situ hybridization (ISH) when IHC is equivocal. As well-differentiated breast cancers are rarely HER2-positive, we assessed the value of routine reflex HER2 ISH testing for this subset of breast cancers. METHODS AND RESULTS: We collected HER2 IHC 2+ cases and fluorescence in-situ hybridization (FISH) data from primary breast cancers with well-differentiated tumour types (grade 1 ductal carcinomas, classic lobular carcinomas, tubular, cribriform and pure mucinous carcinomas) at our centre from 2010 to 2015. Haematoxylin and eosin (H&E) and IHC slides were reviewed to confirm tumour type, grade and IHC score based on ASCO/CAP 2013 guidelines and their recent revisions. Of 4633 invasive carcinomas, 1133 had a well-differentiated tumour type; 177 of these were HER2 IHC equivocal, three of which were low-level amplified by FISH (0.3% of all well-differentiated tumours). One amplified case was classic invasive lobular carcinoma and two were invasive ductal carcinomas, grade 1. One amplified case had chromosome 17 monosomy, and one was rescored as HER2 IHC 1+ upon review. 'Basolateral' staining was noted in one amplified case and in 65 of 174 (37.4%) non-amplified cases. This incomplete membranous staining pattern was observed in the majority of invasive ductal carcinomas that were rescored as 1+ according to the revised 2013 guidelines. CONCLUSIONS: The rate of HER2 amplification among well-differentiated breast cancers is very low. Basolateral staining in well-differentiated tumours may be overinterpreted as HER2 IHC 2+, but is rarely associated with HER2 amplification.

Detecting intratumoral heterogeneity in routine breast-HER2 testing: low yield of testing multiple blocks.

BACKGROUND: Intratumoral heterogeneity can lead to uncertainty in breast carcinoma HER2 testing, both with respect to pathology reporting and clinical significance. The standard practice is to perform breast biomarker testing on a single representative section of tumor; however, concern over heterogeneity often leads to testing on additional tissue blocks. Our objective was to assess the diagnostic yield of testing multiple blocks of a single invasive breast carcinoma. METHODS: We performed a retrospective review of 139 consecutive cases (between 2006 and 2012) in which clinical HER2 testing was performed in multiple blocks. Tumor characteristics and HER2 studies (both immunohistochemistry and data from in situ hybridization) were reviewed. Regional differences in morphology and HER2 immunoreactivity were recorded. In situ hybridization was performed in 25 of 139 of the cases; patterns of genetic heterogeneity were reviewed. We audited discordances in HER2 result between blocks. RESULTS: Testing of multiple blocks yielded no additional HER2 information in 134 (96.4%) of 139 cases. Morphologic differences or heterogeneity in HER2 expression was observed in 22 (15.8%) of 139 of cases. Only 5 of these showed differences in HER2 between blocks, of which 4 were associated with equivocal HER2 immunohistochemistry, and 4 were high-grade. CONCLUSIONS: In the vast majority of cases, even those with heterogeneity, testing of a single block is sufficient for an accurate HER2 determination. High-grade tumors with equivocal HER2 status and observable heterogeneity are more likely to yield a different result on testing of additional blocks.

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.

HER2 testing of multifocal invasive breast carcinoma: how many blocks are enough?

OBJECTIVES: To determine the diagnostic yield of testing multiple blocks for HER2 in cases of multifocal breast carcinoma. METHODS: We identified 246 consecutive cases of multifocal invasive breast carcinoma in which HER2 was tested on more than 1 tumor focus. We performed an audit of all cases with respect to tumor size, grade, and histologic type. RESULTS: HER2 status was concordant between multiple foci in 230 (93.5%) of 246 cases, with the largest focus having the most positive HER2 result in 242 (98.4%) of 246 cases. We did not find a single case in which a smaller focus demonstrated a more positive HER2 status unless this focus was either higher grade or different histologically. CONCLUSIONS: Our findings support the evaluation of HER2 on the largest focus, with additional testing on smaller foci having a different histologic type or higher grade.

Clinical and pathologic factors of prognostic significance in penile squamous cell carcinoma in a North American population.

OBJECTIVE: To analyze, in a clinicopathologic correlation study, a small population of primarily white men with invasive squamous cell carcinoma of the penis for potential prognostic predictors. Penile squamous cell carcinoma is an uncommon cancer in North America. It has a wide spectrum of clinical behavior and an understudied pathogenesis. METHODS: The data from 43 patients with invasive squamous cell carcinoma of the penis were studied retrospectively. Extensive chart reviews were conducted, glass slides were reviewed, and tissue microarrays were constructed for analysis of immunohistochemical stains p16(INK4a), p53, and Ki-67. Univariate and multivariate logistic regression analyses were performed to elucidate any clinical or pathologic factors that would predict overall survival. RESULTS: The mean age at diagnosis was 63 years. Most cases (63%) were invasive squamous cell carcinoma, not otherwise specified, and presented as pathologic stage T1 or T2 tumors. Of the 43 patients, 23% died of their disease; 53% of the cases stained for p16(INK4a). Higher pathologic tumor stage and a lack of p16(INK4a) staining were independent predictors of worse overall survival (P = .014) and cancer-specific survival (P = .010). CONCLUSION: Our results have shown that 53% of the invasive penile squamous cell carcinoma cases in this population were associated with human papillomavirus, using p16(INK4a) as a surrogate marker of human papillomavirus infection. These patients had a statistically significant survival advantage, independent of other prognostic factors.

Epidermal growth factor receptor (EGFR) in lung cancer: an overview and update.

Epidermal growth factor receptor is a trans-membrane glycoprotein with an extracellular epidermal growth factor binding domain and an intracellular tyrosine kinase domain that regulates signaling pathways to control cellular proliferation. Epidermal growth factor receptor binding to its ligand results in autophosphorylation by intrinsic tyrosine/kinase activity, triggering several signal transduction cascades. Constitutive or sustained activation of these sequences of downstream targets is thought to yield more aggressive tumor phenotypes. Mutations in epidermal growth factor receptor have been discovered in association with some lung cancers. Lung adenocarcinomas with mutated epidermal growth factor receptor have significant responses to tyrosine kinase inhibitors, although for unselected patients it does not appear to have a survival benefit. However, in a subset of patients (non-smoking Asian women with adenocarcinoma, particularly with a bronchioloalveolar carcinoma), there appears to be a significant survival advantage. Both EGFR mutation and gene amplification status may be important in determining which tumors will respond to tyrosine kinase inhibitors.