Adjuvant enzalutamide for the treatment of early-stage androgen-receptor positive, triple-negative breast cancer: a feasibility study.

PURPOSE: Chemotherapy with or without immunotherapy remains the mainstay of treatment for triple-negative breast cancer (TNBC). A subset of TNBCs express the androgen receptor (AR), representing a potential new therapeutic target. This study assessed the feasibility of adjuvant enzalutamide, an AR antagonist, in early-stage, AR-positive (AR +) TNBC. METHODS: This study was a single-arm, open-label, multicenter trial in which patients with stage I-III, AR ≥ 1% TNBC who had completed standard-of-care therapy were treated with enzalutamide 160 mg/day orally for 1 year. The primary objective of this study was to evaluate the feasibility of 1 year of adjuvant enzalutamide, defined as the treatment discontinuation rate of enzalutamide due to toxicity, withdrawal of consent, or other events related to tolerability. Secondary endpoints included disease-free survival (DFS), overall survival (OS), safety, and genomic features of recurrent tumors. RESULTS: Fifty patients were enrolled in this study. Thirty-five patients completed 1 year of therapy, thereby meeting the prespecified trial endpoint for feasibility. Thirty-two patients elected to continue with an optional second year of treatment. Grade ≥ 3 treatment-related adverse events were uncommon. The 1-year, 2-year, and 3-year DFS were 94%, 92% , and 80%, respectively. Median OS has not been reached. CONCLUSION: This clinical trial demonstrates that adjuvant enzalutamide is a feasible and well-tolerated regimen in patients with an early-stage AR + TNBC. Randomized trials in the metastatic setting may inform patient selection through biomarker development; longer follow-up is needed to determine the effect of anti-androgens on DFS and OS in this patient population.

Digital validation of breast biomarkers (ER, PR, AR, and HER2) in cytology specimens using three different scanners.

Progression in digital pathology has yielded new opportunities for a remote work environment. We evaluated the utility of digital review of breast cancer immunohistochemical prognostic markers (IHC) using whole slide images (WSI) from formalin fixed paraffin embedded (FFPE) cytology cell block specimens (CB) using three different scanners.CB from 20 patients with breast cancer diagnosis and available IHC were included. Glass slides including 20 Hematoxylin and eosin (H&E), 20 Estrogen Receptor (ER), 20 Progesterone Receptor (PR), 16 Androgen Receptor (AR), and 20 Human Epidermal Growth Factor Receptor 2 (HER2) were scanned on 3 different scanners. Four breast pathologists reviewed the WSI and recorded their semi-quantitative scoring for each marker. Kappa concordance was defined as complete agreement between glass/digital pairs. Discordances between microscopic and digital reads were classified as a major when a clinically relevant change was seen. Minor discordances were defined as differences in scoring percentages/staining pattern that would not have resulted in a clinical implication. Scanner precision was tabulated according to the success rate of each scan on all three scanners.In total, we had 228 paired glass/digital IHC reads on all 3 scanners. There was strong concordance kappa ≥0.85 for all pathologists when comparing paired microscopic/digital reads. Strong concordance (kappa ≥0.86) was also seen when comparing reads between scanners.Twenty-three percent of the WSI required rescanning due to barcode detection failures, 14% due to tissue detection failures, and 2% due to focus issues. Scanner 1 had the best average precision of 92%. HER2 IHC had the lowest intra-scanner precision (64%) among all stains.This study is the first to address the utility of WSI in breast cancer IHC in CB and to validate its reporting using 3 different scanners. Digital images are reliable for breast IHC assessment in CB and offer similar reproducibility to microscope reads.

Stromal MED12 exon 2 mutations in complex fibroadenomas of the breast.

AIMS: Here we explore the presence of mediator complex subunit 12 (MED12) exon 2 and telomerase reverse transcriptase (TERT) promoter hotspot mutations in complex fibroadenomas (CFAs) of the breast. METHODS: The stromal components from 18 CFAs were subjected to Sanger sequencing of MED12 exon 2 and the TERT promoter hotspot loci. The epithelial and stromal components of two MED12 mutated CFAs were subjected to laser capture microdissection, and Sanger sequencing of MED12 exon 2, TERT promoter and PIK3CA exons 9 and 20, separately. RESULTS: MED12 exon 2 mutations were identified in the stroma of 17% of CFAs. The analyses of epithelial and stromal components, microdissected separately, revealed that MED12 mutations were restricted to the stroma. No TERT promoter or PIK3CA mutations in exons 9 and 20 were detected in analysed CFAs. CONCLUSIONS: Like conventional fibroadenomas, MED12 exon 2 mutations appear to be restricted to the stromal component of CFAs, supporting the notion that CFAs are stromal neoplasms.

Nodal Recurrence in Patients With Node-Positive Breast Cancer Treated With Sentinel Node Biopsy Alone After Neoadjuvant Chemotherapy-A Rare Event.

IMPORTANCE: Prospective trials have demonstrated sentinel lymph node (SLN) false-negative rates of less than 10% when 3 or more SLNs are retrieved in patients with clinically node-positive breast cancer rendered clinically node-negative with neoadjuvant chemotherapy (NAC). However, rates of nodal recurrence in such patients treated with SLN biopsy (SLNB) alone are unknown because axillary lymph node dissection (ALND) was performed in all patients, limiting adoption of this approach. OBJECTIVE: To evaluate nodal recurrence rates in a consecutive cohort of patients with clinically node-positive (cN1) breast cancer receiving NAC, followed by a negative SLNB using a standardized technique, and no further axillary surgery. DESIGN, SETTING, AND PARTICIPANTS: From November 2013 to February 2019, a cohort of consecutively identified patients with cT1 to cT3 biopsy-proven N1 breast cancer rendered cN0 by NAC underwent SLNB with dual tracer mapping and omission of ALND if 3 or more SLNs were identified and all were pathologically negative. Metastatic nodes were not routinely clipped, and localization of clipped nodes was not performed. The study was performed in a single tertiary cancer center. INTERVENTION: Omission of ALND in patients with cN1 breast cancer after NAC if 3 or more SLNs were pathologically negative. MAIN OUTCOME AND MEASURES: The primary outcome was the rate of nodal recurrence among patients with cN1 breast cancer treated with SLNB alone after NAC. RESULTS: Of 610 patients with cN1 breast cancer treated with NAC, 555 (91%) converted to cN0 and underwent SLNB; 234 (42%) had 3 or more negative SLNs and had SLNB alone. The median (IQR) age of these 234 patients was 49 (40-58) years; median tumor size was 3 cm; 144 (62%) were ERBB2 (formerly HER2)-positive, and 43 (18%) were triple negative. Most (212 [91%]) received doxorubicin-based NAC; 205 (88%) received adjuvant radiotherapy (RT), and 164 (70%) also received nodal RT. At a median follow-up of 40 months, there was 1 axillary nodal recurrence synchronous with local recurrence in a patient who refused RT. Among patients who received RT (n = 205), there were no nodal recurrences. CONCLUSIONS AND RELEVANCE: This cohort study found that in patients with cN1 disease rendered cN0 with NAC, with 3 or more negative SLNs with SLNB alone, nodal recurrence rates were low, without routine nodal clipping. These findings potentially support omitting ALND in such patients.

Is Residual Nodal Disease at Axillary Dissection Associated with Tumor Subtype in Patients with Low Volume Sentinel Node Metastasis After Neoadjuvant Chemotherapy?

BACKGROUND: In patients with a positive sentinel lymph node (SLN) after neoadjuvant chemotherapy (NAC), the likelihood of residual nodal disease at axillary dissection (ALND) is high. Whether non-SLN metastasis frequency varies based on tumor subtype and SLN metastasis size is uncertain. We examined the association between tumor subtype and frequency of non-SLN metastases in patients with SLN micro- vs macrometastases after NAC. METHODS: Patients with invasive breast cancer and a positive SLN biopsy after NAC between July 2008 and July 2019 were identified. Associations between tumor subtype, SLN disease volume, and frequency of non-SLN metastases were examined. RESULTS: Among 273 patients with ≥ 1 positive SLN and a completion ALND, mean age was 51 years, 87% of tumors were ductal, 80% were clinically node-positive at presentation, and 85% were cT2-3. The frequency of non-SLN metastases was non-significantly higher in HR+/HER2- (61%) vs. HER2+ (52%) and triple negative tumors (45%) (p = 0.09). Frequency of SLN micrometastasis was 9% for triple negative tumors compared with 17% for HR+/HER2- and 34% for HER2+ tumors (p = 0.015). Size of SLN metastasis (micro- vs. macrometastases) was not associated with non-SLN metastasis frequency or number within any subtype. CONCLUSIONS: In patients with a positive SLN after NAC, the likelihood of non-SLN metastasis at ALND was high across all tumor subtypes and did not vary significantly for SLN micro- versus macrometastases. ALND is recommended for SLN micro- and macrometastases after NAC, irrespective of tumor subtype.

Leptomeningeal disease in pancreas ductal adenocarcinoma: A manifestation of longevity.

BACKGROUND: /Objectives: Pancreatic adenocarcinoma (PDAC) metastatic to the leptomeninges is a rare and lethal event. Leptomeningeal disease (LMD) research is limited in PDAC, and insights into clinical descriptors, possible disease predictors, and treatment strategies is necessitated. METHODS: Memorial Sloan Kettering databases were queried with Institutional Review Board approval to identify patients with LMD and PDAC treated between January 2000 and June 2020. Medical record review was used to abstract clinical, genomic, pathologic, and radiographic data. Overall survival was calculated from date of PDAC diagnosis to date of death. Previously published literature on LMD from PDAC was reviewed. RESULTS: Four patients with LMD from PDAC were identified, two males and two females. Age at diagnosis ranged from 57 to 68 years. All four patients had predominant lung metastasis and a relatively low burden of intra-abdominal disease. Somatic testing indicated alterations typical of PDAC and no PDAC defining pathogenic germline mutations were identified. An extended clinical course prior to LMD diagnosis was observed in all patients, ranging from 16 to 148 months. Upon diagnosis of LMD, three patients elected for supportive care and one patient received a limited course of craniospinal radiation. The median survival following diagnosis of LMD was 1.6 months (range 0.5-2.8 months). CONCLUSIONS: LMD from PDAC is a rare occurrence that may be more frequent in patients with lung metastasis and/or a more indolent clinical course. Following diagnosis of LMD, prognosis is poor, and survival is short. New treatment strategies for this manifestation of PDAC are needed.

Triple-Positive Breast Carcinoma: Histopathologic Features and Response to Neoadjuvant Chemotherapy.

CONTEXT.­: It is unclear whether HER2+ tumors expressing both estrogen receptor (ER) and progesterone receptor (PR), that is, triple-positive breast carcinomas (TPBCs), show unique morphologic and clinical features and response to neoadjuvant chemotherapy (NAC). OBJECTIVE.­: To study the morphologic and immunohistochemical features of TPBCs from patients who underwent NAC. DESIGN.­: We retrospectively reviewed core biopsy and post-NAC slides of 85 TPBCs. H-scores were calculated for ER and PR. HER2 slides and fluorescence in situ hybridization (FISH) reports were reviewed. Residual cancer burden was calculated for post-NAC specimens. RESULTS.­: Eighty-one of the 85 tumors (95.3%) showed ductal histology, 3 (3.5%) were invasive lobular carcinomas, and 1 (1.2%) showed mixed ductal and lobular features. A subset showed mucinous (n = 7, 8.2%), apocrine (n = 5, 5.9%), and/or micropapillary (n = 4, 4.7%) differentiation. Fifty-four TPBCs (63.5%) showed high ER expression (H-score >200), including 27 (31.8%) with high expression of ER and PR. Fifty-two tumors (61.1%) showed HER2 3+ staining. Mean HER2/CEP17 ratio by FISH was 3.6 (range, 2-12.2) and mean HER2 signals per cell was 8 (range, 3.7-30.4). Pathologic complete response (pCR) rate was 35.3% (30 of 85). HER2 3+ staining was the only significant predictor of pCR on multivariate analysis (odds ratio = 9.215; 95% CI, 2.401-35.371; P < .001). The ER/PR expression did not correlate with response to therapy. CONCLUSIONS.­: TPBCs are heterogeneous with some showing mucinous, lobular, or micropapillary differentiation. The pCR rate of TPBCs is similar to that reported for ER+/PR-/HER2+ tumors. HER2 overexpression by IHC was associated with significantly better response to therapy and may help select patients for treatment in the neoadjuvant setting.

Correction to: Is Low-Volume Disease in the Sentinel Node After Neoadjuvant Chemotherapy an Indication for Axillary Dissection?

The following are corrections to the original article.

Immunohistochemical assessment of HRAS Q61R mutations in breast adenomyoepitheliomas.

AIMS: Breast adenomyoepitheliomas (AMEs) are uncommon tumours. Most oestrogen receptor (ER)-positive AMEs have mutations in phosphoinositide 3-kinase (PI3K) pathway genes, whereas ER-negative AMEs usually harbour concurrent mutations affecting the HRAS Q61 hotspot and PI3K pathway genes. Here, we sought to determine the sensitivity and specificity of RAS Q61R immunohistochemical (IHC) analysis for detection of HRAS Q61R mutations in AMEs. METHODS AND RESULTS: Twenty-six AMEs (14 ER-positive; 12 ER-negative) previously subjected to massively parallel sequencing (n = 21) or Sanger sequencing (n = 5) of the HRAS Q61 hotspot locus were included in this study. All AMEs were subjected to IHC analysis with a monoclonal (SP174) RAS Q61R-specific antibody, in addition to detailed histopathological analysis. Nine ER-negative AMEs harboured HRAS mutations, including Q61R (n = 7) and Q61K (n = 2) mutations. Five of seven (71%) AMEs with HRAS Q61R mutations were immunohistochemically positive, whereas none of the AMEs lacking HRAS Q61R mutations (n = 17) were immunoreactive. RAS Q61R immunoreactivity was restricted to the myoepithelium in 80% (4/5) of cases, whereas one case showed immunoreactivity in both the epithelial component and the myoepithelial component. RAS Q61R immunohistochemically positive AMEs were associated with infiltrative borders (P < 0.001), necrosis (P < 0.01) and mitotic index in the epithelial (P < 0.05) and myoepithelial (P < 0.01) components. RAS Q61R IHC assessment did not reveal Q61K mutations (0/2). CONCLUSIONS: IHC analysis of RAS Q61R shows high specificity (100%) and moderate sensitivity (71%) for detection of HRAS Q61R mutations in breast AMEs, and appears not to detect HRAS Q61K mutations. IHC analysis of RAS Q61R may constitute a useful technique in the diagnostic workup of ER-negative AMEs.

Hormone receptor and HER2 assessment in breast carcinoma metastatic to bone: A comparison between FNA cell blocks and decalcified core needle biopsies.

BACKGROUND: Estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) guide the clinical management of breast cancer metastases. Decalcification of bone core needle biopsies (CNBs) can affect IHC. In the current study, the authors sought to define whether fine-needle aspiration (FNA) would be a better alternative to CNB for reliable IHC. METHODS: Patients with breast cancer metastases to bone that were sampled by both CNB and FNA were selected. ER, PR, and HER2 were performed in FNA cell blocks (FNA-CBs) and concurrent decalcified CNBs. Discrepancies were classified as minor when there was a difference of up to 30% nuclear staining in IHC for ER and PR between paired samples and as major when a clinically relevant change was observed (ie, positive vs negative). Quantitative reverse transcriptase-polymerase chain reaction of ESR1 messenger RNA levels was performed on FNA/CNB pairs with discrepancies for ER IHC. IHC status of the primary breast carcinoma was recorded. RESULTS: Concordance rates for ER, PR, and HER2 were 89%, 67%, and 93%, respectively, between FNA-CB and CNB pairs from 27 patients. Major discrepancies were noted in approximately 11% of FNA/CNB pairs for ER IHC and in 33% of FNA/CNB pairs for PR. ESR1 messenger RNA levels of FNA/CNB matched samples were similar and did not explain the differences in ER IHC expression in the majority of cases. Two of 27 FNA/CNB pairs had different results for HER2 IHC that changed from negative on CNB to equivocal (2+) on FNA-CB. Both cases had prior HER2 amplification by fluorescence in situ hybridization. CONCLUSIONS: FNA-CB and CNB appear to constitute acceptable methods for the assessment of ER, PR, and HER2 for clinical decision making.

Microscopic Extracapsular Extension in Sentinel Lymph Nodes Does Not Mandate Axillary Dissection in Z0011-Eligible Patients.

BACKGROUND: In the ACOSOG (American College of Surgeons Oncology Group) Z0011 trial and the AMAROS (After Mapping of the Axilla: Radiotherapy or Surgery?) trial, matted nodes with gross extracapsular extension (ECE), a risk factor for locoregional recurrence, were an indication for axillary lymph node dissection (ALND), but the effect of microscopic ECE (mECE) in the sentinel lymph nodes (SLNs) on recurrence was not examined. METHODS: Between 2010 and 2017, 811 patients with cT1-2N0 breast cancer and SLN metastasis were prospectively managed according to Z0011 criteria, with ALND for those with more than two positive SLNs or gross ECE. Management of mECE was not specified. In this study, we compare outcomes of patients with one to two positive SLNs with and without mECE, treated with SLN biopsy alone (n = 685). RESULTS: Median patient age was 58 years, and median tumor size was 1.7 cm. mECE was identified in 210 (31%) patients. Patients with mECE were older, had larger tumors, and were more likely to be hormone receptor positive and HER2 negative, have two positive SLNs, and receive nodal radiation. At a median follow-up of 41 months, no isolated axillary failures were observed. There were 11 nodal recurrences; two supraclavicular ± axillary, four synchronous with breast, and five with distant failure. The five-year rate of any nodal recurrence was 1.6% and did not differ by mECE (2.3% vs. 1.3%; p = 0.84). No differences were observed in local (p = 0.08) or distant (p = 0.31) recurrence rates by mECE status. CONCLUSIONS: In Z0011-eligible patients, nodal recurrence rates in patients with mECE are low after treatment with SLN biopsy alone, even in the absence of routine nodal radiation. The presence of mECE should not be considered a routine indication for ALND.

Automatic quantification of HER2 gene amplification in invasive breast cancer from chromogenic in situ hybridization whole slide images.

Human epidermal growth factor receptor 2 (HER2), a transmembrane tyrosine kinase receptor encoded by the ERBB2 gene on chromosome 17q12, is a predictive and prognostic biomarker in invasive breast cancer (BC). Approximately 20% of BC are HER2-positive as a result of ERBB2 gene amplification and overexpression of the HER2 protein. Quantification of HER2 is performed routinely on all invasive BCs, to assist in clinical decision making for prognosis and treatment for HER2-positive BC patients by manually counting gene signals. We propose an automated system to quantify the HER2 gene status from chromogenic in situ hybridization (CISH) whole slide images (WSI) in invasive BC. The proposed method selects untruncated and nonoverlapped singular nuclei from the cancer regions using color unmixing and machine learning techniques. Then, HER2 and chromosome enumeration probe 17 (CEP17) signals are detected based on the RGB intensity and counted per nucleus. Finally, the HER2-to-CEP17 signal ratio is calculated to determine the HER2 amplification status following the ASCO/CAP 2018 guidelines. The proposed method reduced the labor and time for the quantification. In the experiment, the correlation coefficient between the proposed automatic CISH quantification method and pathologist manual enumeration was 0.98. The p -values larger than 0.05 from the one-sided paired t -test ensured that the proposed method yields statistically indifferent results to the reference method. The method was established on WSI scanned by two different scanners. Through the experiments, the capability of the proposed system has been demonstrated.

Extramammary Paget disease shows differential expression of B7 family members B7-H3, B7-H4, PD-L1, PD-L2 and cancer/testis antigens NY-ESO-1 and MAGE-A.

Extramammary Paget disease (EMPD) is a rare cutaneous adenocarcinoma of the anogenital region most commonly treated with surgical excision. Surgical margin clearance is often problematic and recurrence rates remain high indicating the need for additional therapeutic options. Topical immunomodulators have been used with reported success suggesting EMPD may respond to other immunotherapies. This study investigates EMPD protein expression of targetable B7 family members and cancer/testis antigens (CTAs) B7-H3, B7-H4, PD-L1, PD-L2, MAGE-A, and NY-ESO-1 and components of antigen presenting machinery B2M and MHC-I. Fifty-seven specimens from 48 patients (31 female and 17 male), representing in situ, invasive, and metastatic disease of primary and secondary origin were stained and scored (627 total slides). The percentage of cases expressing each immune regulatory molecule in the in situ followed by invasive tumor components was: B7-H3 (94, 90), B7-H4 (82, 78), PD-L1 (6, 10), MAGE-A (39, 50), NY-ESO-1 (16, 20), B2M (100, 89), and MHC-I (78, 79). PD-L2 was negative in all cases. There was high correlation between marker expression within the in situ and invasive tumor components of the same case. B7-H4 was preferentially expressed in primary cutaneous EMPD. Co-expression of B7 family members B7-H3 and B7-H4 was found within the in situ and invasive tumor components of 74% and 48% of cases, respectively. These findings provide an initial characterization of EMPD tumor cell expression of B7-H3, B7-H4, PD-L1, PD-L2, MAGE-A, and NY-ESO-1 and indicate the potential for new immunotherapeutic options for patients with EMPD.

HER2 assessment by bright-field dual in situ hybridization in cell blocks of recurrent and metastatic breast carcinoma.

BACKGROUND: Breast cancer recurrences or metastases often are diagnosed using cytology material. Cell blocks (CBs) with adequate cellularity are crucial for the determination of accurate hormonal and human epidermal growth factor receptor 2 (HER2) status and to guide treatment. In the current study, the authors evaluated the concordance of HER2 status between bright-field dual in situ hybridization (DISH), fluorescence in situ hybridization (FISH), and HER2 immunohistochemistry (IHC) performed on formalin-fixed CBs of recurrent and metastatic breast cancers. METHODS: The authors searched for patients who had breast carcinoma recurrences or metastases diagnosed between 2010 and 2018 by fine-needle aspiration or by the drainage of body cavity fluids with HER2 IHC and/or FISH performed on formalin-fixed CBs. Cases with adequate tumor cellularity (>50 cells) were selected. HER2 DISH was performed on all CBs. HER2 status of the primary breast carcinoma was recorded. RESULTS: Formalin-fixed CBs were identified from 30 patients with breast cancer recurrences and metastases in axillary lymph nodes (LNs) (5 patients), mediastinal LNs (8 patients), internal mammary LNs (1 patient), supraclavicular LNs (2 patients), portocaval LNs (1 patient), chest wall (3 patients), pleural fluid (3 patients), bone (4 patients), liver (2 patients), and lung (1 patient). All cases had HER2 IHC performed at the study institution and were scored by breast pathologists according to the American Society of Clinical Oncology/College of American Pathologists guidelines. The HER2 DISH results demonstrated 100% concordance (30 of 30 cases) with the concurrent IHC and/or FISH. CONCLUSIONS: All methods of HER2 evaluation were found to accurately identify the amplification status. DISH can be used in tandem with IHC as a reflex assay instead of FISH and is an efficient and reliable method with which to determine HER2 amplification in formalin-fixed CBs.

Pathology Services in Nigeria: Cross-Sectional Survey Results From Three Cancer Consortia.

PURPOSE: Cancer incidence is increasing in sub-Saharan Africa, yet there is little information on the capacity of pathology laboratories in this region. We aimed to assess the current state of pathology services in Nigeria to guide strategies to ensure best practices and improve the quality of surgical specimen handling. METHODS: We developed structured pathology survey to assess tissue handling, sample processing, and immunohistochemistry (IHC) capabilities. The survey was distributed electronically to 22 medical centers in Nigeria that are part of established cancer consortia. Data were collected between September and October 2017. RESULTS: Sixteen of 22 centers completed the survey in full. All 16 institutions had at least one board-certified pathologist and at least one full-time laboratory scientist/technologist. The majority of responding institutions (75%) reported processing fewer than 3,000 samples per year. For sample processing, 38% of institutions reported manual tissue processing and 75% processed biopsies and surgical specimens together. The average tissue fixation time ranged from 5 to more than 72 hours before processing and paraffin embedding. Half of the institutions reported having no quality assurance processes to evaluate hematoxylin and eosin-stained slides, and 25% reported having no written operating procedures. Half of the participating institutions have a facility for routine IHC staining, and among these there was considerable variability in processes and validation procedures. External proficiency testing was not common among surveyed sites (38%). CONCLUSION: Data from 16 Nigerian medical institutions indicate deficiencies in standardization, quality control, and IHC validation that could affect the reliability of pathology results. These findings highlight addressable gaps in pathology services that can ensure accurate diagnosis and follow-up for the growing number of patients with cancer in this region.

CytoLyt fixation significantly inhibits MIB1 immunoreactivity whereas alternative Ki-67 clone 30-9 is not susceptible to the inhibition: Critical diagnostic implications.

BACKGROUND: The Ki-67 proliferation marker has multiple diagnostic and prognostic applications. Although several clones to the Ki-67 antigen are commercially available, the MIB1 clone is widely recommended in the surgical pathology literature for neuroendocrine tumors. In our cytopathology practice, we have encountered unexpectedly low MIB1 immunoreactivity in CytoLyt-fixed cell blocks (CBs). The current study evaluated the impact of fixatives, CB processing, and immunocytochemical (ICC) procedures on Ki-67 immunoreactivity. METHODS: Test CBs were prepared from freshly resected tumors, and multiple variables in the MIB1 ICC procedure were tested, including CytoLyt versus formalin collection media, MIB1 versus other Ki-67 clones including 30-9, and other variables. MIB1 versus Ki-67 30-9 clones were tested in parallel on CytoLyt-fixed CBs from clinical samples of small cell lung carcinoma (SCLC). RESULTS: In the test CBs (n = 10), the mean MIB1 labeling index was 10% in CytoLyt versus 47% in formalin (P = .0116), with a mean loss of reactivity in matched CBs of 37% (up to 70%). None of the procedure modifications tested in 223 individual ICC reactions recovered MIB1 reactivity in CytoLyt except for switching to the Ki-67 30-9 antibody. In CytoLyt-fixed SCLC samples (n = 14), the Ki-67 30-9 antibody demonstrated expected ranges of reactivity (mean, 83%; range, 60%-100%), whereas MIB1 demonstrated markedly inhibited labeling (mean, 60%; range, 10%-95%) (P = .0058). CONCLUSIONS: CytoLyt fixation substantially inhibits MIB1 immunoreactivity, whereas the Ki-67 30-9 clone is not susceptible to inhibition. Markedly discrepant MIB1 reactivity may present a pitfall in the diagnosis of SCLC and may lead to the incorrect prognostic stratification of other tumor types. For laboratories using CytoLyt, we recommend using the Ki-67 30-9 antibody rather than the MIB1 antibody.

Accuracy of Intraoperative Frozen Section of Sentinel Lymph Nodes After Neoadjuvant Chemotherapy for Breast Carcinoma.

False-negative (FN) intraoperative frozen section (FS) results of sentinel lymph nodes (SLN) have been reported to be more common after neoadjuvant chemotherapy (NAC) in the primary surgical setting. We evaluated SLN FS assessment in breast cancer patients treated with NAC to determine the FN rate and the histomorphologic factors associated with FN results. Patients who had FS SLN assessment following NAC from July 2008 to July 2017 were identified. Of the 711 SLN FS cases, 522 were negative, 181 positive, and 8 deferred. The FN rate was 5.4% (28/522). There were no false-positive results. Of the 8 deferred cases, 5 were positive on permanent section and 3 were negative. There was a higher frequency of micrometastasis and isolated tumor cells in FN cases (P<0.001). There was a significant increase in tissue surface area present on permanent section slides compared with FS slides (P<0.001), highlighting the inherent technical limitations of FS and histologic under-sampling of tissue which leads to most FN results. The majority (25/28, 89%) of FN cases had metastatic foci identified exclusively on permanent sections and were not due to a true diagnostic interpretation error. FN cases were more frequently estrogen receptor positive (P<0.001), progesterone receptor positive (P=0.001), human epidermal growth factor receptor-2 negative (P=0.009) and histologic grade 1 (P=0.015), which most likely reflects the lower rates of pathologic complete response in these tumors. Despite its limitations, FS is a reliable modality to assess the presence of SLN metastases in NAC treated patients.

The repertoire of genetic alterations in salivary duct carcinoma including a novel HNRNPH3-ALK rearrangement.

Salivary duct carcinoma (SDC) is a rare, aggressive malignancy with limited treatment options and poor outcome. Twenty-nine primary resected SDC, including 15 SDC de novo (SDCDN), and 14 SDC ex pleomorphic adenoma (SDCXPA) were subjected to the massive parallel sequencing assay (MSK-IMPACT) targeting 287 to 468 cancer-related genes. TP53 was the most frequently altered gene (69%). TP53 mutations and ERBB2 amplification were more frequent in SDCXPA than in SDCDN (P = .0007 and P = .01, respectively). Potentially targetable mutations were detected in 79% (23/29) of SDC involving ERBB2 (31%), PIK3CA (28%), HRAS (21%), ALK (7%) and BRAF (3%), and 22% (5/23) of those cases harbored possible primary resistance mutations involving CCNE1, NF1 and PTEN. A novel HNRNPH3-ALK rearrangement was found in one SDCDN. In another case, EML4-ALK fusion detected in the primary tumor was associated with ALK G1202R secondary resistance mutation in the post-treatment metastasis. A germline analysis of the DNA repair genes revealed a case with a pathogenic BRCA1 E23fs germline variant. SDCDN and SDCXPA are genetically distinct. Although the majority of SDC may be amenable to molecular targeted therapy, concurrent possible resistance mutations may be found in a significant minority of cases. A broad genomic profiling is necessary to ensure detection of rare but clinically actionable somatic alterations in SDC.

Assessment of HMGA2 and PLAG1 rearrangements in breast adenomyoepitheliomas.

Breast adenomyoepitheliomas (AMEs) are rare epithelial-myoepithelial neoplasms that may occasionally produce myxochondroid matrix, akin to pleomorphic adenomas (PAs). Regardless of their anatomic location, PAs often harbor rearrangements involving HMGA2 or PLAG1. We have recently shown that the repertoire of somatic genetic alterations of AMEs varies according to their estrogen receptor (ER) status; whilst the majority of ER-positive AMEs display mutually exclusive PIK3CA or AKT1 hotspot mutations, up to 60% of ER-negative AMEs harbor concurrent HRAS Q61 hotspot mutations and mutations affecting either PIK3CA or PIK3R1. Here, we hypothesized that a subset of AMEs lacking these somatic genetic alterations could be underpinned by oncogenic fusion genes, in particular those involving HMGA2 or PLAG1. Therefore, we subjected 13 AMEs to RNA-sequencing for fusion discovery (n = 5) and/or fluorescence in situ hybridization (FISH) analysis for HMGA2 and PLAG1 rearrangements (n = 13). RNA-sequencing revealed an HMGA2-WIF1 fusion gene in an ER-positive AME lacking HRAS, PIK3CA and AKT1 somatic mutations. This fusion gene, which has been previously described in salivary gland PAs, results in a chimeric transcript composed of exons 1-5 of HMGA2 and exons 3-10 of WIF1. No additional in-frame fusion genes or HMGA2 or PLAG1 rearrangements were identified in the remaining AMEs analyzed. Our results demonstrate that a subset of AMEs lacking mutations affecting HRAS and PI3K pathway-related genes may harbor HMGA2-WIF1 fusion genes, suggesting that a subset of breast AMEs may be genetically related to PAs or that a subset of AMEs may originate in the context of a PA.

Immunohistochemical analysis of estrogen receptor in breast cancer with ESR1 mutations detected by hybrid capture-based next-generation sequencing.

Estrogen receptor-α (ER-α), encoded by ESR1, is detected by immunohistochemistry in approximately 70% of invasive breast cancers and serves as a strong predictive biomarker. ESR1-activating mutations in the ligand-binding domain have been reported in up to 35-40% of ER-positive metastatic breast cancers and are associated with endocrine therapy resistance and disease progression. At present, it is unclear whether ESR1 mutations alter the immunohistochemical detection of ER performed in routine clinical practice. In this study, ESR1 mutations in breast cancer were identified utilizing Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT), a Food and Drug Administration-approved hybridization capture-based next-generation sequencing assay. Five hundred and eighty-six breast cancers from patients with locally advanced or metastatic disease were analyzed using MSK-IMPACT in the study period. ESR1 somatic alterations were identified in 67 breast cancer samples from 66 patients. Immunohistochemical analysis of ER, progesterone receptor, and human epidermal growth factor receptor 2 was performed on the primary and treated breast cancers from these patients at the time of diagnosis. Twenty unique ESR1 mutations were identified involving the ligand-binding domain, all in breast cancer samples from patients previously treated with endocrine therapy. The most frequent mutations were D538G (n = 22), Y537S (n = 7), and E380Q (n = 7). All breast cancer samples with an ESR1 mutation were ER-positive by immunohistochemistry. Review of the ER immunohistochemistry in the paired untreated primary tumor and treated tumor from 34 patients showed no detectable change in the ER-positive immunohistochemical status (median percentage of invasive tumor cells with nuclear staining: untreated primary tumor 90%, treated tumor 95%). We conclude that ESR1 mutations do not appreciably diminish ER-positive staining by immunohistochemistry. In addition to standard biomarker testing by immunohistochemistry, the assessment of ESR1 mutations by molecular testing can help guide the clinical management of patients with ER-positive breast cancer in the setting of endocrine resistance and progression of disease.