Receptor Immunohistochemistry Research Articles

Contrast-enhanced cone beam breast CT features of breast cancers: correlation with immunohistochemical receptors and molecular subtypes.

To investigate the association of contrast-enhanced cone beam breast CT (CE-CBBCT) features, immunohistochemical (IHC) receptors, and molecular subtypes in breast cancer. In this retrospective study, patients who underwent preoperative CE-CBBCT and received complete IHC results were analyzed. CE-CBBCT features were evaluated by two radiologists. Observer reproducibility and feature reliability were assessed. The association between CE-CBBCT features, IHC receptors, and molecular subtypes was analyzed using the chi-square, Mann-Whitney, and Kruskal-Wallis tests. Multivariate logistic regression was performed to assess the ability of combined imaging features to discriminate molecular subtypes. ROC curve was used to evaluate prediction performance. A total of 240 invasive cancers identified in 211 women were enrolled. Molecular subtypes of breast cancer were significantly associated with focality number of lesions, lesion type, tumor size, lesion density, internal enhancement pattern, degree of lesion enhancement (ΔHU), mass shape, spiculation, calcifications, calcification distribution, and increased peripheral vascularity of lesion (all p < 0.005), some of which also helped to differentiate IHC receptor status. A multivariate logistic regression model showed that tumor size (odds ratio, OR = 1.244), mass shape (OR = 0.311), spiculation (OR = 0.159), and internal enhancement pattern (OR = 0.227) were associated with differentiation between luminal and non-luminal subtypes (AUC = 0.809). Combined CE-CBBCT features, including lesion type (OR = 0.118), calcifications (OR = 0.181), and ΔHU (OR = 0.962), could be significant indicators of triple-negative versus HER-2-enriched subtypes (AUC = 0.913). CE-CBBCT features have the potential to help predict IHC receptor status and distinguish molecular subtypes of breast cancer, which could in turn help to develop individual treatment decisions and prognosis predictions. • A total of 11 CE-CBBCT features were associated with molecular subtypes, some of which also helped to differentiate IHC receptor status. • Tumor size, irregular mass shape, spiculation, and internal enhancement pattern could help identify luminal subtype. • Lesion type, calcification, and ΔHU could be significant indicators of HER-2- enriched versus triple-negative breast cancers.

Congenital mesoblastic nephroma is characterised by kinase mutations including EGFR internal tandem duplications, the ETV6-NTRK3 fusion, and the rare KLHL7-BRAF fusion.

Congenital mesoblastic nephroma (CMN) is histologically classified into classic, cellular and mixed subtypes. The aims of this study were to characterise the clinical, pathological and molecular features of a series of CMNs, and to determine the utility of pan-Trk and epidermal growth factor receptor (EGFR) immunohistochemistry as surrogate markers for NTRK gene fusions and EGFR internal tandem duplications (ITDs). Twenty-two archival CMN cases (12 classic, five cellular, and five mixed) were tested for the ETV6-NTRK3 fusion and EGFR ITD transcripts by the use of reverse transcriptase polymerase chain reaction (PCR), and next-generation sequencing-based anchored multiplex PCR. All 12 classic CMNs had EGFR ITD. Of the five cellular CMNs, four had the ETV6-NTRK3 fusion and one had the KLHL7-BRAF fusion. Of the five mixed CMNs, four had EGFR ITD, and one had the ETV6-NTRK3 fusion. Pan-Trk immunoreactivity was 100% sensitive and 94.1% specific for the presence of NTRK rearrangement. However, EGFR staining was only 62.5% sensitive and 33.3% specific for EGFR ITD. EGFR ITD is a consistent genetic event in classic CMN. A majority of cellular CMNs have the ETV6-NTRK3 fusion. Rare cellular CMNs may harbour non-canonical mutations such as the KLHL7-BRAF fusion, which was found in one case. Mixed CMNs may have either EGFR ITD or the ETV6-NTRK3 fusion. Pan-Trk immunohistochemistry is a sensitive, albeit not perfectly specific, marker for NTRK rearrangement. EGFR immunohistochemistry is not helpful as a marker of EGFR ITD.

Wnt/β-Catenin and Hippo Pathway Deregulation in Mammary Tumors of Humans, Dogs, and Cats.

Mammary cancer is a common neoplasm in women, dogs, and cats that still represents a therapeutic challenge. Wnt/β-catenin and Hippo pathways are involved in tumor progression, cell differentiation, and metastasis. The aim of this study was to evaluate mRNA and protein expression of molecules involved in these pathways in human (HBC), canine (CMT), and feline mammary tumors (FMT). Real-time quantitative polymerase chain reaction (qPCR) for β-catenin, CCND1, YAP, TAZ, CTGF, and ANKRD1, western blotting for YAP, TAZ, and β-catenin, and immunohistochemistry for estrogen receptor (ER), progesterone receptor (PR), ERBB2, β-catenin, and YAP/TAZ were performed on mammary tumor tissues. The protein expression of active β-catenin was higher in tumors than in healthy tissues in all 3 species. The mRNA expression of the downstream gene CCND1 was increased in HBC ER+ and CMTs compared to healthy tissues. Membranous and cytoplasmic protein expression of β-catenin were strongly negatively correlated in all 3 species. Tumors showed an increased protein expression of YAP/TAZ when compared to healthy tissues. Notably, YAP/TAZ expression was higher in triple negative breast cancers when compared to HBC ER+ and in FMTs when compared to CMTs. The mRNA expression of β-catenin, YAP, TAZ, CTGF, and ANKRD1 was not different between tumors and healthy mammary gland in the 3 species. This study demonstrates deregulation of Wnt/β-catenin and Hippo pathways in mammary tumors, which was more evident at the protein rather than the mRNA level. Wnt/β-catenin and Hippo pathways seem to be involved in mammary carcinogenesis and therefore represent interesting therapeutic targets that should be further investigated.

Molecular and pathological basis of HPV-negative cervical adenocarcinoma seen in a global study.

International surveys find HPV-negativity in up to 30% of cervical adenocarcinomas. We investigated the pathological diagnosis by expert consensus with immunohistochemistry and the presence of somatic mutations in recognised tumour genes in HPV-positive and negative cervical adenocarcinomas (CADC). A sample was selected of 45 paraffin-embedded cervical blocks diagnosed locally as usual cervical adenocarcinoma from a global study. These represented different diagnoses made at previous diagnostic review and HPV status. All were suitable for analysis for somatic tumour associated gene mutations. Three pathologists examined H/E slides and immunohistochemistry for p16, progesterone receptor and p53 and classified the cases. L1 genes from high-risk HPVs and low-risk HPVs were analysed by SPF10 PCR-DEIA-LiPA25 version 1 in whole tissue sections and microdissected tumour and retested by PCR for E6/E7 genes of hrHPVs if negative. Cases were analysed for microsatellite instability and next-generation sequencing mutation analysis. From the 45 cases, 20 cases of usual CADC were confirmed of which 17 (85%) were HPV-positive in tumour cells. The other 25 cases were reclassified as endometrial, serous, clear-cell and gastric-type adenocarcinomas and all were HPV-negative in tumour cells. Careful retesting for HPV DNA and IHC leads to more accurate identification of HPV-positive usual cervical adenocarcinomas. Endometrioid endometrial adenocarcinomas, other uterine adenocarcinoma with multiple somatic mutations were important in misclassification of HPV-negative cases locally managed as cervical adenocarcinoma, as was gastric-type adenocarcinoma with germline STK11 mutation in East Asia. Few consensuses confirmed HPV-negative usual cervical adenocarcinomas showed somatic tumorigenic mutations also seen in some HPV-positive usual CADC.

Molecular approaches underlying the oogenic cycle of the scleractinian coral, Acropora tenuis

This study aimed to elucidate the physiological processes of oogenesis in Acropora tenuis. Genes/proteins related to oogenesis were investigated: Vasa, a germ cell marker, vitellogenin (VG), a major yolk protein precursor, and its receptor (LDLR). Coral branches were collected monthly from coral reefs around Sesoko Island (Okinawa, Japan) for histological observation by in situ hybridisation (ISH) of the Vasa (AtVasa) and Low Density Lipoprotein Receptor (AtLDLR) genes and immunohistochemistry (IHC) of AtVasa and AtVG. AtVasa immunoreactivity was detected in germline cells and ooplasm, whereas AtVG immunoreactivity was detected in ooplasm and putative ovarian tissues. AtVasa was localised in germline cells located in the retractor muscles of the mesentery, whereas AtLDLR was localised in the putative ovarian and mesentery tissues. AtLDLR was detected in coral tissues during the vitellogenic phase, whereas AtVG immunoreactivity was found in primary oocytes. Germline cells expressing AtVasa are present throughout the year. In conclusion, Vasa has physiological and molecular roles throughout the oogenic cycle, as it determines gonadal germline cells and ensures normal oocyte development, whereas the roles of VG and LDLR are limited to the vitellogenic stages because they act in coordination with lipoprotein transport, vitellogenin synthesis, and yolk incorporation into oocytes.

Developing a breast cancer tissue biobank in low and middle income countries (LMICs): A research program-based approach for improving molecular profiling.

e12585 Background: Breast cancer incidence and mortality is increasing in LMICs especially in sub-Saharan Africa, attributed to changes in lifestyle and possible biological differences. To study putative relationships among lifestyle factors, tumor biology, and the microenvironment, we aimed to develop a breast cancer tissue biobank linked with clinicopathologic and patient reported data in this resource constrained setting. Methods: We prospectively enrolled patients undergoing mastectomy at Obafemi Awolowo University Teaching hospital in Ile-Ife, Nigeria. Pre-operatively, a locally validated survey tool assessed lifestyle factors (diet and exercise); BMI and body composition (by bioimpedence) were measured. Fresh breast tissue was frozen or preserved in 10% neutral buffered formalin, or RNA later for future metabolomic work. Immunohistochemistry (IHC) for ER/PR and HER2 status was performed and read in Nigeria by a locally-trained pathologist, and Memorial Sloan Kettering. The primary outcome was feasibility assessed by successful collection of tissue, clinicopathologic data, and survey completion. Secondary outcomes were completion rate of tumor receptor IHC compared to historic rates and concordance of local vs MSK IHC. Results: From April 2018 to June 2019, 50 patients were enrolled (49 treatment and 1 risk reduction mastectomy). Clinicopathologic, body composition, and survey data, along with breast tissue were collected from all 50 patients. Patient characteristics in table below. Receptor IHC was complete in all patients (100%) compared to a historic rate of 29% (83/286) between 2010-17. Concordance of IHC between sites was 86%, 81% and 84% for subtypes (ER/PR/HER2). Conclusions: Establishing a tissue biobank with clinicopathologic and lifestyle data using locally-adapted protocols is feasible in Nigeria. Patient enrolment is ongoing and updated data will be presented. This initiative also increased local capacity for breast tumor phenotyping, which has critical implications for improving patient care. [Table: see text]

Recurrent or metastatic salivary gland tumor (MSGT) patients treated with selinexor, a first in class selective exportin-1 (XPO1) inhibitor.

6586 Background: MSGT are rare with limited systemic treatments. This single institution, prospective study in recurrent or metastatic (RM) MSGT involved 2 phases: genomic profiling followed by treatment with either genomically-matched or unmatched therapy. Here we present the results of the unmatched arm for patients (pts) treated with S an oral selective inhibitor of XPO1 that leads to activation of tumor suppressor proteins and retention of oncoprotein mRNAs in the cell nucleus, inducing cancer cell apoptosis. Methods: Patients (pts) with RM-MSGT had archived paraffin embedded tumor samples profiled with targeted next generation sequencing, immunohistochemistry for androgen receptor (AR) and fluorescent in-situ hybridization for HER-2 and ALK. If no actionable mutations were identified or if no matched agents were available, pts with progressive disease could receive S (60mg given twice weekly Q28 days). The study had a simon-2 stage design; 1 partial response in the first 18 pts treated with S, would trigger an additional 7pts to receive S in stage 2. Results: Between July 2014 and April 2019 85 pts were enrolled on study: 73 had sequencing which identified 41 with no actionable mutations and 32 with actionable mutations. 18 pts (10F/8M, median age 61 years [40-79]) were treated with S and included adenoid cystic (n = 8), salivary duct (n = 4), acinic cell (n = 2) and other subtypes (n = 4). Of these 18, 4 pts had actionable aberrations: AR amplification (n = 2), mutations in SMARCB1 (n = 1) and CDKN2A (n = 1). 13pts were treatment naïve, 3pts and 2pts received 1 and 2 or more lines of treatment respectively prior to enrollment: androgen deprivation therapy (n = 2), chemotherapy (n = 3), early phase clinical trials (n = 3). The median number of cycles of S received were 3 (range: 1-19). The best response by RECIST was SD in 13pts (72%) (SD &gt; 6 months (range: 6-18 months) in 5pts (28%); tumor reduction measured in 7pts (39%)), no PRs, PD in 3pts (17%), and 2pts (11%) were not evaluable for response due to insufficient duration of treatment coming off early due to toxicity. The median PFS (95% CI) was 7.6 (3.5-NA) months and the median OS (95% CI) was 15.4 (7.3-NA) months. The most common drug-related toxicities were grade 1-2 fatigue 14pts (78%), nausea 13pts (72%) and dysguesia 10pts (56%). 5 (28%) pts had a dose reduction and 6 (33%) in total had a dose interruption due to toxicity. Conclusions: Single agent antitumor activity was limited and the side effect profile was tolerable. No specific genomic aberration was associated with response to S. Clinical trial information: NCT02069730 .

Translational relevance of androgen receptor immunohistochemistry scoring systems for data harmonization in triple negative breast cancer (TNBC).

1078 Background: Androgen receptor (AR) expressing triple negative breast cancer (TNBC) is a sub-set of TNBC with an evolving prognostic and predictive behaviour. AR immunohistochemical threshold for positivity has not been standardized and a wide range of cut-offs have been used across studies ( &gt; 0% to 75%). In this study we explored AR immunohistochemistry thresholds in relation to disease free survival (DFS) and clinical outcomes in non-metastatic TNBC using the Allred and H-Score systems. Increasing interest in AR as a therapeutic target for TNBC and the use of digital tissue image analysis makes it important to standardize AR immunohistochemistry reporting. Methods: 100 FFPE (formalin-fixed paraffin-embedded) tumour blocks were retrieved for non-metastatic TNBCs diagnosed between January 2015 and May 2017 and immunostained using AR441 (IgG1) mouse monoclonal antibody. Clinical follow-up ranged from 59 to 31 months and DFS was calculated. Cut-off scores were explored using Evaluate Cutpoints ( R maxstat package) and X-tile software. The score with maximum split in DFS (based on log-rank statistics and lowest p-value) was chosen as the cut-off. Descriptive and survival statistics was performed. Results: The median age was 51 (SD 11.262; range 28 to 82) years. Using Evaluate Cutpoints ≥3 was found as the threshold for AR by Allred Score. 36% cases were AR positive using Allred score (HR 0.508; CI 0.234 - 1.11; p-value 0.08). Using Evaluate Cutpoints ≥30 was found as the threshold for AR by H-Score (HR 0.624, CI 0.306 - 1.27; p-value 0.19). 35% cases were AR positive using H-Score. X-tile analysis also found the cut-offs as ≥3 and ≥30 for Allred and H-Score respectively (p &lt; 0.05). A significant correlation was seen between the two scoring systems (Pearson Correlation 0.935; p &lt; 0.01). A significantly higher number of grade III TNBCs were AR negative (n = 55/76) compared to grade II (n = 9/24) (p = 0.002). Cut-off for Ki67 was 75 (HR 1.61, CI 0.85-3.04, p-value 0.141) with a significantly higher number of AR negatives in the Ki67≥75 group (21/26; p &lt; 0.05). The overall median DFS was 51.9 months. There was no significant difference in DFS for the AR negative (median: 47.4 months; mean: 39.39 months) and AR positive (Median survival not reached; mean: 41.3 months) groups(p = 0.23). Conclusions: AR immunohistochemistry cut-offs using the Allred (≥3) and H-Score (≥30) are close to the ones used for ER/PR immunohistochemistry as per ASCO/CAP guidelines, making a strong case for universal application of these systems for harmonization of AR data.

Can pathology diagnostic services for cancer be stratified and serve global health?

Before initiating cancer therapy, a diagnostic tumor tissue sample evaluated within a pathology laboratory by a pathologist is essential to confirm the malignancy type and provide key prognostic factors that direct the treatment offered. Pathology evaluation includes multiple expensive reagents, complex equipment, and both laboratory and pathologist technical skills. By using breast cancer as an example, at a minimum, key tumor prognostic information required before the initiation of treatment includes subtype, tumor grade, tumor size, lymph node status when possible, and biomarker expression determined by immunohistochemistry for estrogen receptor. The additional determination of biomarker expression of progesterone receptor and human epidermal growth factor receptor (HER2) is the standard of care in high-resource settings, but assays may not be affordable in low-income and middle-income countries. With positive tests, patients are eligible for either tamoxifen (for estrogen receptor-positive/progesterone receptor-positive cancers) or monoclonal antibody therapy (for HER2-positive cancers). For settings in which endocrine therapy and/or HER2-targeted therapy is unavailable, biomarker studies have no utility, and high-resource setting standards for pathology evaluation and reporting are unachievable. Resource-stratified pathology evaluation guidelines in cancer diagnosis have not been developed, in contrast to excellent comprehensive, resource-stratified clinical guidelines for use in low-income and middle-income countries, and these are long overdue. The challenges of pathology evaluation in the context of global health are being met by innovative solutions, which may change the face of pathology practice.

Integrated immunohistochemical and molecular analysis improves diagnosis of high-grade carcinoma in the urinary bladder of patients with prior radiation therapy for prostate cancer.

Prostatic adenocarcinoma and urothelial carcinoma typically demonstrate distinct morphologic and immunohistochemical features. However, high-grade prostate and urothelial carcinomas sometimes show significant morphologic and immunohistochemical overlap, which can result in misdiagnosis and mistreatment. This diagnostic dilemma is particularly acute in patients previously treated with radiation and/or hormone therapy for prostate cancer, who later present with high-grade carcinoma in the urinary bladder. To address the diagnostic utility of integrated immunohistochemical and molecular analysis in this setting, we evaluated 25 high-grade carcinomas of the bladder for which morphologic features were deemed indeterminate. Our analysis included immunohistochemistry for urothelial markers (GATA3, p63, uroplakin II), prostate markers (NKX3.1, prostate specific antigen, P501S), androgen receptor (AR) and ERG, along with molecular characterization using capture-based next generation DNA sequencing. Immunohistochemical findings were concordant with the final integrated diagnosis in 21 (84%) cases. However, in three (12%) cases, immunohistochemistry supported a diagnosis of urothelial carcinoma, but molecular analysis identified the correct diagnosis of prostate cancer based on the presence of a TMPRSS2-ERG fusion. One case remained unclassifiable even after this integrated analysis. Notably, in 1 of 21 cases, the presence of a TERT promoter mutation and the absence of a TMPRSS2-ERG fusion would typically favor a diagnosis of urothelial carcinoma, but the aggregate immunohistochemical and molecular findings instead supported a diagnosis of microsatellite unstable prostatic adenocarcinoma with deep deletion of MSH2 and MSH6. Our findings highlight the importance of considering prostatic origin in high-grade carcinoma of the urinary bladder of patients with a history of treated prostate cancer, even when the immunohistochemical findings favor urothelial carcinoma. In a subset of cases, an approach that integrates immunophenotypic and molecular data may help correctly assign site of origin and prevent misdiagnosis that can result from overreliance on any individual immunohistochemical or molecular result.

Luminal A versus luminal B breast cancer: MammaTyper mRNA versus immunohistochemical subtyping with an emphasis on standardised Ki67 labelling-based or mitotic activity index-based proliferation assessment.

Proliferation assessment by the use of Ki67 is a crucial component in intrinsic subtyping of luminal breast cancers (BCs), but suffers from variability between laboratories, observers, and methods. MammaTyper is a quantitative molecular tool that measures mRNA levels of ERBB2, ESR1, PGR and MKI67 in BC, and interprets the results according to the St Gallen 2013 consensus recommendations. We compared MammaTyper with immunohistochemistry (IHC)-based subtypes, with a focus on standardised proliferation assessment. We analysed the agreement in assigning subtypes between MammaTyper and receptor IHC in 101 unifocal luminal HER2-negative early BCs of no special type. Two Ki67 counting protocols, Ki67-Global (Ki67-G) and Ki67-HotSpot (Ki67-H), recommended by the International Ki67 in BC Working Group, and the mitotic activity index (MAI) were used for proliferation assessment. The proportions of BCs identified as luminal A and as luminal B were 55% and 45% for MammaTyper, 55% and 45% for IHC+Ki67-G, 36% and 64% for IHC+Ki67-H, and 56% and 44% for IHC+MAI. The levels of agreement between MammaTyper-based and IHC-based subtyping were 84% (κ=0.679) for IHC+Ki67-G, 72% (κ=0.462) for IHC+Ki67-H, and 89% (κ=0.779) for IHC+MAI. High rates of agreement between mRNA-based and IHC-based intrinsic subtyping of luminal HER2-negative BC can be achieved. However, the agreement between IHC-based and MammaTyper-based luminal subtypes depends on the proliferation assessment method, and was highest when the MAI was used. Further comparative clinical studies are needed to determine which method is to be preferred, including analysis of cost-effectiveness.

Expression of Stromal Progesterone Receptor and Differential Methylation Patterns in the Endometrium May Correlate with Response to Progesterone Therapy in Endometrial Complex Atypical Hyperplasia

Progesterone therapy is a viable treatment for complex atypical hyperplasia (CAH) and endometrial adenocarcinoma, though reliable molecular determinants of response are not available. To explore if analysis of pre-therapy endometrial biopsies could yield biomarkers of response to progesterone, patients with CAH or adenocarcinoma undergoing treatment with progestins were included in this cross-sectional study. Immunohistochemistry for progesterone receptor (PR) was performed. Manual PR expression scores (PRES) were first calculated for biopsies by counting PR-positive nuclei in 12 sensitive vs 9 resistant samples. Significant differences in manual PRES were detected in the stroma (p < 0.01) and total endometrium (p < 0.01) for sensitive vs resistant patients. Manual PRES in the stroma had the highest accuracy in segregating sensitive vs resistant patients (96%). Differences in epithelial PRES were not significant. To validate these findings, a correlation between manual PRES and visual PRES was performed in the 21 patients. An additional 11 patients were analyzed to test if visual PRES would be predictive of response to progesterone. Visual PRES in epithelia and stroma in the 32 specimens was calculated. Significant differences in visual PRES were detected in the stroma for sensitive vs resistant samples (p < 0.01), while differences in epithelial and total endometrium were not significant. Whole genome bisulfite sequencing was performed on DNA isolated using pre-therapy biopsies from 6 sensitive and 6 resistant patients in this cohort. Differentially methylated regions were identified in the stroma and epithelium when evaluating sensitive vs resistant samples. Pathways involved in cell adhesion demonstrated the greatest difference in methylation in these samples.

Immuno-genomic landscape of osteosarcoma

Limited clinical activity has been seen in osteosarcoma (OS) patients treated with immune checkpoint inhibitors (ICI). To gain insights into the immunogenic potential of these tumors, we conducted whole genome, RNA, and T-cell receptor sequencing, immunohistochemistry and reverse phase protein array profiling (RPPA) on OS specimens from 48 pediatric and adult patients with primary, relapsed, and metastatic OS. Median immune infiltrate level was lower than in other tumor types where ICI are effective, with concomitant low T-cell receptor clonalities. Neoantigen expression in OS was lacking and significantly associated with high levels of nonsense-mediated decay (NMD). Samples with low immune infiltrate had higher number of deleted genes while those with high immune infiltrate expressed higher levels of adaptive resistance pathways. PARP2 expression levels were significantly negatively associated with the immune infiltrate. Together, these data reveal multiple immunosuppressive features of OS and suggest immunotherapeutic opportunities in OS patients.

Abstract P5-11-01 : Phase 1 dose escalation study of a novel selective androgen receptor modulator (SARM), RAD140, in estrogen receptor positive (ER+), human epidermal growth factor receptor 2 negative (HER2-), metastatic breast cancer

Abstract Introduction: The androgen receptor (AR) is expressed in ~90% of ER+ BC. Androgen-based therapies have demonstrated response rates from 20-25% in advanced/metastatic breast cancer (mBC). RAD140 is an oral nonsteroidal SARM with tissue-selective AR agonist activity in BC cells but attenuated activity in other androgen-responsive tissues. In in vivo and in vitro models recapitulating various stages of AR/ER+ BCs, RAD140 exhibited potent anti-tumor activity as a single agent and in combination with inhibitors (i) of CDK4/6 or mTOR. Methods: This is a first-in-human, phase 1 dose escalation study with a 3+3 design and pharmacokinetic (PK) expansion cohort. The primary endpoint is safety, tolerability, and to establish the maximum tolerated dose (MTD) and/or recommended dose (RD). Secondary endpoints are PK and antitumor activity by RECIST. In the dose escalation phase, patients (pts) were treated with RAD140 once daily (QD) in 28-day cycles. In the subsequent PK expansion (PKE) cohort, pts were treated with a single dose of 100 mg followed by 1 wk of PK sampling before entering continuous 28-day treatment cycles with 100 mg QD dosing. Dose-limiting toxicity (DLT) was assessed during the first 28-day cycle. Key eligibility criteria included: ER+/HER2- and inoperable/mBC, post-menopausal, and ineligible for standard therapy. Archival tumor samples within 2 years or fresh tumor biopsies collected at the time of enrollment were analyzed retrospectively by immunohistochemistry (IHC) for AR, ER, progesterone receptor (PR), HER2 and Ki67. Serum sex hormone-binding globulin (SHBG) and prostate specific antigen (PSA) were used to assess AR engagement. Results: Dose escalation and PKE enrollment (n=22) is complete. Median age = 60 years, 100% stage IV, 86% visceral disease, 95% AR+, 91% AR+/ER+, and 82% AR+/ER+/HER2-. Median number of prior lines of therapy for mBC = 4; prior fulvestrant 86%, aromatase inhibitor 96%, CDK4/6i 91%, mTORi/PI3Ki 46%, chemotherapy 91%. Starting dose levels were 50 mg (n=6), 100 mg (n=13), and 150 mg (n=3) QD. Median time on treatment = 9 wk (range &amp;lt;1-32+ wk). Most frequent (&amp;gt;30%) treatment-emergent adverse events were elevated AST (27% grade [G] 1; 9% G2; 18% G3; 5% G4) and elevated ALT (18% G1; 23% G3). DLTs at 150 mg were hypophosphatemia (n=2). DLTs at 100 mg were hypophosphatemia (n=1), elevated ALT (n=3), and elevated AST (n=1). All DLTs were grade 3 and reversible. No drug-related deaths occurred. PK samples collected for 144 hrs after the 100 mg single dose showed variable absorption with a half-life of approximately 60 hrs. The observed human steady-state PK exposure at 100 mg exceeds the PK exposure of the efficacious dose in mouse of 10 mg/kg. Of 9 evaluable pts at the MTD of 100 mg, there was 1 partial response (PR; ORR=11%) and 3 pts (33%) with stable disease ≥12 wk. Time to PR was 15.9 wk with duration 18.6+ wk. Three pts remain on treatment. SHBG decreased in 18/18 pts and PSA increased in 15/20 pts with paired samples available, indicative of AR target engagement, most notably in the pt with PR. In 1 pt with paired pre- and on-treatment tumor biopsies, a strong induction of AR by IHC, predominantly in the nucleus was seen, further suggesting AR activation by RAD140 at the tissue level. Conclusions: The MTD and recommended dose for RAD140 is 100 mg QD. RAD140 is a novel oral AR-targeted agent for the treatment of AR+/ER+ mBC with an acceptable safety profile and preliminary evidence of target engagement and antitumor activity. This study is ongoing. NCT03088527 Citation Format: Erika Hamilton, Patricia LoRusso, Cynthia Ma, Neelima Vidula, Rebecca G Bagley, Steven Troy, Miriam Annett, Ziyang Yu, Aaron Weitzman, Maureen G Conlan, Amy Weise. Phase 1 dose escalation study of a novel selective androgen receptor modulator (SARM), RAD140, in estrogen receptor positive (ER+), human epidermal growth factor receptor 2 negative (HER2-), metastatic breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-11-01.

Abstract P5-02-08: Androgen receptor pathway activity and the ratio between androgen and estrogen receptor pathway activity in breast cancer subtypes

Abstract Background: Androgen receptor (AR) immunohistochemistry staining in breast cancer has revealed frequent AR expression in all breast cancer subtypes. Activity of the AR signaling pathway can be either tumor suppressive or tumor promoting, depending on tumor context (Pharmacol Ther., In press, https://doi.org/10.1016/j.pharmthera.2019.05.005). For this reason, in an individual patient, AR phenotypic activity needs to be defined in order to choose the proper AR targeted therapy. The AR/ER protein ratio has been suggested as a biomarker to define this AR function and enable the appropriate therapy choice, i.e. androgen therapy in case of a low AR/ER protein expression ratio, and anti-androgen therapy in case of a high ratio. However, AR and ER expression are not necessarily associated with an active signaling pathway. Therefore we investigated the ratio between functional AR and ER pathway activities as this may have an advantage in guiding therapy choice. Methods: AR and ER pathway activity and AR/ER pathway activity ratio were determined on public Affymetrix HG-U133 Plus 2.0 gene expression microarray data of clinical breast cancer studies (available in the GEO database: GSE12276, GSE21653, GSE20685, GSE42568, GSE6532, GSE9195, GSE45827, GSE7307, GSE10780, GSE17907, GSE21422, GSE5764, GSE31192, GSE54002 and Array Express: E-MTAB-365; total n=2090). For this, we used previously described tests that enable quantification of functional pathway activity, based on Bayesian computational model inference of pathway activity from measurements of mRNA levels of target genes of the ER and AR transcription factor (Cancer Res., 2014, vol. 74, no. 11, pp. 2936-2945; Sci Rep., 2019, vol. 9, no. 1, p. 1603). Intrinsic subtyping of all samples has been done using the same methodology as described elsewhere (Am J Pathol, 2018, vol. 188, no. 9, pp 1956-1972).Results: AR pathway activity was increased in all breast cancer subtypes compared to normal breast tissue (p&amp;lt;0.0001); highest AR activity scores were observed in the HER2-enriched subtype. As shown before, ER activity scores were highest in luminal A and B subtypes. The AR/ER pathway activity ratio was low in luminal A and B, and comparable with normal tissue, but high in HER2 and basal breast cancer subtypes (p&amp;lt;0.0001). Discussion: Results are compatible with currently available evidence and show that the AR pathway can be activated in all breast cancer subtypes. We hypothesize that its function depends on the level of co-existent ER pathway activity, i.e. a tumor suppressive function in ER pathway active breast cancer, and a tumor promoting function in ER pathway inactive breast cancer. Upon future clinical conformation, the AR/ER pathway activity ratio as determined on an individual patient tissue sample may be an improved biomarker to guide the choice of (complementary) AR pathway targeted therapy in breast cancer, i.e. androgen receptor pathway inhibitors in case of a high ratio and potential use of androgen receptor modulators in case of a low ratio. Citation Format: Anja van de Stolpe, Yvonne Wesseling-Rozendaal, Marcia Alves de Inda, Henk van Ooijen, Wim Verhaegh. Androgen receptor pathway activity and the ratio between androgen and estrogen receptor pathway activity in breast cancer subtypes [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-02-08.

Low-grade Apocrine Intraductal Carcinoma: Expanding the Morphologic and Molecular Spectrum of an Enigmatic Salivary Gland Tumor.

Intraductal carcinoma (IDC) is the current designation for a salivary gland neoplasm previously referred to as "low-grade salivary duct carcinoma" and "low-grade cribriform cystadenocarcinoma," among others. IDC is conceptually believed to be similar to ductal carcinoma in-situ of the breast. Although IDC is one entity in the current WHO Classification of Head and Neck Tumors, recent studies have suggested that at least three subtypes exist: a low-grade, intercalated duct-like variant with frequent RET rearrangements; a high-grade apocrine variant with complex, salivary duct carcinoma-like genetics; and a mixed variant. We sought to characterize an unusual form of low-grade, purely apocrine IDC. Three cases of apocrine-type IDC with low-grade histology were retrieved from the authors' consultation files. Immunohistochemistry for androgen receptor, GCDFP-15, S100, smooth muscle actin, and p40 was performed. A custom, targeted next generation sequencing (NGS) panel including 1425 cancer-related genes was also done on all cases. All three cases developed in the parotid glands of men, aged 51, 63, and 73years (mean, 62years). All cases consisted of large, rounded macrocysts surrounded by smaller nests which were lined by cells with abundant granular eosinophilic cytoplasm and large round nuclei with prominent nucleoli. Pleomorphism was mild, the mitotic rate was low, and necrosis was absent. No cases had any invasive foci or areas of intercalated duct-like morphology. By immunohistochemistry, all cases were diffusely positive for androgen receptor and GCDFP-15, surrounded entirely by an intact layer of small myoepithelial cells positive for S100, smooth muscle actin, and p40. Targeted NGS results were obtained from two cases: both harbored HRAS mutations and copy number losses in TP53, while one case each harbored mutations in PIK3CA, SPEN, and ATM. Fusions were absent in both cases. All three patients were treated by surgery alone, and are currently free of disease (follow up 12-190months). This study confirms the existence of a low-grade, purely apocrine form of IDC. In its pure form, i.e., without an intercalated duct-type component, low-grade apocrine IDC is genetically similar to high-grade salivary duct carcinoma, with frequent HRAS and PI3K pathway mutations. Despite its molecular similarities to the aggressive salivary duct carcinoma, low-grade apocrine IDC appears to behave in a very indolent manner, supporting is classification as a non-invasive neoplasm, and underscoring the need to distinguish these tumors from each other.

Androgen Receptor Expression in Adenoid Cystic Carcinoma of Breast: A Subset of Seven Cases.

Adenoid cystic carcinoma (ACC) of the breast is an uncommon salivary type of breast carcinoma. It is a triple negative breast carcinoma with a basal-like phenotype that behaves in an indolent manner. Herein, we aimed to document clinicopathologic findings and hormone receptor status of ACC in the breast diagnosed in our institution during an eleven-year period. Medical data of cases diagnosed as adenoid cystic carcinoma in the breast between January 2006 and December 2016 were retrospectively reviewed from hospital data base. Paraffin blocks of seven cases were retrieved from the archive of Pathology Department and androgen receptor (AR) immunohistochemistry was applied to each case. All of the cases diagnosed as ACC were females with a mean age 56.2. Solid growth pattern was present in two cases. P63 was constantly expressed in the whole group, and at least one additional myoepithelial marker (calponin, caldesmon, etc.) was co-expressed in tumors. While weak estrogen receptor expression was detected only in one patient, AR was strikingly expressed in majority (%85.7) of the tumors. To our knowledge, our series is the first to report such high levels of AR expression. This new finding, in turn, suggests considering hormonal therapy as an option in the management of ACC of the breast.

Clinicopathological significance of immunohistochemical expression of Filamin A in breast cancer.

BACKGROUND:Filamin A is an actin-crosslinking protein expressed in many malignancies, although its prognostic and therapeutic role in breast cancer is not studied. There is enigma regarding its dual role in cancer, the tumor-progressing or tumor-suppressing effects depending on the site to which it localizes in the cell. The current study aimed to detect Filamin A expression in breast cancer and its association with other biomarkers and other clinicopathological parameters and established risk factors in breast cancer so that it can be a potential site for targeted therapy.MATERIALS AND METHODS:One hundred female patients of histologically proven breast cancer who presented to our hospital over a 2-year period were included in the study. None of the patients received prior radiotherapy, chemotherapy, or immunotherapy. Patients with recurrent breast cancer are not included in the study. All study cases are subjected to immunohistochemistry for estrogen receptor, progesterone receptor, Her2 neu, and ki-67 from core biopsy tissue of cases diagnosed as breast carcinoma. Tissue sections were subjected to immunohistochemistry with anti-Filamin A.RESULTS:Filamin A is expressed in 69% of cases of invasive breast cancer in our study. There was no statistically significant relationship of Filamin A immunoexpression with histological grade, age, parity, oral contraceptive use, smokeless tobacco use, TNM staging, clinical staging, clinical prognostic staging, and also ER, PR, Her2 neu, and ki-67 status (P > 0.05). Thus, it appears to be an independent biomarker in breast carcinoma. Filamin A was expressed only in the cytoplasm in all our study cases. Filamin A expression can be observed in adjacent normal breast tissue and benign fibroadenoma tissues also, but the pattern of expression is mainly membranous with cytoplasmic positivity. The cytoplasmic expression is seen in malignant cells as well as normal breast and benign tumor sections implicating the dual role of Filamin A in breast cancer.CONCLUSION:No significant correlation could be found between Filamin A expression and clinicopathological parameters in our study. The cytoplasmic expression is seen in malignant cells as well as normal breast and benign tumor sections implicating the dual role of Filamin A in breast cancer. Filamin A immunoexpression should be further correlated with metastasis-free survival period of breast cancer patients

Edible Bird's Nest Attenuates Menopause-Related Bone Degeneration in Rats via Increaing Bone Estrogen-Receptor Expression.

To investigate the mechanistic basis for the attenuation of bone degeneration by edible bird's nest (EBN) in ovariectomized rats. Forty-two female Sprage-Dawley rats were randomized into 7 groups (6 in each group). The ovariectomized (OVX) and OVX + 6%, 3%, and 1.5% EBN and OVX +estrogen groups were given standard rat chow alone, standard rat chow +6%, 3%, and 1.5% EBN, or standard rat chow +estrogen therapy (0.2mg/kg per day), respectively. The sham-operation group was surgically opened without removing the ovaries. The control group did not have any surgical intervention. After 12 weeks of intervention, blood samples were taken for serum estrogen, osteocalcin, and osteoprotegerin, as well as the measurement of magnesium, calcium abd zinc concentrations. While femurs were removed from the surrounding muscles to measure bone mass density using the X-ray edge detection technique, then collected for histology and estrogen receptor (ER) immunohistochemistry. Ovariectomy altered serum estrogen levels resulting in increased food intake and weight gain, while estrogen and EBN supplementation attenuated these changes. Ovariectomy also reduced bone ER expression and density, and the production of osteopcalcin and osteorotegerin, which are important pro-osteoplastic hormones that promote bone mineraliztion and density. Conversely, estrogen and EBN increased serum estrogen levels leading to increased bone ER expression, pro-osteoplastic hormone production and bone density (all P<0.05). EBN could be used as a safe alternative to hormone replacement therapys for managing menopausal complications like bone degeneration.

Abstract B44: Clinical utility of semiquantitative evaluation of progesterone receptor immunohistochemistry in neuroendocrine tumors of the pancreas

Abstract Purpose: Progesterone receptor (PR) is expressed strongly in a subset of neuroendocrine cells in the pancreas. Tamoxifen has long been used in adjuvant endocrine therapy of steroid hormone receptor–positive breast cancer, and currently clinical trials are assessing its use in pancreatic neuroendocrine tumors (NET) that show variable PR expression. Prior studies have reported receptor status as “positive” or “negative,” using 1% or 10% positive nuclei as a cutoff. In this study, we assessed PR immunohistochemical expression in a cohort of NET and other pancreatic nonexocrine carcinomas. Theoretically, tumors with highest PR expression are more likely to respond to adjuvant tamoxifen. Experimental Design: Immunohistochemical staining for PR was performed on tissue microarrays of 54 well-differentiated neuroendocrine tumors of the pancreas and 8 poorly differentiated carcinomas of the pancreas, including 4 acinar cell carcinomas, 2 neuroendocrine carcinomas, and 2 small-cell neuroendocrine carcinomas. A minimum of 4 separate 2-mm tumor cores were sampled on each case. Results: PR expression in non-neoplastic pancreatic islets was used for positive control. PR staining was evaluated using a semiquantitative H-score method. Positive PR staining (defined as greater than 1%) was identified in 61% (33 of 54 cases) of well-differentiated NET (mean H-score 100) and 0% (0 of 8 cases) of poorly differentiated carcinomas of the pancreas. Of the 33 PR positive cases of well-differentiated NET, 3 cases had less than 10% nuclear staining (mean H-score 7), 8 cases had 10% to less than 50% nuclear staining (mean H-score 44), and 22 cases had 50% or greater nuclear staining (mean H-score 229). Conclusions: Based on these findings, we suggest analysis of NET tumor PR expression should include a quantitative or semiquantitative score rather than dichotomized immunohistochemical evaluation of hormone receptors, particularly in the evaluation of patients’ response to clinical trials with tamoxifen. High-grade endocrine and acinar carcinomas showed no PR expression and are less likely to derive benefit from adjuvant endocrine therapy. Citation Format: Sonya Purushothaman, Simona De Michele, Dorothy Low, Helen E. Remotti. Clinical utility of semiquantitative evaluation of progesterone receptor immunohistochemistry in neuroendocrine tumors of the pancreas [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B44.