Maspin (SerpinB5) is an epithelial-specific tumor suppressor gene product that displays context-dependent cellular functions. Maspin-deficient mouse models created to date have not definitively established maspin functions critical for cancer suppression. In this study, we generated a mouse strain in which exon 4 of the Maspin gene was deleted, confirming its essential role in development but also enabling a breeding scheme to bypass embryonic lethality. Phenotypic characterization of this viable strain established that maspin deficiency was associated with a reduction in maximum body weight and a variety of context-dependent epithelial abnormalities. Specifically, maspin-deficient mice exhibited pulmonary adenocarcinoma, myoepithelial hyperplasia of the mammary gland, hyperplasia of luminal cells of dorsolateral and anterior prostate, and atrophy of luminal cells of ventral prostate and stratum spinosum of epidermis. These cancer phenotypes were accompanied by increased inflammatory stroma. These mice also displayed the autoimmune disorder alopecia aerate. Overall, our findings defined context-specific tumor suppressor roles for maspin in a clinically relevant model to study maspin functions in cancer and other pathologies. Cancer Res; 77(4); 886-96. ©2017 AACR.

Cervical carcinoma is the second most common female malignancy worldwide. It usually spreads by direct local extension or the lymphatic vessels. Hematogenous dissemination with distant skeletal muscle metastases is a rare phenomenon. We report here 2 patients whose recurrent squamous cell carcinomas of the cervix presented with symptomatic skeletal muscle metastases affecting the muscles of the thoracic wall and forearm, respectively. We also discuss the differential diagnosis and comprehensively review the previously published literature on this rare presentation of cervical carcinoma.

Apocrine carcinoma of the breast is a rare, primary breast cancer characterized by the apocrine morphology, estrogen receptor-negative and androgen receptor-positive profile with a frequent overexpression of Her-2/neu protein (~30%). Apart from the Her-2/neu target, advanced and/or metastatic apocrine carcinomas have limited treatment options. In this review, we briefly describe and discuss the molecular features and new theranostic biomarkers for this rare mammary malignancy. The importance of comprehensive profiling is highlighted due to synergistic and potentially antagonistic molecular events in the individual patients.

Targeted immunotherapy based on PD-1/PD-L1 suppression has revolutionized the treatment of various solid tumors. A remarkable improvement has also been observed in the treatment of patients with refractory/relapsing classical Hodgkin lymphoma (cHL). We investigated PD-L1 status in a variety of treatment resistant lymphomas. Tumor samples from 78 patients with therapy resistant lymphomas were immunohistochemically (IHC) investigated for the expression of PD-L1 using two antibody clones (SP142 and SP263, Ventana). Thirteen PD-L1+ cases were further analyzed for gene copy number variations (CNV) by NGS and for PD-L1/JAK2/PD-L2 co-amplification using fluorescent in-situ hybridization assay (FISH). PD-L1 positivity (≥5% positive cancer cells, IHC) was present in 32/77 (42%) and 33/71 cases (46%) using SP142 and SP263 antibodies, respectively. Concordance between the two anti-PD-L1 clones was high with only three (4%) discrepant cases. The strongest and consistent (10/11 cases) expression was observed in cHL and primary mediastinal B-cell lymphomas (3/3). Diffuse large B-cell lymphomas (DLBCL) were frequently positive (13/26) irrespective of subtype. Follicular (1/8), peripheral T-cell (3/11) and mantle cell (1/8) lymphomas were rarely positive, while small lymphocytic lymphoma/CLL and marginal zone lymphomas were consistently negative (3/3). Co-amplification/CNVs of PD-L1/JAK2/PD-L2 were observed in 3 cases of DLBCL and cHL, respectively. Of note, all three cHL-amplified cases were positive by FISH, but not by NGS. Since only a fraction of the IHC positive lymphoma cases were positive by FISH and NGS assays, other mechanisms are involved in PD-L1 upregulation, especially in DLBCL. FISH assay may be more suitable than NGS assay for determination of PD-L1 alterations in cHL.

Background: Olfactory neuroblastoma (esthesioneuroblastoma) [ONB] is a rare malignant neoplasm arising from the olfactory epithelium in the nasal vault. It usually takes an aggressive clinical course for which there are no specific treatment guidelines. Pursuing the goals of personalized medicine, weinvestigated acohort of recurrent and/ or metastatic ONBs using multiplatform molecular profiling approach. Methods: Formalin-fixed paraffin-embedded tissue samples of twenty (10 male, 10 female patients, age range: 29-84 years) ONBs were profiled at Caris Life Sciences (Phoenix, Arizona) using DNA sequencing (Sanger sequencing, massively parallel sequencing [Illumina NGS] and gene fusions [Archer FusionPlex]), whole genome RNA microarray (HumanHT-12 v4 beadChip, Illumina), gene copy numberassays (chromogenic and fluorescent in situ hybridization) and immunohistochemistry. Results: Mutations were detected in 8/14 (57%) ONBs including TP53 (3 cases), CTNNB1 (2 cases), APC, cKIT, cMETand PDGFRA, and SMAD4 gene (single cases, respectively). When compared with control tissues, 21 genes were overexpressed and 19 genes under expressed by microarrayassay (>10x). Some of the upregulated genes included stem cell marker CD24, SCG2 (Secretogranin II) and Insulin-Like Growth Factor Binding Protein 2 (IGFBP-2). None of the cases harbored copy number variations of EGFR, HER2 and cMET genes, and no gene fusions were identified. No case expressed PD-L1 (0/6) or IDO-1 (0/3). Multiple protein biomarkers of response or resistance to classic chemotherapy drugs were identified: low ERCC1 [cisplatin sensitivity] in 82% (9/11), high TOPO1 [irinotecan sensitivity] in 63% [12/19], high TUBB3 [vincristine resistance] in 92% (12/13) and high MRP1 (multidrug resistance) in 100% (6/6). Conclusions: Our study indicatesthata subset of ONBs exhibits molecularalterations, notably in the Wnt and cKIT/PDGFRA pathways, that are potentially treatable using novel targeted therapies. Optimization of cytotoxic chemotherapyapproaches based on protein expression may beworthy offurther investigation. study: Disclosure: ofCaris All declaredno ofinterest.

Aims Metaplastic breast carcinoma (MBC) is a rare subtype of breast carcinoma less responsive to conventional chemotherapy than ductal carcinoma. In molecular terms, MBCs usually cluster with triple-negative breast cancers (TNBCs), but have a worse prognosis than TNBCs. Studies investigating MBCs for specific biomarkers of therapy response are rare and limited by the methodological approaches. The aim of the present study was to characterise MBCs on a molecular level and test programmed death-ligand 1 (PD-L1) biomarker expression in MBCs for future therapeutic interventions. Methods We profiled 297 samples (MBC (n=75), TNBC (n=106), human epidermal growth factor receptor 2 (HER2)-positive breast cancers (n=32) and hormone-positive breast cancers (n=84)) by next-generation sequencing. Immunohistochemistry for PD-L1 and programmed cell death 1 (PD-1) expression was performed using automated procedures. Results The most commonly mutated genes in MBCs included TP53 (56%) and PIK3CA (23%). Pathogenic mutations in other genes, including HRAS, FBXW7, PTEN, AKT1 and SMAD4, were rare. PD-L1 expression was detected in a significantly higher proportion of MBCs (46%) than in other subtypes (6% each in hormone-positive and HER2-positive breast cancers, and 9% in TNBC, not otherwise specified, p<0.001). PD-1-positive tumour infiltrating lymphocytes (TILs) varied greatly in MBCs. Conclusions Comprehensive profiling of a large cohort of this rare subtype of breast carcinoma highlighted the predominance of TP53 mutation and increased PD-L1 expression in carcinoma cells. These results can be exploited in clinical trials using immune checkpoint inhibitors.

Mammary analogue secretory carcinoma (MASC) is a recently described low-grade malignant tumor of the salivary glands, biologically and morphologically equivalent to secretory breast carcinoma. We give a brief overview of this new entity, including morphological, immunohistochemical, molecular-genetic, clinical, epidemiologic features, differential diagnosis, and outcome results.

Germ cell tumors of the testis are a heterogeneous group of neoplasms that affect male adolescents and young adults. Wnt signaling pathway components have been shown to be actively involved in normal and malignant germ cell differentiation and progression. In this study, we aimed to explore the expression patterns of the secreted frizzled‐related protein (SFRP) and Disheveled protein family (DVL) in a subset of testicular germ cell tumors. Eighty‐five formalin‐fixed, paraffin‐embedded tissue samples of the primary germ cell tumors of the testis were stained against SFRP1, SFRP3, DVL1, and DVL2 proteins using immunohistochemistry. SFRP1 and SFRP3 exhibited lower expression in both seminomas and mixed/non‐seminomatous tumors, compared with atrophic/benign tissue (p < 0.001). SFRP3 expression was lower than SFRP1 expression within the seminoma group (p = 0.004), but not within the mixed/non‐seminomatous group (p = 0.409). The majority of the tested cases (27/28, 96%) exhibited low DVL1 protein expression (median 0%, range 0–90%). In contrast, 20 out of 22 tested cases (91%) exhibited strong expression of DVL2 protein (median 80%, range 0–100%). No significant difference in DVL1 and DVL2 protein expression was observed between seminomas and mixed/non‐seminomatous tumors (p = 0.68 and 0.29). The secreted frizzled‐related protein and disheveled protein family members appear to be actively involved in the pathogenesis of primary testicular germ cell tumors.

Introduction: Caveolin-1 (Cav1) is associated with basal-like triple-negative (ER-/PR-/Her2-) breast cancers (TNBC). Its biological contribution to this subtype has not been fully explored and controversy persists regarding the molecular role of Cav1 in carcinogenesis. Experimental Procedures: Thirty-four TNBC (17 Cav1+/17 Cav1-) patients molecularly-profiled with a commercial assay (Caris Life Sciences, AZ) were evaluated retrospectively. Cav1 status was determined by immunohistochemistry (caveolin-1 polyclonal; ≥2+ ≥50%). The majority of specimens (28/34) used for profiling were from primary breast sites and contained ≥50% neoplastic cells. The transcriptomes were profiled using Illumina9s HumanHT-12 microarray (v4). Data were normalized using mean normalization procedure. Differential expression analysis was performed using R9s Limma package. Pathway analysis was carried out using R9s signaling pathway impact analysis (SPIA) package with 69 cancer, immunity, and cell signaling related KEGG pathways. Results: Using a cutoff of two-fold and adjusted p-value of 0.05, we identified 954 genes differentially expressed between Cav1+/- TNBC patients. Included in these were 31 genes which were found to be up-regulated by over five- fold and 3 genes down-regulated by over five fold in Cav1+ TNBC. Genes of notable interest for their role in cell signaling, cell adhesion, tumor invasion and metastasis, included an up-regulation of TGFBR2, SPARC, integrins (ITGA11, ITGB5, ITGBL1), cell adhesion proteins (LAMB3, COL5A3) and molecules which facilitate tumor invasion (LAMB3, MMP1, MMP2, MMP9). In addition, genes found to be down-regulated in Cav1+ patients and notable for their roles in promoting epithelial-mesenchymal-transition (EMT) included Claudin 3(CLD3) and CA125/MUC16 (Mucin 16). We also detected an approximately two-fold down-regulation of CDKN2A in Cav1+ patients. Using SPIA pathway analysis, 12 pathways were found to be differentially activated in Cav1+ vs. Cav1- TNBC. The most differentially activated pathways were the focal adhesion pathway (p = 4.51E-18), PI3k-Akt signaling pathway (p = 2.01E-6) and TGF-β and MAPK signaling pathways (p = 0.005, 0.014, respectively). Conclusions: Differential gene expression patterns and pathway analyses provide evidence for distinct profiles for gene expression between Cav1+/- TNBC. Cav1+ TNBC patients exhibit up-regulation of genes important for cell signaling, extracellular matrix remodeling and tumor invasion, and down-regulation of genes that may facilitate EMT and loss of cell cycle control. The focal adhesion pathway, as well as TGF-β, PI3K and MAPK signaling pathways, were identified as differentially activated among Cav1+/- TNBC. Taken together, these data support the role of Cav1+ in identifying a subtype of TNBC that may have a greater risk for invasion and metastasis. The correlation of this subtype with prognosis and drug response should be investigated in future studies. Citation Format: Rebecca A. Feldman, Zoran Gatalica, Semir Vranic, Ryan Bender, Sandeep Reddy, Anatole Ghazalpour. Caveolin-1: Beyond a marker for basal-like breast cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3928.

MET amplification has been implicated in signaling pathways that promote cell proliferation, invasion, and survival. It has been identified as an oncogenic driver in various malignancies and is currently being investigated as a potential therapeutic target. To date, MET exon 14 skipping by sequencing and MET amplification by FISH have been found to have potential clinical utility in predicting those patients who may derive benefit from MET-targeted therapy. However, little research has been conducted on alternative technologies to FISH such as CISH, which does not require a dark room and can be interpreted by a board-certified pathologist. The purpose of this study is to report our experience with MET amplification across solid tumors using CISH. A retrospective analysis was done on 26,619 specimens analyzed for MET amplification by CISH at a CLIA-certified lab (Caris Life Sciences). The validated CISH assay, previously validated against a FISH assay, utilized a gene copy number > 5 to assess amplification. Concordance and correlative studies were done in MET-amplified, non-small cell lung cancer (NSCLC) specimens analyzed using a cMET IHC (SP44, 2+ or 3+ staining intensity in 50% or more tumor cell membrane) analyzing protein expression. Correlative studies involving co-existing aberrations, including PD-L1 (SP142, any intensity in at least 50% of tumor cells), in this MET-amplified, NSCLC cohort were also performed. MET amplification utilizing CISH was 0.7% (188/26,619) overall. MET-amplified solid tumors included carcinomas such as NSCLC (3.1%, 87/2767), gastric adenocarcinoma (3.8%, 11/293), esophageal and esophagogastric junction adenocarcinoma (3.3%, 11/338), and endometrial carcinoma (0.4%, 9/2020) along with non-carcinomas, including glioblastoma multiforme (1.0%, 5/510), uterine sarcoma (1.3%, 5/400), melanoma (0.4%, 2/538), and rare tumors such as placental-site trophoblastic tumor (100%, 1/1) and prostatic neuroendocrine tumor (100%, 1/1). A sub-analysis of MET-amplified, NSCLC specimens demonstrated co-occurring protein overexpression in 92.6% (75/81) of cases. These same MET-amplified, NSCLC specimens were found to have EGFR pathogenic/presumed pathogenic mutations (19.7%, 15/76), ALK rearrangements (2.5%, 2/80), and PD-L1 overexpression (27.5%, 14/51). ROS1 rearrangements were not detected in this NSCLC cohort (0%, 0/76). Our data suggest MET amplification detection utilizing CISH is a viable option for identifying MET-driven cancers. The presence of MET across various solid tumors contrasts with biomarkers like HER2, which are exclusive to carcinomas. A sub-analysis of our NSCLC population shows MET-amplified tumors contains a similar molecular distribution to the general NSCLC population. Future studies should incorporate MET CISH in clinical trials utilizing MET-targeted agents to determine its potential as a predictive test for evaluating who may derive the most benefit. Citation Format: David Arguello, Zoran Gatalica, Semir Vranic, Sandeep Reddy, Ari VanderWalde. Analysis of MET-amplified solid tumors using chromogenic in situ hybridization (CISH). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 396.