It remains unclear to what extent tumor heterogeneity impacts on protein biomarker discovery. Here, we quantified proteome intra-tissue heterogeneity (ITH) based on a multi-region analysis of prostate tissues using pressure cycling technology and Sequential Windowed Acquisition of all THeoretical fragment ion mass spectrometry. We quantified 6,873 proteins and analyzed the ITH of 3,700 proteins. The level of ITH varied depending on proteins and tissue types. Benign tissues exhibited more complex ITH patterns than malignant tissues. Spatial variability of 10 prostate biomarkers was validated by immunohistochemistry in an independent cohort (n = 83) using tissue microarrays. Prostate-specific antigen was preferentially variable in benign prostatic hyperplasia, whereas growth/differentiation factor 15 substantially varied in prostate adenocarcinomas. Furthermore, we found that DNA repair pathways exhibited a high degree of variability in tumorous tissues, which may contribute to the genetic heterogeneity of tumors. This study conceptually adds a new perspective to protein biomarker discovery: it suggests that recent technological progress should be exploited to quantify and account for spatial proteome variation to complement biomarker identification and utilization.

Purpose: The clinical standard treatment for patients with malignant pleural mesothelioma (MPM) includes a cisplatin-based chemotherapy, leading to reduction of tumor size in only a minority of patients. Predicting response to chemotherapy in patients with MPM by using a genetic marker would, therefore, enable patient stratification. Experimental Design: In this retrospective biomarker study, eligible patients had resectable MPM, measurable disease, and available primary MPM tissue. All patients underwent first-line treatment with cisplatin and pemetrexed, followed by surgery. Thorough molecular analysis was performed (whole-exome and targeted deep sequencing, and copy-number analyses), and also mechanistic in vitro data (viability assays, Western blots, and immunoprecipitation) using mesothelioma cell lines with and without siRNA-mediated BRCA1-associated protein 1 (BAP1) knockdown were provided. Results: In a training cohort of patients with MPM (n = 28), mutations or deletions of BAP1 each predicted resistance to chemotherapy in patients with primary MPM. The negative predictive value of BAP1 loss in patients with MPM was confirmed by amplicon sequencing and copy-number array technology in an independent test cohort (n = 39). Preliminary mechanistic studies using siRNA-based knockdown of BAP1 in MPM cell culture models along with immunoprecipitation assays confirmed chemoresistance in vitro, possibly through inhibition of apoptosis and transcriptional regulation of the BAP1/HCF1/E2F1 axis. Conclusions: Alterations in BAP1 in MPM were a negative predictor for response to chemotherapy and could possibly be used as a companion biomarker for treatment decision.

CDKN2A deletion is a common alteration in pleural mesothelioma (PM) and frequently associated with co-deletion of MTAP. Since the standard detection method for CDKN2A deletion and FISH analysis is relatively expensive, we here investigated the suitability of inexpensive p16 and MTAP IHC by comparing concordance between IHC and OncoScan CNV arrays on samples from 52 PM patients. Concordance was determined using Cohen’s kappa statistics. Loss of CDKN2A was associated with co-deletion of MTAP in 71% of cases. CDKN2A-MTAP copy-number normal cases were also IHC positive in 93% of cases for p16 and 100% for MTAP, while homozygous deletion of CDKN2A-MTAP was always associated with negative IHC for both proteins. In cases with heterozygous CDKN2A-MTAP loss, IHC expression of p16 and MTAP was negative in 100% and 71%, respectively. MTAP and p16 IHC showed high sensitivity (MTAP 86.5%, p16 100%) and specificity (MTAP 100%, p16 93.3%) for the detection of any gene loss. Loss of MTAP expression occurred exclusively in conjunction with loss of p16 labeling. Both p16 and MTAP IHC showed high concordance with Oncoscan CNV arrays (kappa = 0.952, p < 0.0001, and kappa = 0.787, p < 0.0001 respectively). We recommend combined MTAP and p16 immunohistochemistry to confirm the diagnosis of PM.

The engraftment potential of myeloproliferative neoplasms in immunodeficient mice is low. We hypothesized that the physiological expression of human cytokines (macrophage colony-stimulating factor, interleukin-3, granulocyte-macrophage colony-stimulating factor, and thrombopoietin) combined with human signal regulatory protein α expression in Rag2−/−Il2rγ−/− (MISTRG) mice might provide a supportive microenvironment for the development and maintenance of hematopoietic stem and progenitor cells (HSPC) from patients with primary, post–polycythemia or post–essential thrombocythemia myelofibrosis (MF). We show that MISTRG mice, in contrast to standard immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ and Rag2−/−Il2rγ−/− mice, supported engraftment of all patient samples investigated independent of MF disease stage or risk category. Moreover, MISTRG mice exhibited significantly higher human MF engraftment levels in the bone marrow, peripheral blood, and spleen and supported secondary repopulation. Bone marrow fibrosis development was limited to 3 of 14 patient samples investigated in MISTRG mice. Disease-driving mutations were identified in all xenografts, and targeted sequencing revealed maintenance of the primary patient sample clonal composition in 7 of 8 cases. Treatment of engrafted mice with the current standard-of-care Janus kinase inhibitor ruxolitinib led to a reduction in human chimerism. In conclusion, the established MF patient-derived xenograft model supports robust engraftment of MF HSPCs and maintains the genetic complexity observed in patients. The model is suited for further testing of novel therapeutic agents to expedite their transition into clinical trials.

Oncogenic rearrangements leading to targetable gene fusions are well-established cancer driver events in lung adenocarcinoma. Accurate and reliable detection of these gene fusions is crucial to select the appropriate targeted therapy for each patient. We compared the targeted next-generation-sequencing Oncomine Focus Assay (OFA; Thermo Fisher Scientific) with conventional ALK FISH and anti-Alk immunohistochemistry in a cohort of 52 lung adenocarcinomas (10 ALK rearranged, 18 non-ALK rearranged, and 24 untested cases). We found a sensitivity and specificity of 100% for detection of ALK rearrangements using the OFA panel. In addition, targeted next generation sequencing allowed us to analyze a set of 23 driver genes in a single assay. Besides EML4-ALK (11/52 cases), we detected EZR-ROS1 (1/52 cases), KIF5B-RET (1/52 cases) and MET-MET (4/52 cases) fusions. All EML4-ALK, EZR-ROS1 and KIF5B-RET fusions were confirmed by multiplexed targeted next generation sequencing assay (Oncomine Solid Tumor Fusion Transcript Kit, Thermo Fisher Scientific). All cases with EML4-ALK rearrangement were confirmed by Alk immunohistochemistry and all but one by ALK FISH. In our experience, targeted next-generation sequencing is a reliable and timesaving tool for multiplexed detection of targetable rearrangements. Therefore, targeted next-generation sequencing represents an efficient alternative to time-consuming single target assays currently used in molecular pathology.

Sinonasal melanoma is a rare subtype of melanoma and little is known about its molecular fingerprint. Systemic treatment options are limited, as targetable BRAF mutations are rare compared to cutaneous melanoma. Currently, metastatic sinonasal melanoma is being treated according to the guidelines of cutaneous melanoma. In this study, we investigated the molecular profile of 19 primary sinonasal melanomas, using a novel customized melanoma-specific next generation sequencing (NGS) panel (MelArray) of 190 genes. Results were correlated to histological and clinical features to further characterize this rare, aggressive type of melanoma and screen for prognostic markers and possible treatment options. Molecular profiles encompassed predominantly mutations in NRAS (25%), whereas KIT or BRAF p.V600 mutations were not detected. Tumor mutational burden was overall low. High level of copy number variations (CNVs) were associated with alterations in DNA-repair genes and shorter distant metastasis-free survival (p = 0.005). Monomorphic (vs. pleomorphic) morphology was found to be significantly associated with worse disease-specific survival (p < 0.001), however no correlation between morphology and molecular aberrations was found. A variety of alterations in different pathways were detected, justifying molecular testing and opening potential personalized treatment options in current study or compassionate use settings.