Risk SNP-Mediated Promoter-Enhancer Switching Drives Prostate Cancer through lncRNA PCAT19.

The prostate cancer (PCa) risk-associated SNP rs11672691 is positively associated with aggressive disease at diagnosis. We showed that rs11672691 maps to the promoter of a short isoform of long noncoding RNA PCAT19 (PCAT19-short), which is in the third intron of the long isoform (PCAT19-long). The risk variant is associated with decreased and increased levels of PCAT19-short and PCAT19-long, respectively. Mechanistically, the risk SNP region is bifunctional with both promoter and enhancer activity. The risk variants of rs11672691 and its LD SNP rs887391 decrease binding of transcription factors NKX3.1 and YY1 to the promoter of PCAT19-short, resulting in weaker promoter but stronger enhancer activity that subsequently activates PCAT19-long. PCAT19-long interacts with HNRNPAB to activate a subset of cell-cycle genes associated with PCa progression, thereby promoting PCa tumor growth and metastasis. Taken together, these findings reveal a risk SNP-mediated promoter-enhancer switching mechanism underlying both initiation and progression of aggressive PCa.

Early innate and adaptive immune perturbations determine long-term severity of chronic virus and Mycobacterium tuberculosis coinfection.

Chronic viral infections increase severity of Mycobacterium tuberculosis (Mtb) coinfection. Here, we examined how chronic viral infections alter the pulmonary microenvironment to foster coinfection and worsen disease severity. We developed a coordinated system of chronic virus and Mtb infection that induced central clinical manifestations of coinfection, including increased Mtb burden, extra-pulmonary dissemination, and heightened mortality. These disease states were not due to chronic virus-induced immunosuppression or exhaustion; rather, increased amounts of the cytokine TNFα initially arrested pulmonary Mtb growth, impeding dendritic cell mediated antigen transportation to the lymph node and subverting immune-surveillance, allowing bacterial sanctuary. The cryptic Mtb replication delayed CD4 T cell priming, redirecting T helper (Th) 1 toward Th17 differentiation and increasing pulmonary neutrophilia, which diminished long-term survival. Temporally restoring CD4 T cell induction overcame these diverse disease sequelae to enhance Mtb control. Thus, Mtb co-opts TNFα from the chronic inflammatory environment to subvert immune-surveillance, avert early immune function, and foster long-term coinfection.

Pembrolizumab plus chemotherapy versus chemotherapy in untreated advanced pleural mesothelioma in Canada, Italy, and France: a phase 3, open-label, randomised controlled trial.

BACKGROUND: Pleural mesothelioma usually presents at an advanced, incurable stage. Chemotherapy with platinum-pemetrexed is a standard treatment. We hypothesised that the addition of pembrolizumab to platinum-pemetrexed would improve overall survival in patients with pleural mesothelioma. METHODS: We did this open-label, international, randomised phase 3 trial at 51 hospitals in Canada, Italy, and France. Eligible participants were aged 18 years or older, with previously untreated advanced pleural mesothelioma, with an Eastern Cooperative Oncology Group performance status score of 0 or 1. Patients were randomly assigned (1:1) to intravenous chemotherapy (cisplatin [75 mg/m2] or carboplatin [area under the concentration-time curve 5-6 mg/mL per min] with pemetrexed 500 mg/m2, every 3 weeks for up to 6 cycles), with or without intravenous pembrolizumab 200 mg every 3 weeks (up to 2 years). The primary endpoint was overall survival in all randomly assigned patients; safety was assessed in all randomly assigned patients who received at least one dose of study therapy. This trial is registered with ClinicalTrials.gov, NCT02784171, and is closed to accrual. FINDINGS: Between Jan 31, 2017, and Sept 4, 2020, 440 patients were enrolled and randomly assigned to chemotherapy alone (n=218) or chemotherapy with pembrolizumab (n=222). 333 (76 %) of patients were male, 347 (79%) were White, and median age was 71 years (IQR 66-75). At final analysis (database lock Dec 15, 2022), with a median follow-up of 16·2 months (IQR 8·3-27·8), overall survival was significantly longer with pembrolizumab (median overall survival 17·3 months [95% CI 14·4-21·3] with pembrolizumab vs 16·1 months [13·1-18·2] with chemotherapy alone, hazard ratio for death 0·79; 95% CI 0·64-0·98, two-sided p=0·0324). 3-year overall survival rate was 25% (95% CI 20-33%) with pembrolizumab and 17% (13-24%) with chemotherapy alone. Adverse events related to study treatment of grade 3 or 4 occurred in 60 (27%) of 222 patients in the pembrolizumab group and 32 (15%) of 211 patients in the chemotherapy alone group. Hospital admissions for serious adverse events related to one or more study drugs were reported in 40 (18%) of 222 patients in the pembrolizumab group and 12 (6%) of 211 patients in the chemotherapy alone group. Grade 5 adverse events related to one or more drugs occurred in two patients on the pembrolizumab group and one patient in the chemotherapy alone group. INTERPRETATION: In patients with advanced pleural mesothelioma, the addition of pembrolizumab to standard platinum-pemetrexed chemotherapy was tolerable and resulted in a significant improvement in overall survival. This regimen is a new treatment option for previously untreated advanced pleural mesothelioma. FUNDING: The Canadian Cancer Society and Merck & Co.

Canadian Multicentric Pan-TRK (CANTRK) Immunohistochemistry Harmonization Study.

Tumor-agnostic testing for NTRK1-3 gene rearrangements is required to identify patients who may benefit from TRK inhibitor therapies. The overarching objective of this study was to establish a high-quality pan-TRK immunohistochemistry (IHC) screening assay among 18 large regional pathology laboratories across Canada using pan-TRK monoclonal antibody clone EPR17341 in a ring study design. TRK-fusion positive and negative tumor samples were collected from participating sites, with fusion status confirmed by panel next-generation sequencing assays. Each laboratory received: (1) unstained sections from 30 cases of TRK-fusion-positive or -negative tumors, (2) 2 types of reference standards: TRK calibrator slides and IHC critical assay performance controls (iCAPCs), (3) EPR17341 antibody, and (4) suggestions for developing IHC protocols. Participants were asked to optimize the IHC protocol for their instruments and detection systems by using iCAPCs, to stain the 30 study cases, and to report the percentage scores for membranous, cytoplasmic, and nuclear staining. TRK calibrators were used to assess the analytical sensitivity of IHC protocols developed by using the 2 reference standards. Fifteen of 18 laboratories achieved diagnostic sensitivity of 100% against next-generation sequencing. The diagnostic specificity ranged from 40% to 90%. The results did not differ significantly between positive scores based on the presence of any type of staining vs the presence of overall staining in ≥1% of cells. The median limit of detection measured by TRK calibrators was 76,000 molecules/cell (range 38,000 to >200,000 molecules/cell). Three different patterns of staining were observed in 19 TRK-positive cases, cytoplasmic-only in 7 samples, nuclear and cytoplasmic in 9 samples, and cytoplasmic and membranous in 3 samples. The Canadian multicentric pan-TRK study illustrates a successful strategy to accelerate the multicenter harmonization and implementation of pan-TRK immunohistochemical screening that achieves high diagnostic sensitivity by using laboratory-developed tests where laboratories used centrally developed reference materials. The measurement of analytical sensitivity by using TRK calibrators provided additional insights into IHC protocol performance.

Multivalent assembly of KRAS with the RAS-binding and cysteine-rich domains of CRAF on the membrane.

Membrane anchoring of farnesylated KRAS is critical for activation of RAF kinases, yet our understanding of how these proteins interact on the membrane is limited to isolated domains. The RAS-binding domain (RBD) and cysteine-rich domain (CRD) of RAF engage KRAS and the plasma membrane, unleashing the kinase domain from autoinhibition. Due to experimental challenges, structural insight into this tripartite KRAS:RBD-CRD:membrane complex has relied on molecular dynamics simulations. Here, we report NMR studies of the KRAS:CRAF RBD-CRD complex. We found that the nucleotide-dependent KRAS-RBD interaction results in transient electrostatic interactions between KRAS and CRD, and we mapped the membrane interfaces of the CRD, RBD-CRD, and the KRAS:RBD-CRD complex. RBD-CRD exhibits dynamic interactions with the membrane through the canonical CRD lipid-binding site (CRD ß7-8), as well as an alternative interface comprising ß6 and the C terminus of CRD and ß2 of RBD. Upon complex formation with KRAS, two distinct states were observed by NMR: State A was stabilized by membrane association of CRD ß7-8 and KRAS α4-α5 while state B involved the C terminus of CRD, ß3-5 of RBD, and part of KRAS α5. Notably, α4-α5, which has been proposed to mediate KRAS dimerization, is accessible only in state B. A cancer-associated mutation on the state B membrane interface of CRAF RBD (E125K) stabilized state B and enhanced kinase activity and cellular MAPK signaling. These studies revealed a dynamic picture of the assembly of the KRAS-CRAF complex via multivalent and dynamic interactions between KRAS, CRAF RBD-CRD, and the membrane.

Integration of multiomic annotation data to prioritize and characterize inflammation and immune-related risk variants in squamous cell lung cancer.

Clinical trial results have recently demonstrated that inhibiting inflammation by targeting the interleukin-1ß pathway can offer a significant reduction in lung cancer incidence and mortality, highlighting a pressing and unmet need to understand the benefits of inflammation-focused lung cancer therapies at the genetic level. While numerous genome-wide association studies (GWAS) have explored the genetic etiology of lung cancer, there remains a large gap between the type of information that may be gleaned from an association study and the depth of understanding necessary to explain and drive translational findings. Thus, in this study we jointly model and integrate extensive multiomics data sources, utilizing a total of 40 genome-wide functional annotations that augment previously published results from the International Lung Cancer Consortium (ILCCO) GWAS, to prioritize and characterize single nucleotide polymorphisms (SNPs) that increase risk of squamous cell lung cancer through the inflammatory and immune responses. Our work bridges the gap between correlative analysis and translational follow-up research, refining GWAS association measures in an interpretable and systematic manner. In particular, reanalysis of the ILCCO data highlights the impact of highly associated SNPs from nuclear factor-κB signaling pathway genes as well as major histocompatibility complex mediated variation in immune responses. One consequence of prioritizing likely functional SNPs is the pruning of variants that might be selected for follow-up work by over an order of magnitude, from potentially tens of thousands to hundreds. The strategies we introduce provide informative and interpretable approaches for incorporating extensive genome-wide annotation data in analysis of genetic association studies.

A drug discovery platform to identify compounds that inhibit EGFR triple mutants.

Receptor tyrosine kinases (RTKs) are transmembrane receptors of great clinical interest due to their role in disease. Historically, therapeutics targeting RTKs have been identified using in vitro kinase assays. Due to frequent development of drug resistance, however, there is a need to identify more diverse compounds that inhibit mutated but not wild-type RTKs. Here, we describe MaMTH-DS (mammalian membrane two-hybrid drug screening), a live-cell platform for high-throughput identification of small molecules targeting functional protein-protein interactions of RTKs. We applied MaMTH-DS to an oncogenic epidermal growth factor receptor (EGFR) mutant resistant to the latest generation of clinically approved tyrosine kinase inhibitors (TKIs). We identified four mutant-specific compounds, including two that would not have been detected by conventional in vitro kinase assays. One of these targets mutant EGFR via a new mechanism of action, distinct from classical TKI inhibition. Our results demonstrate how MaMTH-DS is a powerful complement to traditional drug screening approaches.

A renewed model of pancreatic cancer evolution based on genomic rearrangement patterns.

Pancreatic cancer, a highly aggressive tumour type with uniformly poor prognosis, exemplifies the classically held view of stepwise cancer development. The current model of tumorigenesis, based on analyses of precursor lesions, termed pancreatic intraepithelial neoplasm (PanINs) lesions, makes two predictions: first, that pancreatic cancer develops through a particular sequence of genetic alterations (KRAS, followed by CDKN2A, then TP53 and SMAD4); and second, that the evolutionary trajectory of pancreatic cancer progression is gradual because each alteration is acquired independently. A shortcoming of this model is that clonally expanded precursor lesions do not always belong to the tumour lineage, indicating that the evolutionary trajectory of the tumour lineage and precursor lesions can be divergent. This prevailing model of tumorigenesis has contributed to the clinical notion that pancreatic cancer evolves slowly and presents at a late stage. However, the propensity for this disease to rapidly metastasize and the inability to improve patient outcomes, despite efforts aimed at early detection, suggest that pancreatic cancer progression is not gradual. Here, using newly developed informatics tools, we tracked changes in DNA copy number and their associated rearrangements in tumour-enriched genomes and found that pancreatic cancer tumorigenesis is neither gradual nor follows the accepted mutation order. Two-thirds of tumours harbour complex rearrangement patterns associated with mitotic errors, consistent with punctuated equilibrium as the principal evolutionary trajectory. In a subset of cases, the consequence of such errors is the simultaneous, rather than sequential, knockout of canonical preneoplastic genetic drivers that are likely to set-off invasive cancer growth. These findings challenge the current progression model of pancreatic cancer and provide insights into the mutational processes that give rise to these aggressive tumours.

Collagen Type XI Inhibits Lung Cancer-Associated Fibroblast Functions and Restrains the Integrin Binding Site Availability on Collagen Type I Matrix.

The tumor microenvironment, including cancer-associated fibroblast (CAF), plays an active role in non-small cell lung cancer (NSCLC) development and progression. We previously reported that collagen type XI and integrin α11, a collagen receptor, were upregulated in NSCLC; the latter promotes tumor growth and metastasis. We here explored the role of collagen type XI in NSCLC stroma. We showed that the presence of collagen type XI in collagen type I matrices inhibits CAF-mediated collagen remodeling and cell migration. This resulted in the inhibition of CAF-dependent lung-tumor cell invasion. Among the collagen receptors expressed on CAF, we determined that DDR2 and integrin α2ß1, but not integrin α11ß1, mediated the high-affinity binding to collagen type XI. We further demonstrated that collagen type XI restrained the integrin binding site availability on collagen type I matrices, thus limiting cell interaction with collagen type I. As a consequence, CAFs failed to activate FAK, p38 and Akt one hour after they interacted with collagen type I/XI. We concluded that collagen type XI may have a competitive negative feedback role on the binding of collagen type I to its receptors.

Surgery for malignant pleural mesothelioma after radiotherapy (SMART): final results from a single-centre, phase 2 trial.

BACKGROUND: A novel approach for managing malignant pleural mesothelioma, surgery for mesothelioma after radiotherapy (SMART), consisting of a short accelerated course of high-dose, hemithoracic, intensity modulated radiotherapy (IMRT) followed by extrapleural pneumonectomy was developed. The aim of this study was to evaluate the clinical feasibility of the SMART protocol. METHODS: In this single-centre, phase 2 trial, patients aged 18 years or older with an Eastern Cooperative Oncology Group performance status of 0-2, with histologically proven, resectable, cT1-3N0M0 disease who had previously untreated malignant pleural mesothelioma were eligible for inclusion. Patients received 25 Gy in five daily fractions over 1 week to the entire ipsilateral hemithorax with a concomitant 5 Gy boost to high risk areas followed by extrapleural pneumonectomy within 1 week. Adjuvant chemotherapy was offered to patients with ypN+ disease on final pathology. The primary endpoint was feasibility, which was defined as the number of patients with 30-day perioperative treatment-related death (grade 5 events) or morbidity (grade 3 or 4 events). A key secondary endpoint was cumulative incidence of distant recurrence. The final analysis was done on an intention-to-treat basis (including all eligible patients). This trial is registered with ClinicalTrials.gov, NCT00797719. FINDINGS: Between Nov 1, 2008, and Oct 31, 2019, 102 patients were enrolled onto the trial and 96 eligible patients were treated with SMART on protocol and included in the analysis. Extrapleural pneumonectomy was done at a median of 5 days (range 2-12) after completing IMRT. 47 (49%) patients had 30-day perioperative grade 3-4 events and one (1%) patient died within 30 days perioperatively (grade 5 event; pneumonia). After a median follow-up of 46·8 months (IQR 13·4-61·2), the 5-year cumulative incidence of distant recurrence was 62 (63·3% [95% CI 52·3-74·4]). The most common first sites of recurrence were the contralateral chest (33 [46%] of 72 patients) and the peritoneal cavity (32 [44%]). INTERPRETATION: Results from this study suggest that extrapleural pneumonectomy after radiotherapy can be done with good early and long-term results. However, minimising grade 4 events on the protocol is technically demanding and might affect survival beyond the post-operative period. FUNDING: Princess Margaret Hospital Foundation Mesothelioma Research Fund.

Refined RIP-seq protocol for epitranscriptome analysis with low input materials.

N6-Methyladenosine (m6A) accounts for approximately 0.2% to 0.6% of all adenosine in mammalian mRNA, representing the most abundant internal mRNA modifications. m6A RNA immunoprecipitation followed by high-throughput sequencing (MeRIP-seq) is a powerful technique to map the m6A location transcriptome-wide. However, this method typically requires 300 µg of total RNA, which limits its application to patient tumors. In this study, we present a refined m6A MeRIP-seq protocol and analysis pipeline that can be applied to profile low-input RNA samples from patient tumors. We optimized the key parameters of m6A MeRIP-seq, including the starting amount of RNA, RNA fragmentation, antibody selection, MeRIP washing/elution conditions, methods for RNA library construction, and the bioinformatics analysis pipeline. With the optimized immunoprecipitation (IP) conditions and a postamplification rRNA depletion strategy, we were able to profile the m6A epitranscriptome using 500 ng of total RNA. We identified approximately 12,000 m6A peaks with a high signal-to-noise (S/N) ratio from 2 lung adenocarcinoma (ADC) patient tumors. Through integrative analysis of the transcriptome, m6A epitranscriptome, and proteome data in the same patient tumors, we identified dynamics at the m6A level that account for the discordance between mRNA and protein levels in these tumors. The refined m6A MeRIP-seq method is suitable for m6A epitranscriptome profiling in a limited amount of patient tumors, setting the ground for unraveling the dynamics of the m6A epitranscriptome and the underlying mechanisms in clinical settings.

The role of FOXOs and autophagy in cancer and metastasis-Implications in therapeutic development.

Autophagy is a highly conserved intracellular degradation process that plays a crucial role in cell survival and stress reactions as well as in cancer development and metastasis. Autophagy process involves several steps including sequestration, fusion of autophagosomes with lysosomes and degradation. Forkhead box O (FOXO) transcription factors regulate the expression of genes involved in cellular metabolic activity and signaling pathways of cancer growth and metastasis. Recent evidence suggests that FOXO proteins are also involved in autophagy regulation. The relationship among FOXOs, autophagy, and cancer has been drawing attention of many who work in the field. This study summarizes the role of FOXO proteins and autophagy in cancer growth and metastasis and analyzes their potential roles in cancer disease management.

Amplifying Outcomes: Checkpoint Inhibitor Combinations in First-Line Non-Small Cell Lung Cancer.

PURPOSE: Lung cancer is one of the most common types of cancer, resulting in approximately 1.8 million deaths worldwide. Immunotherapy using checkpoint inhibitors has become standard of care in advanced non-small cell lung cancer (NSCLC), and there is increasing interest in further improving outcomes through combination with other therapeutics. This systematic review evaluates emerging phase III data on the efficacy and safety of checkpoint inhibitor combinations as first-line treatment for advanced NSCLC. MATERIALS AND METHODS: Published and presented literature was searched using the key search terms "non-small cell lung cancer" AND "checkpoint-inhibitors" (OR respective aliases) AND phase III trials. Seven randomized phase III clinical trials reporting outcomes on checkpoint inhibitor combinations in first-line advanced NSCLC were identified. RESULTS: Four first-line trials reported outcomes for checkpoint inhibitor combinations in nonsquamous NSCLC. Pembrolizumab-chemotherapy, atezolizumab-chemotherapy, and atezolizumab-bevacizumab-chemotherapy showed significantly improved overall survival compared with controls in patients with advanced nonsquamous epidermal growth factor receptor-negative (EGFR-)/ anaplastic lymphoma kinase gene (ALK)- NSCLC. Two trials reported outcomes for squamous NSCLC, with pembrolizumab-chemotherapy reporting significantly improved overall survival (OS) compared with chemotherapy. The combination of nivolumab-ipilimumab in all-comer histology failed to improve OS compared with histology appropriate chemotherapy in patients regardless of their tumor mutational burden status. Based on improved survival and safety, either pembrolizumab monotherapy or pembrolizumab-chemotherapy administered based on PD-L1 status and histology is a preferred treatment option. Outcomes for atezolizumab-bevacizumab-chemotherapy in EGFR+/ALK+ patients are promising and require further exploration. CONCLUSION: First-line checkpoint inhibitors added to standard therapies improve overall survival for nonsquamous EGFR-/ALK- and squamous advanced NSCLC. IMPLICATIONS FOR PRACTICE: Single-agent immune checkpoint inhibitors are now standard of care for advanced non-small cell lung cancer (NSCLC), and emerging data show that combining these agents with established chemotherapy further improves outcomes. The phase III KEYNOTE-189 and IMPower-130 trials showed significantly improved survival using this strategy for nonsquamous NSCLC, and the phase III KEYNOTE-407 trial showed similar results in squamous disease. Checkpoint inhibitor combinations are therefore an important new treatment option for first-line NSCLC. Programmed death ligand-1 expression may inform the use of checkpoint inhibitor combination therapy, and overall tumor mutation burden is also an emerging biomarker for this new treatment strategy.

Recapitulating pancreatic tumor microenvironment through synergistic use of patient organoids and organ-on-a-chip vasculature.

Tumor progression relies heavily on the interaction between the neoplastic epithelial cells and their surrounding stromal partners. This cell cross-talk affects stromal development, and ultimately the heterogeneity impacts drug efflux and efficacy. To mimic this evolving paradigm, we have micro-engineered a three-dimensional (3D) vascularized pancreatic adenocarcinoma tissue in a tri-culture system composed of patient derived pancreatic organoids, primary human fibroblasts and endothelial cells on a perfusable InVADE platform situated in a 96-well plate. Uniquely, through synergistic engineering we combined the benefits of cellular fidelity of patient tumor derived organoids with the addressability of a plastic organ-on-a-chip platform. Validation of this platform included demonstrating the growth of pancreatic tumor organoids by monitoring the change in metabolic activity of the tissue. Investigation of tumor microenvironmental behavior highlighted the role of fibroblasts in symbiosis with patient organoid cells, resulting in a six-fold increase of collagen deposition and a corresponding increase in tissue stiffness in comparison to fibroblast free controls. The value of a perfusable vascular network was evident in drug screening, as perfusion of gemcitabine into a stiffened matrix did not show the dose-dependent effects on tumor viability as those under static conditions. These findings demonstrate the importance of studying the dynamic synergistic relationship between patient cells with stromal fibroblasts, in a 3D perfused vascular network, to accurately understand and recapitulate the tumor microenvironment.

Malignant mesothelioma in situ: morphologic features and clinical outcome.

The existence of an in situ phase of malignant mesothelioma has long been postulated but until recently has been impossible to prove. Here we describe ten patients with mesothelioma in situ, defined by a single layer of surface mesothelial cells showing loss of BAP1 nuclear immunostaining, no evidence of tumor by imaging and/or by direct examination of the pleura/peritoneum, and no invasive mesothelioma developing for at least 1 year. Nine cases were pleural and one peritoneal. Most patients were biopsied for repeated effusions of unknown etiology; in two patients mesothelioma in situ was found incidentally in lung cancer resections. In addition to surface mesothelium with BAP1 loss, one case had a surface papillary proliferation with BAP1 loss, and two cases had a small (few millimeter) nodule with BAP1 loss. CDKN2A was deleted by FISH in one of eight cases. Methylthioadenosine phosphorylase showed partial loss in the surface mesothelium by immunohistochemistry in three cases. Invasive malignant mesothelioma developed in seven patients with time between biopsy and invasive disease from 12 to 92 (median 60) months. Invasive mesothelioma has not developed in the other three patients at 12, 57, and 120 months, but the latter patient, who has pleural plaques, still has repeated pleural effusions, probably representing a so-called "benign asbestos effusion." We conclude that mesothelioma in situ, as diagnosed using the criteria outlined above, is associated with a high risk of developing invasive mesothelioma, but typically over a relatively protracted time, so that curable interventions maybe possible.

Minimalist approaches to cancer tissue-of-origin classification by DNA methylation.

Classification of cancers by tissue-of-origin is fundamental to diagnostic pathology. While the combination of clinical data, tissue histology, and immunohistochemistry is usually sufficient, there remains a small but not insignificant proportion of difficult-to-classify cases. These challenging cases provide justification for ancillary molecular testing, including high-throughput DNA methylation array profiling, which promises cell-of-origin information and compatibility with formalin-fixed specimens. While diagnostically powerful, methylation profiling platforms are costly and technically challenging to implement, particularly for less well-resourced laboratories. To address this, we simulated the performance of "minimalist" methylation-based tests for cancer classification using publicly-available and internal institutional profiling data. These analyses showed that small and focused sets of the most informative CpG biomarkers from the arrays are sufficient for accurate diagnoses. As an illustrative example, one classifier, using information from just 53 out of about 450,000 available CpG probes, achieved an accuracy of 94.5% on 2575 fresh primary validation cases across 28 cancer types from The Cancer Genome Atlas Network. By training minimalist classifiers on formalin-fixed primary and metastatic cases, generally high accuracies were also achieved on additional datasets. These results support the potential of minimalist methylation testing, possibly via quantitative PCR and targeted next-generation sequencing platforms, in cancer classification.

Localized malignant mesothelioma, an unusual and poorly characterized neoplasm of serosal origin: best current evidence from the literature and the International Mesothelioma Panel.

Localized malignant mesotheliomas (LMM) is an uncommon and poorly recognized neoplasm. Its pathologic diagnosis is often surprising in patients with serosal/subserosal based localized tumors that are clinically suspicious for metastatic lesions or primary sarcomas. Once a tumor is diagnosed as "mesothelioma", LMM is often mistaken for diffuse malignant mesothelioma (DMM). Best currently available evidence about LMM was collected from the literature and cases diagnosed by members of the International Mesothelioma Panel (IMP). One hundred and one (101) LMM have been reported in the English literature. Patients had localized tumors with identical histopathologic features to DMM. Patients ranged in age from 6 to 82 years; 75% were men. Most (82%) of the tumors were intrathoracic. Others presented as intrahepatic, mesenteric, gastric, pancreatic, umbilical, splenic, and abdominal wall lesions. Tumors varied in size from 0.6 to 15 cm. Most patients underwent surgical resection and/or chemotherapy or radiation therapy. Median survival in a subset of patients was 29 months. Seventy two additional LMM from IMP institutions ranged in age from 28 to 95 years; 58.3% were men. Sixty tumors (83.3%) were intrathoracic, others presented in intraabdominal sites. Tumors varied in size from 1.2 to 19 cm. Median survival for 51 cases was 134 months. Best evidence was used to formulate guidelines for the diagnosis of LMM. It is important to distinguish LMM from DMM as their treatment and prognosis is different. A multidisciplinary approach is needed for the diagnosis of LMM as it shows identical histopathology and immunophenotype to DMM.

"Interchangeability" of PD-L1 immunohistochemistry assays: a meta-analysis of diagnostic accuracy.

Different clones, protocol conditions, instruments, and scoring/readout methods may pose challenges in introducing different PD-L1 assays for immunotherapy. The diagnostic accuracy of using different PD-L1 assays interchangeably for various purposes is unknown. The primary objective of this meta-analysis was to address PD-L1 assay interchangeability based on assay diagnostic accuracy for established clinical uses/purposes. A systematic search of the MEDLINE database using PubMed platform was conducted using "PD-L1" as a search term for 01/01/2015 to 31/08/2018, with limitations "English" and "human". 2,515 abstracts were reviewed to select for original contributions only. 57 studies on comparison of two or more PD-L1 assays were fully reviewed. 22 publications were selected for meta-analysis. Additional data were requested from authors of 20/22 studies in order to enable the meta-analysis. Modified GRADE and QUADAS-2 criteria were used for grading published evidence and designing data abstraction templates for extraction by reviewers. PRISMA was used to guide reporting of systematic review and meta-analysis and STARD 2015 for reporting diagnostic accuracy study. CLSI EP12-A2 was used to guide test comparisons. Data were pooled using random-effects model. The main outcome measure was diagnostic accuracy of various PD-L1 assays. The 22 included studies provided 376 2×2 contingency tables for analyses. Results of our study suggest that, when the testing laboratory is not able to use an Food and Drug Administration-approved companion diagnostic(s) for PD-L1 assessment for its specific clinical purpose(s), it is better to develop a properly validated laboratory developed test for the same purpose(s) as the original PD-L1 Food and Drug Administration-approved immunohistochemistry companion diagnostic, than to replace the original PD-L1 Food and Drug Administration-approved immunohistochemistry companion diagnostic with a another PD-L1 Food and Drug Administration-approved companion diagnostic that was developed for a different purpose.

A phase Ib study of a PI3Kinase inhibitor BKM120 in combination with panitumumab in patients with KRAS wild-type advanced colorectal cancer.

Background Resistance to Epidermal Growth Factor inhibition (EGFRi) in patients with KRAS wild-type (wt) Colorectal Cancer (CRC) may occur as a result of PI3K/AKT/mTOR signaling. We conducted a study to establish the recommended phase II dose (RP2D) and response rate of panitumumab, an EGFRi, plus BKM120, a PI3K inhibitor, in advanced CRC. Methods Patients with chemotherapy refractory KRAS wt CRC, who were EGFRi naive were enrolled. A 3 + 3 dose escalation design was utilized. The starting dose of panitumumab was 6 mg/kg iv every 2 weeks with BKM120 at 60 mg oral daily. Results Nineteen patients were treated and 17 were evaluable for response. The starting dose was not tolerable (mucositis, fatigue). At dose level (DL) 1, three of six patients discontinued treatment due to toxicity, DL - 1 had no significant toxicity. Panitumumab 6 mg/kg iv q 2 weeks with BKM120 60 mg given 5 out of 7 days per week was declared the RP2D. One patient (5.9%) who was PTEN and PIK3CA negative by IHC had a partial response, seven had stable disease, and nine had disease progression. Conclusion Panitumumab (6 mg/kg iv q 2 weeks) with BKM120 60 mg given 5 out of 7 days per week was declared the RP2D. Toxicities including fatigue, rash and mucositis. There was little evidence of activity in this biomarker unselected cohort.

Whole genomes define concordance of matched primary, xenograft, and organoid models of pancreas cancer.

Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis among solid malignancies and improved therapeutic strategies are needed to improve outcomes. Patient-derived xenografts (PDX) and patient-derived organoids (PDO) serve as promising tools to identify new drugs with therapeutic potential in PDAC. For these preclinical disease models to be effective, they should both recapitulate the molecular heterogeneity of PDAC and validate patient-specific therapeutic sensitivities. To date however, deep characterization of the molecular heterogeneity of PDAC PDX and PDO models and comparison with matched human tumour remains largely unaddressed at the whole genome level. We conducted a comprehensive assessment of the genetic landscape of 16 whole-genome pairs of tumours and matched PDX, from primary PDAC and liver metastasis, including a unique cohort of 5 'trios' of matched primary tumour, PDX, and PDO. We developed a pipeline to score concordance between PDAC models and their paired human tumours for genomic events, including mutations, structural variations, and copy number variations. Tumour-model comparisons of mutations displayed single-gene concordance across major PDAC driver genes, but relatively poor agreement across the greater mutational load. Genome-wide and chromosome-centric analysis of structural variation (SV) events highlights previously unrecognized concordance across chromosomes that demonstrate clustered SV events. We found that polyploidy presented a major challenge when assessing copy number changes; however, ploidy-corrected copy number states suggest good agreement between donor-model pairs. Collectively, our investigations highlight that while PDXs and PDOs may serve as tractable and transplantable systems for probing the molecular properties of PDAC, these models may best serve selective analyses across different levels of genomic complexity.