Predicting programmed death-ligand 1 (PD-L1) expression with fluorine-18 fluorodeoxyglucose ([18F]FDG) positron emission tomography/computed tomography (PET/CT) metabolic parameters in resectable non-small cell lung cancer.

BACKGROUND: Programmed death-ligand 1 (PD-L1) expression is a predictive biomarker for immunotherapy in non-small cell lung cancer (NSCLC). PD-L1 and glucose transporter 1 expression are closely associated, and studies demonstrate correlation of PD-L1 with glucose metabolism. AIM: The aim of this study was to investigate the association of fluorine-18 fluorodeoxyglucose positron emission tomography/computed tomography ([18F]FDG-PET/CT) metabolic parameters with PD-L1 expression in primary lung tumour and lymph node metastases in resected NSCLC. METHODS: We conducted a retrospective analysis of 210 patients with node-positive resectable stage IIB-IIIB NSCLC. PD-L1 tumour proportion score (TPS) was determined using the DAKO 22C3 immunohistochemical assay. Semi-automated techniques were used to analyse pre-operative [18F]FDG-PET/CT images to determine primary and nodal metabolic parameter scores (including max, mean, peak and peak adjusted for lean body mass standardised uptake values (SUV), metabolic tumour volume (MTV), total lesional glycolysis (TLG) and SUV heterogeneity index (HISUV)). RESULTS: Patients were predominantly male (57%), median age 70 years with non-squamous NSCLC (68%). A majority had negative primary tumour PD-L1 (TPS < 1%; 53%). Mean SUVmax, SUVmean, SUVpeak and SULpeak values were significantly higher (p < 0.05) in those with TPS ≥ 1% in primary tumour (n = 210) or lymph nodes (n = 91). However, ROC analysis demonstrated only moderate separability at the 1% PD-L1 TPS threshold (AUCs 0.58-0.73). There was no association of MTV, TLG and HISUV with PD-L1 TPS. CONCLUSION: This study demonstrated the association of SUV-based [18F]FDG-PET/CT metabolic parameters with PD-L1 expression in primary tumour or lymph node metastasis in resectable NSCLC, but with poor sensitivity and specificity for predicting PD-L1 positivity ≥ 1%. CLINICAL RELEVANCE STATEMENT: Whilst SUV-based fluorine-18 fluorodeoxyglucose positron emission tomography/computed tomography metabolic parameters may not predict programmed death-ligand 1 positivity ≥ 1% in the primary tumour and lymph nodes of resectable non-small cell lung cancer independently, there is a clear association which warrants further investigation in prospective studies. TRIAL REGISTRATION: Non-applicable KEY POINTS: • Programmed death-ligand 1 immunohistochemistry has a predictive role in non-small cell lung cancer immunotherapy; however, it is both heterogenous and dynamic. • SUV-based fluorine-18 fluorodeoxyglucose positron emission tomography/computed tomography ([18F]FDG-PET/CT) metabolic parameters were significantly higher in primary tumour or lymph node metastases with positive programmed death-ligand 1 expression. • These SUV-based parameters could potentially play an additive role along with other multi-modal biomarkers in selecting patients within a predictive nomogram.

Global Ring Study to Investigate the Comparability of Total Assay Performance of Commercial Claudin 18 Antibodies for Evaluation in Gastric Cancer.

Claudin 18.2 (CLDN18.2), the dominant isoform of CLDN18 in gastric tissues, is a highly specific tight junction protein of the gastric mucosa with variably retained expressions in gastric and gastroesophageal junction cancers. Additionally, CLDN18.2-targeted treatment with zolbetuximab, in combination with chemotherapy, has recently been assessed in 2 phase-III studies of patients with HER2-negative, locally advanced, unresectable, or metastatic gastric or gastroesophageal junction adenocarcinoma. These trials used the investigational VENTANA CLDN18 (43-14A) RxDx immunohistochemistry (IHC) assay on the Ventana BenchMark platform to identify patients eligible for CLDN18.2-targeted treatment. We report the findings of a global ring study evaluating the analytical comparability of concordance of the results of 3 CLDN18 antibodies (Ventana, LSBio, and Novus) stained on 3 IHC-staining platforms (Ventana, Dako, and Leica). A tissue microarray (TMA), comprising 15 gastric cancer cases, was stained by 27 laboratories across 11 countries. Each laboratory stained the TMAs using at least 2 of the 3 evaluated CLDN18 antibodies. Stained TMAs were assessed and scored using an agreed IHC-scoring algorithm, and the results were collated for statistical analysis. The data confirmed a high level of concordance for the VENTANA CLDN18 (43-14A; Ventana platform only) and LSBio antibodies on both the Dako and Leica platforms, with accuracy, precision, sensitivity, and specificity rates all reaching a minimum acceptable ≥85% threshold and good-to-excellent levels of concordance as measured by Cohen's kappa coefficient. The Novus antibody showed the highest level of variability against the reference central laboratory results for the same antibody/platform combinations. It also failed to meet the threshold for accuracy and sensitivity when used on either the Dako or Leica platform. These results demonstrated the reliability of IHC testing for CLDN18 expression in gastric tumor samples when using commercially available platforms with an appropriate methodology and primary antibody selection.

Adenoid cystic carcinoma of the thymus gland.

BACKGROUND: Thymic carcinomas are rare and aggressive tumours. They constitute a heterogeneous group of tumours with various histological patterns and subtypes resembling epithelial tumours arising from other organs. CASE PRESENTATION: We hereby represent a case of primary thymic carcinoma with adenoid cystic carcinoma-like features (TCACC) which is an extremely rare variant of thymic adenocarcinoma. To date and to the best of our knowledge, there are nine reported cases in literature and ours is the tenth. Our case was treated surgically but the implementation of adjuvant chemoradiotherapy has been reported in few of the published cases. CONCLUSIONS: TCACC constitutes a rare entity of thymic adenocarcinoma with limited available literature. The current data is derived from few case reports and case series. The histological overlap of these tumours and primary ACC of salivary glands poses a diagnostic challenge. Radiological investigations, immunohistochemical phenotyping and genetic analysis are crucial in establishing the diagnosis.

MHC Class II is Induced by IFNγ and Follows Three Distinct Patterns of Expression in Colorectal Cancer Organoids.

Tumor-specific MHC class II (tsMHC-II) expression impacts tumor microenvironmental immunity. tsMHC-II positive cancer cells may act as surrogate antigen-presenting cells and targets for CD4+ T cell-mediated lysis. In colorectal cancer, tsMHC-II negativity is common, in cell lines due to CIITA promoter methylation. To clarify mechanisms of tsMHC-II repression in colorectal cancer, we analyzed colorectal cancer organoids which are epigenetically faithful to tissue of origin. 15 primary colorectal cancer organoids were treated with IFNγ ± epigenetic modifiers: flow cytometry was used for tsMHC-II expression. qRT-PCR, total RNA sequencing, nanopore sequencing, bisulfite conversion/pyrosequencing, and Western blotting was used to quantitate CIITA, STAT1, IRF1, and JAK1 expression, mutations and promoter methylation and chromatin immunoprecipitation to quantitate H3K9ac, H3K9Me2, and EZH2 occupancy at CIITA. We define three types of response to IFNγ in colorectal cancer: strong, weak, and noninducibility. Delayed and restricted expression even with prolonged IFNγ exposure was due to IFNγ-mediated EZH2 occupancy at CIITA. tsMHC-II expression was enhanced by EZH2 and histone deacetylase inhibition in the weakly inducible organoids. Noninducibility is seen in three consensus molecular subtype 1 (CMS1) organoids due to JAK1 mutation. No organoid demonstrates CIITA promoter methylation. Providing IFNγ signaling is intact, most colorectal cancer organoids are class II inducible. Upregulation of tsMHC-II through targeted epigenetic therapy is seen in one of fifteen organoids. Our approach can serve as a blueprint for investigating the heterogeneity of specific epigenetic mechanisms of immune suppression across individual patients in other cancers and how these might be targeted to inform the conduct of future trials of epigenetic therapies as immune adjuvants more strategically in cancer. Significance: Cancer cell expression of MHC class II significantly impacts tumor microenvironmental immunity. Previous studies investigating mechanisms of repression of IFNγ-inducible class II expression using cell lines demonstrate epigenetic silencing of IFN pathway genes as a frequent immune evasion strategy. Unlike cell lines, patient-derived organoids maintain epigenetic fidelity to tissue of origin. In the first such study, we analyze patterns, dynamics, and epigenetic control of IFNγ-induced class II expression in a series of colorectal cancer organoids.

PD-L1 Testing in Urothelial Carcinoma: Analysis of a Series of 1401 Cases Using Both the 22C3 and SP142 Assays.

Immune checkpoint blockade (ICB) drugs are a novel, effective treatment for advanced urothelial carcinoma. Worldwide, several different ICB drugs are approved, each developed and clinically validated with a specific PD-L1 compound diagnostic assay. As a result, PD-L1 testing workflows in routine practice are complex: requiring multiple assays across two platforms, with each assay having a different method of interpretation. Our service tested 1,401 urothelial carcinoma cases for PD-L1 expression, using both the 22C3 PharmDx assay (required prior to Pembrolizumab therapy) and SP142 assay (required prior to Atezolizumab therapy). Of the 1,401 cases tested, 621 cases (44%) were tested with both the 22C3 PharmDx and SP142 assays, 492 cases (35%) with 22C3 PharmDx only, and 288 cases (21%) with SP142 only. Each assay was used and interpreted according to the manufacturer's guidelines. The rate of positivity we observed was 26% with the 22C3 assay and 31% with the SP142 assay, similar to the pre-licensing studies for both drugs. The discrepancy observed between the assays was 11%, which reinforces the requirement for utilisation of the correct assay for each agent, and limits potential cross-utility of assays. This aspect must be considered when setting up a PD-L1 testing strategy in laboratories where both Pembrolizumab and Atezolizumab are available for the treatment of urothelial carcinoma but also has broader implications for testing of other cancers where multiple ICB drugs and their respective assays are approved.

TMP3-NTRK1 rearranged uterine sarcoma: A case report.

INTRODUCTION: Uterine sarcomas are a group of rare tumours with heterogeneous morphological and genetic features. Recent advances in the molecular characterisation of these tumours have identified a novel clinicopathological category underpinned by NTRK gene fusions. CASE REPORT: We present the case of a 42-year-old woman with a polypoid cervical lesion formed of densely cellular, short, haphazard fascicles of monomorphic spindle cells that lacked coagulative necrosis and which showed high mitotic activity. On immunohistochemistry, the tumour was diffusely positive for pan-Trk and weakly positive for CD34 but was negative for a range of other markers, including cytokeratins, smooth muscle markers, hormone receptors and S100. FISH analysis using a NTRK1 break-apart probe was above the threshold for translocation positivity and subsequent next-generation sequencing (NGS) identified a TPM3-NTRK1 fusion. DISCUSSION: NTRK-rearranged uterine sarcomas are a novel subset of gynaecological mesenchymal neoplasms characterised by cytological isomorphism and fibrosarcoma-like morphology. Although distinction from more common mesenchymal neoplasms is possible on the basis of morphology and immunohistochemistry, exclusion of rare differential diagnoses, such as malignant peripheral nerve sheath tumour or the recently described COL1A1-PDGFB fusion sarcoma, requires molecular work-up with FISH or NGS. Identification of these rare tumours is clinically relevant because of their cervical location and the possible role for tropomyosin receptor kinase inhibitors in their treatment.

A review of retroperitoneal liposarcoma genomics.

Retroperitoneal liposarcomas are rare tumours that carry a poorer prognosis than their extremity counterparts. Within their subtypes - well differentiated (WDL), dedifferentiated (DDL), myxoid (MLS) and pleomorphic (PLS) - they exhibit a diverse genomic landscape. With recent advances in next generation sequencing, the number of studies exploring this have greatly increased. The recent literature has deepened our understanding of the hallmark MDM2/CDK4 amplification in WDL/DDL and addressed concerns about toxicity and resistance when targeting this. The FUS-DDIT3 fusion gene remains the primary focus of interest in MLS with additional potential targets described. Whole genome sequencing has driven identification of novel genes and pathways implicated in WDL/DDL outside of the classic 12q13-15 amplicon. Due to their rarity; anatomical location and histologic subtype are infrequently mentioned when reporting the results of these studies. Reports can include non-adipogenic or extremity tumours, making it difficult to draw specific retroperitoneal conclusions. This narrative review aims to provide a summary of retroperitoneal liposarcoma genomics and the implications for therapeutic targeting.

A novel next generation sequencing approach to improve sarcoma diagnosis.

Sarcoma is a rare disease affecting both bone and connective tissue and with over 100 pathologic entities, differential diagnosis can be difficult. Complementing immune-histological diagnosis with current ancillary diagnostic techniques, including FISH and RT-PCR, can lead to inconclusive results in a significant number of cases. We describe here the design and validation of a novel sequencing tool to improve sarcoma diagnosis. A NGS DNA capture panel containing probes for 87 fusion genes and 7 genes with frequent copy number changes was designed and optimized. A cohort of 113 DNA samples extracted from soft-tissue and bone sarcoma FFPE material with clinical FISH and/or RT-PCR results positive for either a translocation or gene amplification was used for validation of the NGS method. Sarcoma-specific translocations or gene amplifications were confirmed in 110 out of 113 cases using FISH and/or RT-PCR as gold-standard. MDM2/CDK4 amplification and a total of 25 distinct fusion genes were identified in this cohort of patients using the NGS approach. Overall, the sensitivity of the NGS panel is 97% with a specificity of 100 and 0% failure rate. Targeted NGS appears to be a feasible and cost-effective approach to improve sarcoma subtype diagnosis with the ability to screen for a wide range of genetic aberrations in one test.

The Clinicopathological and Molecular Associations of PD-L1 Expression in Non-small Cell Lung Cancer: Analysis of a Series of 10,005 Cases Tested with the 22C3 Assay.

PD-L1 expression testing is mandatory prior to pembrolizumab prescription in non-small cell lung cancer. Our service offers PD-L1 testing using the PD-L1 IHC 22C3 pharmDx assay, in parallel with EGFR, ALK, ROS1 and (in some cases) KRAS testing. We correlate PD-L1 expression in 10,005 tumours with patient age and sex, with tumour histological subtypes, with the sampling modality and type of tissue, and with the presence of other molecular alterations. PD-L1 expression testing was performed using the aforementioned assay; tumour proportion scores (TPS) of 1 and 50% were taken as cut-offs for low and high positivity, respectively. EGFR testing was performed using the cobas® EGFR Mutation Test v2. ALK testing was performed using the VENTANA ALK (D5F3) CDx Assay. KRAS testing was performed using pyrosequencing. TPS <1% was seen in 44.4% of tumours, 1-49% in 25.0% and ≥ 50% in 30.6%. We identified no significant relationship with age. Female patients were slightly more likely to express PD-L1. Poorly-differentiated tumour histology and ALK translocation were significantly associated with PD-L1 expression. Rare EGFR mutations tended to be associated with PD-L1 expression. Pleural and nodal metastases were more likely to express PD-L1 than primary tumours, but biopsy and cytological specimens did not show different PD-L1 expression rates. Our data show that the means of acquiring a tumour sample (biopsy versus cytology) does not have a significant impact on PD-L1 expression. However, we found that certain metastatic sites were associated with significantly higher expression rates, which has substantial implications for selection of tissue for testing.

Large-Scale EGFR Mutation Testing in Clinical Practice: Analysis of a Series of 18,920 Non-Small Cell Lung Cancer Cases.

We make use of a very large dataset of non-small cell lung cancer specimens to examine the molecular epidemiology of EGFR mutations, particularly with respect to rare and compound mutations, and to non-adenocarcinoma histological subtypes. We also demonstrate the feasibility of large-scale EGFR mutation screening using the full range of specimens encountered in routine practice. We retrospectively reviewed 18,920 unselected EGFR mutation results from our centre between July 2009 and October 2016, using Qiagen's therascreen EGFR RGQ PCR Kit. Mutation rates were correlated with patient demographics and tumour histology. Our testing success rate was 93.9%, with similar success rates using histological and cytological specimens. Rare, potentially-targetable mutations accounted for 9.5% of all mutations detected. We identified a 2.5% mutation rate in tumours diagnosed as squamous cell carcinomas. There was a trend towards increasing EGFR mutation rates with increasing age, and while Del19 was the commonest mutation in the young, L858R predominated in the elderly. We found that EGFR mutation heterogeneity is rare within tumours and between primary and metastatic deposits. Our data demonstrate that large-scale, reflex EGFR mutation testing is feasible and affordable in the context of a publicly-funded health system. Furthermore, we have shown that the use of techniques sensitive only to classical mutations and selection of patients on the grounds of age, sex and histology denies patients access to potentially beneficial TKI therapy.

Gastrointestinal Stromal Tumor With Multiple Primary Tyrosine Kinase Mutations-Clinicopathologic and Molecular Characterization.

A unique cohort of chemo-naive gastrointestinal stromal tumors (GISTs) with double-primary tyrosine kinase mutations was characterized particularly to determine whether coexistent mutations represent a single mutational event. Up to 2013, 4 UK centers reported 9 GISTs with 2 primary tyrosine kinase mutations. In each of 8 cases validated by next generation sequencing, both mutations were present in the same allele of the same exon (KIT exon 11 or 17, or PDGFRA exon 18). One case showed the second mutation only on some of the mutant alleles. Seven cases showed both mutations in all the reads, but in 2 cases, additional variants were found only in some reads. Clinicopathologic features of the 8 cases were similar to GISTs with single-primary mutations. When GIST genotyping rarely uncovers multiple tyrosine kinase variants in an exon, they occur in the same allele but are likely to represent separate mutational events and lack clinical significance.

Predictive markers for anti-PD-1/PD-L1 therapy in non-small cell lung cancer-where are we?

The selection of patients for immunotherapy remains challenging given the lack of highly specific and highly sensitive biomarkers. Nevertheless, it is essential that testing laboratories are able to fulfil licencing criteria by providing the tests which have been validated as providing useful predictive information. Programmed cell death protein 1 (PD-1) expression assessment is now established in routine practice, although the situation regarding the selection of a particular assay remains complex, and testing protocols are likely to change in future. It is probable that programmed death-ligand 1 (PD-L1) expression assessment will be supplemented in the near future by tumour mutation burden (TMB), but this will require novel solutions to allow testing to be completed using small tissue samples and within narrow timeframes. While DNA mismatch repair (MMR) testing and CD8 T-cell density may also have a role to play in predicting immunotherapy response, their roles are not well-defined at present. Above all, the main challenge facing laboratories will be to perform this multitude of tests alongside the molecular markers already established in clinical practice [e.g., epidermal growth factor receptor (EGFR) mutation, anaplastic lymphoma kinase (ALK) translocation, ROS proto-oncogene 1 (ROS1) translocation]; the challenge for pathologists and clinicians will be to develop algorithms which will integrate the complex set of results from these tests and provide clinically-useful management regimens.

HER2 testing of gastro-oesophageal adenocarcinoma: a commentary and guidance document from the Association of Clinical Pathologists Molecular Pathology and Diagnostics Committee.

The use of biologics targeted to the human epidermal growth factor receptor 2 (HER2) protein is the latest addition to the armamentarium used to fight advanced gastric or gastro-oesophageal junction adenocarcinoma. The decision to treat with the biologic trastuzumab is completely dependent on HER2 testing of tumour tissue. In 2017, the College of American Pathologists, American Society for Clinical Pathology and the American Society of Clinical Oncology jointly published guidelines for HER2 testing and clinical decision making in gastro-oesophageal adenocarcinoma. The Association of Clinical Pathologists Molecular Pathology and Diagnostics Committee has issued the following document as a commentary of these guidelines and, in parallel, to provide guidance on HER2 testing in National Health Service pathology departments within the UK. This guidance covers issues related to case selection, preanalytical aspects, analysis and interpretation of such HER2 testing.

KRAS Mutation and Consensus Molecular Subtypes 2 and 3 Are Independently Associated with Reduced Immune Infiltration and Reactivity in Colorectal Cancer.

Purpose:KRAS mutation is a common canonical mutation in colorectal cancer, found at differing frequencies in all consensus molecular subtypes (CMS). The independent immunobiological impacts of RAS mutation and CMS are unknown. Thus, we explored the immunobiological effects of KRAS mutation across the CMS spectrum.Experimental Design: Expression analysis of immune genes/signatures was performed using The Cancer Genome Atlas (TCGA) RNA-seq and the KFSYSCC microarray datasets. Multivariate analysis included KRAS status, CMS, tumor location, MSI status, and neoantigen load. Protein expression of STAT1, HLA-class II, and CXCL10 was analyzed by digital IHC.Results: The Th1-centric co-ordinate immune response cluster (CIRC) was significantly, albeit modestly, reduced in KRAS-mutant colorectal cancer in both datasets. Cytotoxic T cells, neutrophils, and the IFNγ pathway were suppressed in KRAS-mutant samples. The expressions of STAT1 and CXCL10 were reduced at the mRNA and protein levels. In multivariate analysis, KRAS mutation, CMS2, and CMS3 were independently predictive of reduced CIRC expression. Immune response was heterogeneous across KRAS-mutant colorectal cancer: KRAS-mutant CMS2 samples have the lowest CIRC expression, reduced expression of the IFNγ pathway, STAT1 and CXCL10, and reduced infiltration of cytotoxic cells and neutrophils relative to CMS1 and CMS4 and to KRAS wild-type CMS2 samples in the TCGA. These trends held in the KFSYSCC dataset.Conclusions:KRAS mutation is associated with suppressed Th1/cytotoxic immunity in colorectal cancer, the extent of the effect being modulated by CMS subtype. These results add a novel immunobiological dimension to the biological heterogeneity of colorectal cancer. Clin Cancer Res; 24(1); 224-33. ©2017 AACR.

Endothelial protein C receptor is overexpressed in colorectal cancer as a result of amplification and hypomethylation of chromosome 20q.

Endothelial Protein C Receptor (EPCR) is a Major Histocompatibility Complex homologue, with established roles downregulating coagulation and in endothelial protection. Expressed predominantly on endothelium, EPCR affects inflammatory, apoptotic and cell proliferation pathways by binding to activated protein C (APC). However, EPCR can also be expressed on cancer cells, although the underlying reasons are unclear. Moreover, although EPCR has been linked with chemosensitivity in lung cancer, its clinical significance in many tumours is unknown. Here, we explored its significance in colorectal cancer (CRC). Bioinformatic methods revealed EPCR overexpression in many epithelial cancers, which was confirmed on CRC epithelial tumour cells by immunohistochemistry. EPCR upregulation resulted from gene amplification and DNA hypomethylation, and occurred in concert with a cohort of neighbouring genes on chromosome 20q, a region previously implicated in chemoresistance. As in endothelial cells, EPCR reproducibly mediated ERK pathway activation in a model CRC cell line following APC treatment. However, EPCR knockdown studies failed to highlight compelling EPCR-intrinsic impact on CRC cell phenotype, with limited effects on chemosensitivity and no effect on invasion observed, while EPCR appeared to decrease CRC cell migration. Consistent with these observations, differential EPCR expression did not influence response to chemotherapy in a human CRC cohort. Our results provide a compelling explanation for how EPCR is upregulated in diverse epithelial malignancies. They indicate that the clinical significance of EPCR varies across different tumour types. Furthermore, they raise the possibility that the prognostic significance of EPCR in certain tumours relates significantly to co-upregulation of neighbouring genes on chromosome 20q. Therefore, efforts to exploit EPCR as a prognostic marker should be focussed on specific tumours, and in such scenarios EPCR-co-dysregulated genes may represent potential axes for therapeutic intervention.

Microscopic gastrointestinal stromal tumours: a clinical and molecular study of 13 cases.

AIMS: Recent literature suggests that clinically silent, microscopic gastrointestinal stromal tumours (micro-GISTs) are common incidental findings. The aim of this study was to examine the histological, immunohistochemical and molecular characteristics of these tumours, which we have defined as measuring ≤20 mm, in order to determine whether the rate and spectrum of mutations are similar to those of clinically symptomatic gastrointestinal stromal tumours (GISTs). METHODS AND RESULTS: Thirteen micro-GISTs identified as incidental findings in patients undergoing management of concomitant disease were tested for KIT/PDGFRA mutations. Ten micro-GISTs (77%) were located in the stomach, two (15%) in the duodenum, and one (8%) in the rectum. The mean tumour size was 9.3 mm (range 2-19 mm). All tumours were well-circumscribed lesions showing a predominantly spindle-cell morphology and a very low mitotic rate. Twelve of 13 (92%) tumours carried mutations in either KIT (83%) or PDGFRA (17%), a rate higher than in other published series. A high mutation rate (80%) was also seen in lesions measuring ≤5 mm. CONCLUSIONS: Our results suggest that KIT/PDGFRA mutation is a very common early event in GIST development, that tumour size does not reliably predict the presence of mutation, and that one or more subsequent mutations are required for clinical manifestation.

Genomic complexity of urothelial bladder cancer revealed in urinary cfDNA.

Urothelial bladder cancers (UBCs) have heterogeneous clinical characteristics that are mirrored in their diverse genomic profiles. Genomic profiling of UBCs has the potential to benefit routine clinical practice by providing prognostic utility above and beyond conventional clinicopathological factors, and allowing for prediction and surveillance of treatment responses. Urinary DNAs representative of the tumour genome provide a promising resource as a liquid biopsy for non-invasive genomic profiling of UBCs. We compared the genomic profiles of urinary cellular DNA and cell-free DNA (cfDNA) from the urine with matched diagnostic formalin-fixed paraffin-embedded tumour DNAs for 23 well-characterised UBC patients. Our data show urinary DNAs to be highly representative of patient tumours, allowing for detection of recurrent clinically actionable genomic aberrations. Furthermore, a greater aberrant load (indicative of tumour genome) was observed in cfDNA over cellular DNA (P<0.001), resulting in a higher analytical sensitivity for detection of clinically actionable genomic aberrations (P<0.04) when using cfDNA. Thus, cfDNA extracted from the urine of UBC patients has a higher tumour genome burden and allows greater detection of key genomic biomarkers (90%) than cellular DNA from urine (61%) and provides a promising resource for robust whole-genome tumour profiling of UBC with potential to influence clinical decisions without invasive patient interventions.

KIT Mutation and Loss of 14q May Be Sufficient for the Development of Clinically Symptomatic Very Low-Risk GIST.

The aim of this study was to determine the minimal set of genetic alterations required for the development of a very low risk clinically symptomatic gastro-intestinal stromal tumour within the stomach wall. We studied the genome of a very low-risk gastric gastro-intestinal stromal tumour by whole-genome sequencing, comparative genomic hybridisation and methylation profiling. The studied tumour harboured two typical genomic lesions: loss of the long arm of chromosome 14 and an activating mutation in exon 11 of KIT. Besides these genetic lesions, only two point mutations that may affect tumour progression were identified: A frame-shift deletion in RNF146 and a missense mutation in a zinc finger of ZNF407. Whilst the frameshift deletion in RNF146 seemed to be restricted to this particular tumour, a similar yet germline mutation in ZNF407 was found in a panel of 52 gastro-intestinal stromal tumours from different anatomical sites and different categories. Germline polymorphisms in the mitotic checkpoint proteins Aurora kinase A and BUB1 kinase B may have furthered tumour growth. The epigenetic profile of the tumour matches that of other KIT-mutant tumours. We have identified mutations in three genes and loss of the long arm of chromosome 14 as the so far minimal set of genetic abnormalities sufficient for the development of a very low risk clinically symptomatic gastric stromal tumour.

The anaplastic lymphoma kinase testing conundrum.

Given the excellent results of the clinical trials with anaplastic lymphoma kinase (ALK) inhibitors, the importance of accurately identifying ALK-positive lung carcinoma patients has never been greater. It brings with it a pressing need for harmonized development of companion diagnostics, for economic, scientific and medical reasons. Therefore, it is crucial that ALK testing assays become more standardized both in performance (analytical phase) and interpretation (post-analytical phase). We find that both methods currently recommended by College of American Pathologists/International Association for the Study of Lung Cancer/Association for Molecular Pathology guidelines (FISH and Immunohistochemistry) are reasonable approaches for primary routine ALK testing, if at least 50 tumor cells are scored and protocols are strictly followed. Moreover, due to the high demand to study multiple predictive biomarkers on different assay platforms, quick and reliable approaches to achieve this are essential to guide treatment decisions.