Immuno-oncologic profiling of pediatric brain tumors reveals major clinical significance of the tumor immune microenvironment.

With the success of immunotherapy in cancer, understanding the tumor immune microenvironment (TIME) has become increasingly important; however in pediatric brain tumors this remains poorly characterized. Accordingly, we developed a clinical immune-oncology gene expression assay and used it to profile a diverse range of 1382 samples with detailed clinical and molecular annotation. In low-grade gliomas we identify distinct patterns of immune activation with prognostic significance in BRAF V600E-mutant tumors. In high-grade gliomas, we observe immune activation and T-cell infiltrates in tumors that have historically been considered immune cold, as well as genomic correlates of inflammation levels. In mismatch repair deficient high-grade gliomas, we find that high tumor inflammation signature is a significant predictor of response to immune checkpoint inhibition, and demonstrate the potential for multimodal biomarkers to improve treatment stratification. Importantly, while overall patterns of immune activation are observed for histologically and genetically defined tumor types, there is significant variability within each entity, indicating that the TIME must be evaluated as an independent feature from diagnosis. In sum, in addition to the histology and molecular profile, this work underscores the importance of reporting on the TIME as an essential axis of cancer diagnosis in the era of personalized medicine.

B-Cell Infiltrate in the Tumor Microenvironment Is Associated With Improved Survival in Resected Lung Adenocarcinoma.

Introduction: Relapse is common after resection of lung adenocarcinoma (LUAD). Features of the tumor microenvironment (TME) which influence postsurgical survival outcomes are poorly characterized. Here, we analyzed the TME of more than 1500 LUAD specimens to identify the relationship between B-cell infiltration and prognosis. Methods: Whole exome sequencing and bulk RNA sequencing were performed on LUADs and adjacent normal lung tissue. Relapse-free survival and overall survival (OS) were retrospectively correlated with characteristics of the tumor and TME in three data sets. Results: High B-cell content (defined as >10% B cells) was associated with improved OS in both a The Cancer Genome Atlas-resected LUAD data set (p = 0.01) and a separate institutional stage II LUAD data set (p = 0.04, median not reached versus 89.5 mo). A validation cohort consisting of pooled microarray data representing more than 1400 resected stage I to III LUADs confirmed the association between greater B-cell abundance, specifically higher B-cell expression, and longer postsurgical survival (median OS 90 versus 71 mo, p < 0.01). Relapse-free survival was longer for patients with adenocarcinomas with high B-cell content across data sets, but it did not reach statistical significance. Subcategorization of B-cell subsets indicated that high naive B-cell content was most predictive of survival. There was no correlation between programmed death-ligand 1 expression, lymphoid aggregates, or overall immune infiltrate density and survival outcomes across the cohorts. Conclusions: The growing adjuvant immunotherapy repertoire has increased the urgency for identifying prognostic and predictive biomarkers. Comprehensive profiling of more than 1500 LUADs suggests that high tumor-infiltrating B-cell content is a favorable prognostic marker.

Integrated Molecular and Clinical Analysis of 1,000 Pediatric Low-Grade Gliomas.

Pediatric low-grade gliomas (pLGG) are frequently driven by genetic alterations in the RAS-mitogen-activated protein kinase (RAS/MAPK) pathway yet show unexplained variability in their clinical outcome. To address this, we characterized a cohort of >1,000 clinically annotated pLGG. Eighty-four percent of cases harbored a driver alteration, while those without an identified alteration also often exhibited upregulation of the RAS/MAPK pathway. pLGG could be broadly classified based on their alteration type. Rearrangement-driven tumors were diagnosed at a younger age, enriched for WHO grade I histology, infrequently progressed, and rarely resulted in death as compared with SNV-driven tumors. Further sub-classification of clinical-molecular correlates stratified pLGG into risk categories. These data highlight the biological and clinical differences between pLGG subtypes and opens avenues for future treatment refinement.

Alterations in ALK/ROS1/NTRK/MET drive a group of infantile hemispheric gliomas.

Infant gliomas have paradoxical clinical behavior compared to those in children and adults: low-grade tumors have a higher mortality rate, while high-grade tumors have a better outcome. However, we have little understanding of their biology and therefore cannot explain this behavior nor what constitutes optimal clinical management. Here we report a comprehensive genetic analysis of an international cohort of clinically annotated infant gliomas, revealing 3 clinical subgroups. Group 1 tumors arise in the cerebral hemispheres and harbor alterations in the receptor tyrosine kinases ALK, ROS1, NTRK and MET. These are typically single-events and confer an intermediate outcome. Groups 2 and 3 gliomas harbor RAS/MAPK pathway mutations and arise in the hemispheres and midline, respectively. Group 2 tumors have excellent long-term survival, while group 3 tumors progress rapidly and do not respond well to chemoradiation. We conclude that infant gliomas comprise 3 subgroups, justifying the need for specialized therapeutic strategies.

Iris pigmentation as a quantitative trait: variation in populations of European, East Asian and South Asian ancestry and association with candidate gene polymorphisms.

In this study, we present a new quantitative method to measure iris colour based on high-resolution photographs. We applied this method to analyse iris colour variation in a sample of individuals of East Asian, European and South Asian ancestry. We show that measuring iris colour using the coordinates of the CIELAB colour space uncovers a significant amount of variation that is not captured using conventional categorical classifications, such as 'brown', 'blue' or 'green'. We tested the association of a selected panel of polymorphisms with iris colour in each population group. Six markers showed significant associations with iris colour in the European sample, three in the South Asian sample and two in the East Asian sample. We also observed that the marker HERC2 rs12913832, which is the main determinant of 'blue' versus 'brown' iris colour in European populations, is also significantly associated with central heterochromia in the European sample.

Does KRAS testing in metastatic colorectal cancer impact overall survival? A comparative effectiveness study in a population-based sample.

PURPOSE: Epidermal growth factor receptor (EGFR) inhibitors are approved for treating metastatic colorectal cancer (CRC); KRAS mutation testing is recommended prior to treatment. We conducted a non-inferiority analysis to examine whether KRAS testing has impacted survival in CRC patients. PATIENTS AND METHODS: We included 1186 metastatic CRC cases from seven health plans. A cutpoint of July, 2008, was used to define two KRAS testing time period groups: "pre-testing" (n = 760 cases) and "post-testing" (n = 426 cases). Overall survival (OS) was estimated, and the difference in median OS between the groups was calculated. The lower bound of the one-sided 95% confidence interval (CI) for the difference in survival was used to test the null hypothesis of post-testing inferiority. Multivariable Cox regression models were constructed to adjust for covariates. RESULTS: The median unadjusted OS was 15.4 months (95% CI: 14.0-17.5) and 12.8 months (95% CI: 10.0-15.2) in the pre- and post-testing groups, respectively. The OS difference was -2.6 months with one-sided 95% lower confidence bound of -5.13 months, which was less than the non-inferiority margin (-5.0 months, unadjusted p = 0.06), leading to a failure to reject inferiority of OS in the post-testing period. In contrast, in the adjusted analysis, OS non-inferiority was identified in the post-testing period (p = 0.001). Sensitivity analyses using cutpoints before and after July, 2008, also met the criteria for non-inferiority. CONCLUSION: Implementation of KRAS testing did not influence CRC OS. Our data support the use of KRAS testing to guide administration of EGFR inhibitors for treatment of metastatic CRC without diminished OS.

Reliability of KRAS mutation testing in metastatic colorectal cancer patients across five laboratories.

BACKGROUND: Mutations in the KRAS gene are associated with poor response to epidermal growth factor receptor inhibitors used in the treatment of metastatic colorectal cancer. Factors influencing KRAS test results in tumor specimens include: tumor heterogeneity, sample handling, slide preparation, techniques for tumor enrichment, DNA preparation, assay design and sensitivity. We evaluated comparability and consistency of KRAS test results among five laboratories currently being used to determine KRAS mutation status of metastatic colorectal cancer specimens in a large, multi-center observational study. FINDINGS: Twenty formalin-fixed paraffin-embedded human colorectal cancer samples from colon resections previously tested for KRAS mutations were selected based on mutation status (6 wild type, 8 codon 12 mutations, and 6 codon 13 mutations). We found good agreement across laboratories despite differences in mutation detection methods. Eighteen of twenty samples (90%) were concordant across all five labs. Discordant results are likely not due to laboratory error, but instead to tumor heterogeneity, contamination of the tumor sample with normal tissue, or analytic factors affecting assay sensitivity. CONCLUSIONS: Our results indicate commercial and academic laboratories provide reliable results for the common KRAS gene mutations at codons 12 and 13 when an adequate percentage of tumor cells is present in the sample.

A comparative study of five technologically diverse CFTR testing platforms.

Multiple cystic fibrosis (CF) testing platforms, using diverse and rapidly evolving technologies, are available to clinical laboratories commercially or for evaluation. Considerations when choosing a CF platform may include: sensitivity, specificity, accuracy, signal discrimination, ability to genotype, ability to reflex test, no calls/repeat rate, composition of mutation panel, hands-on time, start-to-finish time, integration into laboratory workflow, data analysis methods, flexibility regarding custom test design, and required instrumentation. Mindful of these considerations, we evaluated five technologically diverse CF platforms: 1) eSensor, an electronic detection assay system; 2) InPlex, a signal amplification methodology using a microfluidics card; 3) oligonucleotide ligation assay, an electrophoretic-based separation of amplicon-derived ligation-generated products; and two liquid bead arrays; 4) Signature, a direct hybridization assay using allele-specific capture probes; and 5) Tag-It, an assay using allele-specific primer extension and a universal microarray. A core of 150 samples, focusing on mutations in the American College of Medical Genetics/American College of Obstetricians and Gynecologists mutation panel, was tested throughout several runs for each platform. All of the platforms performed comparably in respect to sensitivity, specificity, and no-call rate. As our results indicate, consideration of all of the parameters evaluated may be useful when selecting the most appropriate platform for the specific setting.

Mitochondrial localization of human PANK2 and hypotheses of secondary iron accumulation in pantothenate kinase-associated neurodegeneration.

Mutations in the pantothenate kinase 2 gene (PANK2) lead to pantothenate kinase-associated neurodegeneration (PKAN, formerly Hallervorden-Spatz syndrome). This neurodegenerative disorder is characterized by iron accumulation in the basal ganglia. Pantothenate kinase is the first enzyme in the biosynthesis of coenzyme A from pantothenate (vitamin B(5)). PANK2, one of four human pantothenate kinase genes, is uniquely predicted to be targeted to mitochondria. We demonstrate mitochondrial localization of PANK2 and speculate on mechanisms of secondary iron accumulation in PKAN. Furthermore, PANK2 uses an unconventional translational start codon, CUG, which is polymorphic in the general population. The variant sequence, CAG (allele frequency: 0.05), leads to skipping of the mitochondrial targeting signal and cytosolic localization of PANK2. This common variant may cause mitochondrial dysfunction and impart susceptibility to late-onset neurodegenerative disorders with brain iron accumulation, including Parkinson's disease.