Longitudinal monitoring of circulating tumor DNA to detect relapse early and predict outcome in early breast cancer.

Purpose: Detection of molecular residual disease (MRD) allows for the identification of breast cancer patients at high-risk of recurrence, with the potential that early initiation of treatment at early stages of relapse could improve patient outcomes. The Invitae Personalized Cancer Monitoring™ assay (PCM) is a newly developed next-generation sequencing approach that utilizes up to 50 patient-specific, tumor-informed DNA variants, to detect circulating tumor DNA (ctDNA). The ability of the PCM assay to detect MRD before clinical relapse was evaluated.

Personalized Cancer Monitoring Assay for the Detection of ctDNA in Patients with Solid Tumors.

Background: Highly sensitive molecular assays have been developed to detect plasma-based circulating tumor DNA (ctDNA), and emerging evidence suggests their clinical utility for monitoring minimal residual disease and recurrent disease, providing prognostic information, and monitoring therapy responses in patients with solid tumors. The Invitae Personalized Cancer Monitoring assay uses a patient-specific, tumor-informed variant signature identified through whole exome sequencing to detect ctDNA in peripheral blood of patients with solid tumors.

Methods: The assay's tumor whole exome sequencing and ctDNA detection components were analytically validated using 250 unique human specimens and nine commercial reference samples that generated 1349 whole exome sequencing and cell-free DNA (cfDNA)-derived libraries.

Tracking early lung cancer metastatic dissemination in TRACERx using ctDNA.

Circulating tumour DNA (ctDNA) can be used to detect and profile residual tumour cells persisting after curative intent therapy. The study of large patient cohorts incorporating longitudinal plasma sampling and extended follow-up is required to determine the role of ctDNA as a phylogenetic biomarker of relapse in early-stage non-small-cell lung cancer (NSCLC). Here we developed ctDNA methods tracking a median of 200 mutations identified in resected NSCLC tissue across 1,069 plasma samples collected from 197 patients enrolled in the TRACERx study.

Molecular Neuropathology in Practice: Clinical Profiling and Integrative Analysis of Molecular Alterations in Glioblastoma.

Molecular profiling of glioblastoma has revealed complex cytogenetic, epigenetic, and molecular abnormalities that are necessary for diagnosis, prognosis, and treatment. Our neuro-oncology group has developed a data-driven, institutional consensus guideline for efficient and optimal workup of glioblastomas based on our routine performance of molecular testing. We describe our institution's testing algorithm, assay development, and genetic findings in glioblastoma, to illustrate current practices and challenges in neuropathology related to molecular and genetic testing.

Validation of a next-generation sequencing oncology panel optimized for low input DNA.

One caveat of next-generation sequencing (NGS)-based clinical oncology testing is the high amount of input DNA required. We sought to develop a focused NGS panel that could capture hotspot regions in relevant genes requiring 0.5-10 ng input DNA.

Genotypic and clinical heterogeneity within NCCN favorable-risk acute myeloid leukemia.

The National Comprehensive Cancer Network (NCCN) defines the following types of acute myeloid leukemia (AML) as favorable-risk: acute promyelocytic leukemia with t(15;17) (APL); AML with core-binding factor (CBF) rearrangements, including t(8;21) and inv(16) or t(16;16) without mutations in KIT (CBF-KIT); and AML with normal cytogenetics and mutations in NPM1 (NPM1); or biallelic mutations in CEBPA (CEBPA), without FLT3-ITD. Although these AMLs are categorized as favorable risk by NCCN, clinical experience suggests that there are differences in clinical outcome amongst these cytogenetically and molecularly distinct leukemias. This study compared clinical and genotypic characteristics of 60 patients with favorable-risk AML, excluding APL, and demonstrated significant differences between them.

Use of Biosynthetic Controls as Performance Standards for Next-Generation Sequencing Assays of Somatic Tumors: A Multilaboratory Study.

Background: Next-generation sequencing (NGS) assays are highly complex tests that can vary substantially in both their design and intended application. Despite their innumerous advantages, NGS assays present some unique challenges associated with the preanalytical process, library preparation, data analysis, and reporting. According to a number of professional laboratory organization, control materials should be included both during the analytical validation phase and in routine clinical use to guarantee highly accurate results.

BRCA locus-specific loss of heterozygosity in germline BRCA1 and BRCA2 carriers.

Complete loss of BRCA1 or BRCA2 function is associated with sensitivity to DNA damaging agents. However, not all BRCA1 and BRCA2 germline mutation-associated tumors respond. Herein we report analyses of 160 BRCA1 and BRCA2 germline mutation-associated breast and ovarian tumors.

A novel approach for next-generation sequencing of circulating tumor cells.

Background: Next-generation sequencing (NGS) of surgically resected solid tumor samples has become integral to personalized medicine approaches for cancer treatment and monitoring. Liquid biopsies, or the enrichment and characterization of circulating tumor cells (CTCs) from blood, can provide noninvasive detection of evolving tumor mutations to improve cancer patient care. However, the application of solid tumor NGS approaches to circulating tumor samples has been hampered by the low-input DNA available from rare CTCs.

A Modified Integrated Genetic Model for Risk Prediction in Younger Patients with Acute Myeloid Leukemia.

Background: Although cytogenetics-based prognostication systems are well described in acute myeloid leukemia (AML), overall survival (OS) remains highly variable within risk groups. An integrated genetic prognostic (IGP) model using cytogenetics plus mutations in nine genes was recently proposed for patients ≤60 years to improve classification. This model has not been validated in clinical practice.

Building a Robust Tumor Profiling Program: Synergy between Next-Generation Sequencing and Targeted Single-Gene Testing.

Next-generation sequencing (NGS) is a powerful platform for identifying cancer mutations. Routine clinical adoption of NGS requires optimized quality control metrics to ensure accurate results. To assess the robustness of our clinical NGS pipeline, we analyzed the results of 304 solid tumor and hematologic malignancy specimens tested simultaneously by NGS and one or more targeted single-gene tests (EGFR, KRAS, BRAF, NPM1, FLT3, and JAK2).

Rare Complex Mutational Profile in an ALK Inhibitor-resistant Non-small Cell Lung Cancer.

Testing for somatic alterations, including anaplastic lymphoma receptor tyrosine kinase gene (ALK) rearrangements and epidermal growth factor receptor gene (EGFR) mutations, is standard practice in the diagnostic evaluation and therapeutic management of non-small cell lung cancer (NSCLC), where the results of such tests can predict response to targeted-therapy. ALK rearrangements, EGFR mutations and mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) are considered mutually exclusive in NSCLC. Herein we identified a KRAS Q22K mutation and frameshift mutations in the genes encoding serine/threonine kinase 11 (STK11) and ataxia telangiectasia mutated serine/threonine kinase (ATM) by next-generation sequencing in a patient with ALK rearrangement-positive oligo-metastatic NSCLC, whose disease progressed while on two ALK-targeted therapies.

DNMT3A Mutational Status Affects the Results of Dose-Escalated Induction Therapy in Acute Myelogenous Leukemia.

Purpose: DNA methyltransferase 3A (DNMT3A) is one of the commonly mutated genes in acute myelogenous leukemia (AML). Reports on the prognostic significance of DNMT3A mutations have been inconsistent, and most of the data are available only for patients 60 years of age or younger. We hypothesized that this inconsistency is due to an interaction between the dose of anthracycline used in induction therapy and DNMT3A status.

Whole-exome sequencing identifies somatic ATRX mutations in pheochromocytomas and paragangliomas.

Pheochromocytomas and paragangliomas (PCC/PGL) are the solid tumour type most commonly associated with an inherited susceptibility syndrome. However, very little is known about the somatic genetic changes leading to tumorigenesis or malignant transformation. Here we perform whole-exome sequencing on a discovery set of 21 PCC/PGL and identify somatic ATRX mutations in two SDHB-associated tumours.

Development and implementation of a custom integrated database with dashboards to assist with hematopathology specimen triage and traffic.

Background: At some institutions, including ours, bone marrow aspirate specimen triage is complex, with hematopathology triage decisions that need to be communicated to downstream ancillary testing laboratories and many specimen aliquot transfers that are handled outside of the laboratory information system (LIS). We developed a custom integrated database with dashboards to facilitate and streamline this workflow.

Methods: We developed user-specific dashboards that allow entry of specimen information by technologists in the hematology laboratory, have custom scripting to present relevant information for the hematopathology service and ancillary laboratories and allow communication of triage decisions from the hematopathology service to other laboratories.

What you are missing could matter: a rare, complex BRAF mutation affecting codons 599, 600, and 601 uncovered by next generation sequencing.

Testing for somatic mutations in tumor samples is becoming standard practice in a number of tumor types where targeted therapies are available. Since clinical care is dependent on the identification of the presence or absence of specific mutations, it is important that these mutations be identified consistently and accurately. Here we identify in a patient with metastatic melanoma a novel, complex mutation involving BRAF c.

Acquired isochromosome 12p, somatic TP53 and PTEN mutations, and a germline ATM variant in an adolescent male with concurrent acute megakaryoblastic leukemia and mediastinal germ cell tumor.

Previous reports have described an association between hematologic malignancies (HMs) and extragonadal germ cell tumor (GCT). Most patients have been adolescent males with mediastinal nonseminomatous GCT. Although a variety of HMs have been reported, there is a striking predilection toward acute megakaryoblastic leukemia (AMKL).

Cytogenomic and phenotypic analysis in low-level monosomy 7 mosaicism with non-supernumerary ring chromosome 7.

We present the literature review of ring chromosome 7 and clinical, cytogenetic and fine molecular mapping of the first postnatal report of a male child with a non-supernumerary ring chromosome 7, r(7). The patient had dysmorphic features, developmental delay, dermatologic lesions with variable pigmentation, hypogenitalism, lumbar dextroscoliosis, cerebellar and ophthalmological abnormalities, and melanocytic congenital nevi. Cytogenetic analysis of peripheral blood and the nevus sample showed the presence of three different cell lines r(7), monosomy 7, and duplicated r(7) (idic r(7)), while findings on fibroblasts from both light and dark skin showed only mosaicism with r(7) and monosomy 7 cell lines in various proportions.

Understanding the limitations of next generation sequencing informatics, an approach to clinical pipeline validation using artificial data sets.

The advantages of massively parallel sequencing are quickly being realized through the adoption of comprehensive genomic panels across the spectrum of genetic testing. Despite such widespread utilization of next generation sequencing (NGS), a major bottleneck in the implementation and capitalization of this technology remains in the data processing steps, or bioinformatics. Here we describe our approach to defining the limitations of each step in the data processing pipeline by utilizing artificial amplicon data sets to simulate a wide spectrum of genomic alterations.

Trials and Tribulations with VH Replacement.

VH replacement (VHR) is a type of antibody gene rearrangement in which an upstream heavy chain variable gene segment (VH) invades a pre-existing rearrangement (VDJ). In this Hypothesis and Theory article, we begin by reviewing the mechanism of VHR, its developmental timing and its potential biological consequences. Then we explore the hypothesis that specific sequence motifs called footprints reflect VHR versus other processes.

CLL/SLL diagnosed in an adolescent.

Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is a disease of older adults. Pediatric CLL/SLL is vanishingly rare in the literature. We present a case of CLL/SLL diagnosed in a 17-year-old male.

Molecular diagnostics of acute myeloid leukemia: it's a (next) generational thing.

This commentary highlights the article by Spencer et al that outlines a novel next-generation sequencing-based method for the detection of FLT3 mutations.

Ring chromosome 20.

Ring Chromosome 20 syndrome is a rare chromosomal disorder characterized by refractory epilepsy, with seizures in wakefulness and sleep, behavioral problems and mild to severe cognitive impairment. Facial dysmorphism or other congenital malformations are rarely reported making it difficult to diagnose the syndrome based on clinical findings alone. Therefore, diagnosis requires cytogenetic testing.