Cancer cytogenetics in a genomics world: Wedding the old with the new.

Since the discovery of the Philadelphia chromosome in 1960, cytogenetic studies have been instrumental in detecting chromosomal abnormalities that can inform cancer diagnosis, treatment, and risk assessment efforts. The initial expansion of cancer cytogenetics was with fluorescence in situ hybridization (FISH) to assess submicroscopic alterations in dividing or non-dividing cells and has grown into the incorporation of chromosomal microarrays (CMA), and next generation sequencing (NGS). These molecular technologies add additional dimensions to the genomic assessment of cancers by uncovering cytogenetically invisible molecular markers. Rapid technological and bioinformatic advances in NGS are so promising that the idea of performing whole genome sequencing as part of routine patient care may soon become economically and logistically feasible. However, for now cytogenetic studies continue to play a major role in the diagnostic testing and subsequent assessments in leukemia with other genomic studies serving as complementary testing options for detection of actionable genomic abnormalities. In this review, we discuss the role of conventional cytogenetics (karyotyping, chromosome analysis) and FISH studies in hematological malignancies, highlighting the continued clinical utility of these techniques, the subtleties and complexities that are relevant to treating physicians and the unique strengths of cytogenetics that cannot yet be paralleled by the current high-throughput molecular technologies. Additionally, we describe how CMA, optical genome mapping (OGM), and NGS detect abnormalities that were beyond the capacity of cytogenetic studies and how an integrated approach (broad molecular testing) can contribute to the detection of actionable targets and variants in malignancies. Finally, we discuss advances in the field of genomic testing that are bridging the advantages of individual (single) cell based cytogenetic testing and broad genomic testing.

Cytogenetics and genomics of acute myeloid leukemia.

The diversity of genetic and genomic abnormalities observed in acute myeloid leukemia (AML) reflects the complexity of these hematologic neoplasms. The detection of cytogenetic and molecular alterations is fundamental to diagnosis, risk stratification and treatment of AML. Chromosome rearrangements are well established in the diagnostic classification of AML, as are some gene mutations, in several international classification systems. Additionally, the detection of new mutational profiles at relapse and identification of mutations in the pre- and post-transplant settings are illuminating in understanding disease evolution and are relevant to the risk assessment of AML patients. In this review, we discuss recurrent cytogenetic abnormalities, as well as the detection of recurrent mutations, within the context of a normal karyotype, and in the setting of chromosome abnormalities. Two new classification schemes from the WHO and ICC are described, comparing these classifications in terms of diagnostic criteria and entity definition in AML. Finally, we discuss ways in which genomic sequencing can condense the detection of gene mutations and chromosome abnormalities into a single assay.

RAD21 mutations in acute myeloid leukemia.

The cohesin complex is a ring-shaped protein structure involved in DNA repair and chromosomal segregation. Studies have showed that genomic alterations in the cohesin complex members are among the initial occurrences in the development of acute myeloid leukemia (AML). STAG2 is the most commonly mutated and best-studied member of the cohesin complex in AML and mutations in this gene have been associated with adverse outcomes and are diagnostically relevant. However, the exact role of mutations in other members of the cohesin complex in the development of myeloid neoplasia is controversial. In this single institution study, we retrospectively reviewed data from the molecular profiles of 1,381 AML patients and identified 14 patients with mutations in RAD21, another member of the cohesin complex. We evaluated the frequency, mutational profile, clinico-pathologic features, and prognostic impact of RAD21 in this cohort. This study showed that RAD21-mutated AML often associates with monocytic differentiation, CD7 expression, co-existing mutations in epigenetic regulators, a normal karyotype, and poor prognosis. Our findings provide additional insights into the morphologic, immunophenotypic, and genomic profile of RAD21 mutation-positive AML and suggest that RAD21 mutations should be evaluated for independent prognostic significance in AML.

Integrating imaging and genomic data for the discovery of distinct glioblastoma subtypes: a joint learning approach.

Glioblastoma is a highly heterogeneous disease, with variations observed at both phenotypical and molecular levels. Personalized therapies would be facilitated by non-invasive in vivo approaches for characterizing this heterogeneity. In this study, we developed unsupervised joint machine learning between radiomic and genomic data, thereby identifying distinct glioblastoma subtypes. A retrospective cohort of 571 IDH-wildtype glioblastoma patients were included in the study, and pre-operative multi-parametric MRI scans and targeted next-generation sequencing (NGS) data were collected. L21-norm minimization was used to select a subset of 12 radiomic features from the MRI scans, and 13 key driver genes from the five main signal pathways most affected in glioblastoma were selected from the genomic data. Subtypes were identified using a joint learning approach called Anchor-based Partial Multi-modal Clustering on both radiomic and genomic modalities. Kaplan-Meier analysis identified three distinct glioblastoma subtypes: high-risk, medium-risk, and low-risk, based on overall survival outcome (p < 0.05, log-rank test; Hazard Ratio = 1.64, 95% CI 1.17-2.31, Cox proportional hazard model on high-risk and low-risk subtypes). The three subtypes displayed different phenotypical and molecular characteristics in terms of imaging histogram, co-occurrence of genes, and correlation between the two modalities. Our findings demonstrate the synergistic value of integrated radiomic signatures and molecular characteristics for glioblastoma subtyping. Joint learning on both modalities can aid in better understanding the molecular basis of phenotypical signatures of glioblastoma, and provide insights into the biological underpinnings of tumor formation and progression.

T cell lymphoma and secondary primary malignancy risk after commercial CAR T cell therapy.

We report a T cell lymphoma (TCL) occurring 3 months after anti-CD19 chimeric antigen receptor (CAR) T cell immunotherapy for non-Hodgkin B cell lymphoma. The TCL was diagnosed from a thoracic lymph node upon surgery for lung cancer. The TCL exhibited CD8+ cytotoxic phenotype and a JAK3 variant, while the CAR transgene was very low. The T cell clone was identified at low levels in the blood before CAR T infusion and in lung cancer. To assess the overall risk of secondary primary malignancy after commercial CAR T (CD19, BCMA), we analyzed 449 patients treated at the University of Pennsylvania. At a median follow-up of 10.3 months, 16 patients (3.6%) had a secondary primary malignancy. The median onset time was 26.4 and 9.7 months for solid and hematological malignancies, respectively. The projected 5-year cumulative incidence is 15.2% for solid and 2.3% for hematological malignancies. Overall, one case of TCL was observed, suggesting a low risk of TCL after CAR T.

TP53 mutations predict for poor outcomes in patients with newly diagnosed aggressive B-cell lymphomas in the current era.

Genetic subgroups of diffuse large B-cell lymphoma (DLBCL) have been identified through comprehensive genomic analysis; however, it is unclear whether this can be applied in clinical practice. We assessed whether mutations detected by clinical laboratory mutation analysis (CLMA) were predictive of outcomes in patients with newly diagnosed DLBCL/high-grade B-cell lymphoma (HGBL). Patients diagnosed from 2018 to 2022 whose biopsy samples were subjected to CLMA and who received rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone or rituximab plus etoposide, prednisolone, vincristine, cyclophosphamide, and doxorubicin were analyzed for overall/complete response rate (ORR/CRR) and estimated progression-free/overall survival (PFS/OS). CLMA was successfully performed in 117 of 122 patient samples (96%), with a median turnaround time of 17 days. Median duration of follow-up was 31.3 months. Of the mutations detected in ≥10% of the samples, only TP53 was associated with both progression and death at 2 years. TP53 mutations were detected in 36% of tumors, and patients with TP53 mutations experienced significantly lower ORR (71% vs 90%; P = .009), CRR (55% vs 77%; P = .01), 2-year PFS (57% vs 77%; P = .006), 2-year OS (70% vs 91%; P = .001), and median OS after relapse (6.1 months vs not yet reached; P = .001) as than those without TP53 mutations. Furthermore, patients with TP53 loss-of-function (LOF) mutations experienced lower rates of 2-year PFS/OS than those with non-LOF mutations and inferior or near-inferior 2-year PFS if harboring high-risk clinicopathologic features. TP53 mutations identified through CLMA can predict for inferior outcomes in patients with newly diagnosed DLBCL/HGBL. Results of CLMA can be used in real time to inform prognosis and/or identify candidates for clinical trials.

ROS1 Alterations as a Potential Driver of Gliomas in Infant, Pediatric, and Adult Patients.

Gliomas harboring oncogenic ROS1 alterations are uncommon and primarily described in infants. Our goal was to characterize the clinicopathological features and molecular signatures of the full spectrum of ROS1 fusion-positive gliomas across all age groups. Through a retrospective multi-institutional collaboration, we report a collection of unpublished ROS1 fusion gliomas along with the characterization and meta-analysis of new and published cases. A cohort of 32 new and 58 published cases was divided into the following 3 age groups: 19 infants, 40 pediatric patients, and 31 adults with gliomas. Tumors in infants and adults showed uniformly high-grade morphology; however, tumors in pediatric patients exhibited diverse histologic features. The GOPC::ROS1 fusion was prevalent (61/79, 77%) across all age groups, and 10 other partner genes were identified. Adult tumors showed recurrent genomic alterations characteristic of IDH wild-type glioblastoma, including the +7/-10/CDKN2A deletion; amplification of CDK4, MDM2, and PDGFRA genes; and mutations involving TERTp, TP53, PIK3R1, PIK3CA, PTEN, and NF1 genes. Infant tumors showed few genomic alterations, whereas pediatric tumors showed moderate genomic complexity. The outcomes were significantly poorer in adult patients. Although not statistically significant, tumors in infant and pediatric patients with high-grade histology and in hemispheric locations appeared more aggressive than tumors with lower grade histology or those in nonhemispheric locations. In conclusion, this study is the largest to date to characterize the clinicopathological and molecular signatures of ROS1 fusion-positive gliomas from infant, pediatric, and adult patients. We conclude that ROS1 likely acts as a driver in infant and pediatric gliomas and as a driver or codriver in adult gliomas. Integrated comprehensive clinical testing might be helpful in identifying such patients for possible targeted therapy.

The Genomics Organization for Academic Laboratories (GOAL): A vision for a genomics future for academic pathology.

Innovative and self-sustaining clinical genomics laboratories specializing in cutting-edge oncology testing are critical to the success of academic pathology departments and resident and fellow education in molecular pathology. However, the pressures and challenges facing these laboratories are numerous, including the complexities of validating comprehensive cancer next-generation sequencing (NGS) panels, competition from commercial laboratories, and the reimbursement and regulatory hurdles inherent in high-complexity testing. Cross-institutional collaborations, including shared assay content and interpretative frameworks, are a valuable element to academic laboratory success. To address these and other needs, the Genomics Organization for Academic Laboratories (GOAL) was conceived in 2018, incorporated in 2020 and has grown to include 29 participating institutions in 2022. Here, we describe the mission of GOAL, its structure, and the outcomes and projects undertaken in its first years.

Homologous recombination pathway gene variants identified by tumor-only sequencing assays in lung carcinoma patients.

Background: The homologous recombination (HR) repair pathway plays a key role in double-stranded DNA break repair, and germline HR pathway gene variants are associated with increased risk of several cancers, including breast and ovarian cancer. HR deficiency is also a therapeutically targetable phenotype. Methods: Somatic (tumour-only) sequencing was performed on 1,109 cases of lung tumors, and the pathological data were reviewed to filter for lung primary carcinomas. Cases were filtered for variants (disease-associated or of uncertain significance) in 14 HR pathway genes, including BRCA1, BRCA2, and ATM. The clinical, pathological and molecular data were reviewed. Results: Sixty-one HR pathway gene variants in 56 patients with primary lung cancer were identified. Further filtering by variant allele fraction (VAF) of ≥30% identified 17 HR pathway gene variants in 17 patients. ATM gene variants were most the commonly identified (9/17), including two patients with c.7271T>G (p.V2424G), a variant in the germline that is associated with increased familial cancer risk. Four (4/17) patients had a family history of lung cancer, among which three patients had ATM gene variants suspected to be germline in origin. In three other patients with BRCA1/2 or PALB2 gene variants who had undergone germline testing, the variants were confirmed to be germline; lung cancer was the sentinel cancer in two of these patients with a BRCA1 or PALB2 variant. Conclusions: Genomic variants in the HR repair pathway identified in tumor-only sequencing and occurring at higher VAFs (i.e., ≥30%) may suggest a germline origin. Correlating with personal and family history, a subset of these variants is also suggested to be associated with familial cancer risks. Patient age, smoking history and driver mutation status are expected to be a poor screening tool in identifying these patients. Finally, the relative enrichment for ATM variants in our cohort suggests a possible association between ATM mutation and lung cancer risk.

RNA fusion transcript panel identifies diverse repertoire of fusions in adult glioma patients with therapeutic implications.

Background: Recurrent gliomas are therapeutically challenging diseases with few treatment options available. One area of potential therapeutic vulnerability is the presence of targetable oncogenic fusion proteins. Methods: To better understand the clinical benefit of routinely testing for fusion proteins in adult glioma patients, we performed a retrospective review of 647 adult patients with glioma who underwent surgical resection at our center between August 2017 and May 2021 and whose tumors were analyzed with an in-house fusion transcript panel. Results: Fifty-two patients (8%) were found to harbor a potentially targetable fusion with 11 (21%) of these patients receiving treatment with a fusion-targeted inhibitor. The targetable genes found to be involved in a fusion included FGFR3, MET, EGFR, NTRK1, NTRK2, BRAF, ROS1, and PIK3CA. Conclusions: This analysis demonstrates that routine clinical testing for gene fusions identifies a diverse repertoire of potential therapeutic targets in adult patients with glioma and can offer rational therapeutic options for patients with recurrent disease.

The Performance of an Extended Next Generation Sequencing Panel Using Endobronchial Ultrasound-Guided Fine Needle Aspiration Samples in Non-Squamous Non-Small Cell Lung Cancer: A Pragmatic Study.

INTRODUCTION/BACKGROUND: Samples from endobronchial ultrasound-guided fine needle aspiration (EBUS-TBNA) are frequently used for next generation sequencing (NGS) in patients with non-small cell lung cancer (NSCLC) to look for genetic driver mutations. The objective of the current study was to evaluate the performance of extended NGS panels using EBUS-TBNA samples in a real-world setting and identify factors associated with the success of NGS. MATERIALS AND METHODS: This study included all patients who underwent EBUS and were diagnosed with non-squamous NSCLC with mediastinal metastasis from 2016 to 2019 at the University of Pennsylvania. We reviewed demographic information, imaging studies, procedure reports, pathology and NGS reports. Logistic regression was used to analyze factors associated with the success of NGS panels. RESULTS: The success rates of NGS using EBUS-TBNA samples were 92.5%, and 91.5% for DNA and RNA NGS panels respectively. Samples from higher N stage (N2 and N3 lymph nodes) and with higher tumor cellularity (>25%) resulted in higher success rate for DNA NGS. The effect of tumor cellularity remained borderline significant after entering multivariable logistic regression. The short-axis diameter of the sampled lymph node on CT scan, FDG-avidity on PET CT and >3 EBUS passes per lymph node during the procedure were not associated with NGS success. CONCLUSION: Both DNA and RNA extended-panel NGS had high performance using EBUS-TBNA samples. Sampling more advanced nodal stations and obtaining samples with higher tumor cellularity were associated with higher success rate of DNA NGS. Other imaging or procedural factors did not affect NGS performance.

Rapid and Automated Semiconductor-Based Next-Generation Sequencing for Simultaneous Detection of Somatic DNA and RNA Aberrations in Myeloid Neoplasms.

Evaluation of suspected myeloid neoplasms involves testing for recurrent, diagnostically and therapeutically relevant genetic alterations. Current molecular testing requires multiple technologies, different domains of expertise, and unconnected workflows, resulting in variable, lengthy turnaround times that can delay treatment. To address this unmet clinical need, we evaluated the Oncomine Myeloid Assay GX panel on the Ion Torrent Genexus platform, a rapid, integrated nucleic acid to report next-generation sequencing platform for detecting clinically relevant genetic aberrations in myeloid disorders. Specimens included synthetic DNA (101 targets) and RNA (9 targets) controls and real-world nucleic acid material derived from bone marrow or peripheral blood samples (40 patients). Ion Torrent Genexus results and performance indices were compared with those obtained from clinically validated genomic testing workflows in 2 separate clinical laboratories. The Ion Torrent Genexus identified 100% of DNA and RNA control variants. For primary patient specimens, the Ion Torrent Genexus reported 82 of 107 DNA variants and 19 of 19 RNA gene fusions identified on clinically validated assays, yielding an 80% overall detection rate. Reanalysis of exported, unfiltered Ion Torrent Genexus data revealed 15 DNA variants not called by the filtered on-board bioinformatics pipeline, yielding a 92% potential detection rate. These results hold promise for the implementation of an integrated next-generation sequencing system to rapidly detect genetic aberrations, facilitating accurate, genomics-based diagnoses and accelerated time to precision therapies in myeloid neoplasms.

Greater expectations: meeting clinical needs through broad and rapid genomic testing.

Cancer describes a group of diseases driven by genetic and genomic changes that can occur across hundreds of different genes. Knowledge of the specific variants present in a patient's cancer can help to predict response to different treatment options, confirm disease diagnosis, and understand a patient's prognosis and risks, which ultimately leads to improved survival outcomes. The advent of next-generation sequencing (NGS) technology has allowed pathologists to simultaneously profile the sequences of many genes in a single reaction, but not all NGS assays are built the same. While those used for broad genomic profiling are useful to probe large regions of the genome and gather more information about a patient's tumor, it comes at the cost of relatively long turnaround times (TAT), which may be detrimental to patient care. Conversely, NGS assays used for rapid genomic profiling provide faster results, but may miss detection of variants that are clinically informative. Determining which type of genomic profiling to order depends on a number of factors including the severity of a patient's illness, standard of care paradigms, and success or failure of previous therapies. Ultimately, the ideal clinical diagnostic laboratory will be able to offer both options to best meet the clinical needs of its patients.

Fanconi anemia caused by biallelic inactivation of BRCA2 can present with an atypical cancer phenotype in adulthood.

Inherited biallelic pathogenic variants (PVs) in BRCA2 cause Fanconi Anemia complementation group D1 (FA-D1), a severe pediatric bone marrow failure and high-risk cancer syndrome. We identified biallelic BRCA2 PVs in a young adult with multiple basal cell carcinomas, adult-onset colorectal cancer and small cell neuroendocrine carcinoma, without bone marrow failure. No PVs were identified in any other known cancer susceptibility gene, and there was no evidence of reversion mosaicism. The proband's deceased sister had a classic FA-D1 presentation and was shown to carry the same biallelic BRCA2 PVs. A lymphoblastoid cell line derived from the proband demonstrated hypersensitivity to DNA damaging agents, and bone marrow showed aberrant RAD51 staining. Family expansion demonstrated the presence of BRCA2 related cancers in heterozygous family members. Our data highlight the striking phenotypic differences which can be observed within FA-D1 families and expands the clinical spectrum of FA-D1 to include adult presentation with a constellation of solid tumors not previously thought of as characteristic of Fanconi Anemia. Early recognition of this syndrome in a family could prevent further morbidity and mortality by implementation of hereditary breast and ovarian cancer screening and treatment strategies for heterozygous family members.

Mutation analysis performed on tumor biopsies from patients with newly-diagnosed germinal center aggressive B cell lymphomas.

Comprehensive genomic analyses of tumor biopsies from patients with newly-diagnosed germinal center B cell (GCB) diffuse large B cell/high grade B cell lymphoma (DLBCL/HGBL) have identified molecular subtypes predictive of inferior survival, which are characterized by somatic mutations that can be detected through clinical laboratory mutation analysis (CLMA). To determine the frequency and predictive value of individual genetic mutations associated with these experimentally-defined poor-risk subgroups, we reviewed the findings from CLMA performed on tumors from patients with newly-diagnosed GCB DLBCL/HGBL who were previously treated at our institution. CLMA was successfully performed on 58/59 patient tumor biopsies with a median turnaround time of 16 days, and 51 on which CLMA was routinely performed with adequate clinical follow-up were analyzed. Patients whose tumors demonstrated CREBBP mutation experienced a lower estimated rate of 2-year disease free survival (DFS) as compared to those whose tumors did not (45% [95% CI 18-68%] vs. 67% [95% CI 44-83%], P = 0.045). CREBBP mutations may be frequent and predict for inferior DFS in patients with newly-diagnosed GCB DLBCL/HGBL. Furthermore, CLMA may be practically-applied to translate experimental findings into those with more direct application to risk stratification and clinical trial design in subsets of patients with DLBCL/HGBL.

The University of Pennsylvania glioblastoma (UPenn-GBM) cohort: advanced MRI, clinical, genomics, & radiomics.

Glioblastoma is the most common aggressive adult brain tumor. Numerous studies have reported results from either private institutional data or publicly available datasets. However, current public datasets are limited in terms of: a) number of subjects, b) lack of consistent acquisition protocol, c) data quality, or d) accompanying clinical, demographic, and molecular information. Toward alleviating these limitations, we contribute the "University of Pennsylvania Glioblastoma Imaging, Genomics, and Radiomics" (UPenn-GBM) dataset, which describes the currently largest publicly available comprehensive collection of 630 patients diagnosed with de novo glioblastoma. The UPenn-GBM dataset includes (a) advanced multi-parametric magnetic resonance imaging scans acquired during routine clinical practice, at the University of Pennsylvania Health System, (b) accompanying clinical, demographic, and molecular information, (d) perfusion and diffusion derivative volumes, (e) computationally-derived and manually-revised expert annotations of tumor sub-regions, as well as (f) quantitative imaging (also known as radiomic) features corresponding to each of these regions. This collection describes our contribution towards repeatable, reproducible, and comparative quantitative studies leading to new predictive, prognostic, and diagnostic assessments.

Lung Cancer Biomarker Speak: Teach Me the Language.

A vital role of the advanced practitioner is to analyze biomarker testing reports and explain to patients how the results may impact their treatment. At JADPRO Live Virtual 2021, presenters reviewed the importance of molecular testing in non-small cell lung cancer and how to interpret molecular pathology reports.

NTRK point mutations and their functional consequences.

The neurotrophic receptor tyrosine kinase (NTRK) family of genes, including NTRK1, NTRK2, and NTRK3, encodes membrane-bound receptors that normally regulate cell survival and differentiation upon binding of growth factors. Not surprisingly, mutations in these genes are known to contribute to the growth of a diverse number of cancers. With the recent FDA approval of two first-generation tyrosine-kinase inhibitors (TKIs) for adult and pediatric patients with solid tumors harboring NTRK gene fusions, much of the literature has focused on the biology behind these types of NTRK abnormalities; however, point mutations can also contribute to oncogenesis or resistance to TKI therapy, albeit at a lower frequency than fusions. This review focuses on NTRK gene mutations that are associated with resistance to directed therapies, mutations detected in the primary setting that confer increased oncogenic activity, and evidence that suggests that some of these variants may be treated using specific targeted therapies. Finally, this review focuses on the detection of point mutations, including the utility of cell-free DNA (cfDNA) for monitoring the acquisition of resistance mutations.