Evaluation of the Oncomine Comprehensive Assay Plus NGS Panel and the OncoScan CNV Assay for Homologous Recombination Deficiency Detection.

INTRODUCTION: Testing for homologous recombination deficiency (HRD) as a biomarker in relation to poly (ADP-ribose) polymerase inhibitor (PARPi) treatment in ovarian cancer is done by sequencing of the BRCA1/2 genes and/or by assessing a genomic instability signature. Here we present data obtained with two different methods for genomic instability testing: the Oncomine™ Comprehensive Assay Plus (OCA Plus) NGS panel and the OncoScan CNV assay. METHODS: The retrospective analytical study included 80 ovarian cancer samples of patients previously referred to clinical Myriad testing (reference cohort), and 50 ovarian cancer samples from patients collected as part of the Pelvic Mass study. OCA Plus NGS libraries were sequenced with the Ion S5™XL Sequencer and analyzed with the Ion Reporter™ Software v5.20 for calculation of the genomic instability metric (GIM). In addition, all samples were tested with the OncoScan CNV FFPE Assay and analyzed with a previously published R-algorithm for generation of an in-house genomic instability score (in-house GIS). RESULTS: The OCA Plus assay had a concordance to the reference of 89% on samples with a tumor fraction ≥ 30% (auto-calculated or via molecular estimation). A total of 15 samples in the reference cohort had a calculated tumor fraction < 30% in the OCA Plus assay. In these, the concordance to reference was only 60%. For the OncoScan CNV in-house GIS a local cutoff point of ≥ 50 was calculated. This gave a concordance to the reference of 85%, with 91% of the samples in the reference cohort passing quality control (QC) on tumor fraction. Both assays had a high sensitivity for the detection of genomic instability in samples with pathogenic or likely pathogenic BRCA1/2 mutations, with 12/13 being GIM positive (OCA Plus assay) and 13/13 being in-house GIS positive (OncoScan CNV assay). CONCLUSIONS: The OCA Plus assay and the OncoScan CNV assay show a high but not complete concordance to reference standard homologous recombination deficiency (HRD) detection. The main reason for QC failure or non-concordance in our study was a low tumor fraction estimated in the assay, despite the selection of material by a pathologist with an inclusion criterion of > 30% tumor. QC steps should include careful tumor content evaluation, and results on samples with < 30% tumor should not be reported.

Atypical presentation of γ/δ mycosis fungoides with an unusual phenotype and SOCS1 mutation.

Mycosis fungoides is the most frequent subtype of primary cutaneous T-cell lymphomas. The diagnosis is based on a thorough clinic-pathologic correlation, which can, especially in early-stage disease, be challenging due to similarities with several benign skin disorders such as psoriasis and atopic dermatitis. Here, we present a case of an 81-year-old man with a 20-year-long medical history of skin problems treated as psoriasis with limited effect. Since December 2021, the patient experienced worsening of his skin symptoms with rapidly growing tumors and widespread patches and plaques. Positron emission tomography/computed tomography evaluation revealed markedly metabolic activity related to the skin tumors and increased FDG uptake in several retroperitoneal lymph nodes. Histological assessment of skin biopsies demonstrated a highly proliferative T-cell lymphoma with a γ/δ+ and CD8+ cytotoxic phenotype. The morphology of the tumor cells appeared blastic with an abnormal immunephenotype CD3+, CD2-, CD5dim, CD4-, CD8+, CD56-, and CD30-. Next-generation sequencing detected a likely pathogenic SOCS1 mutation with an allele frequency of 72% as well as a STAT3 variant of unknown significance. This case highlights the diagnostic complexity of an indolent skin lymphoma evolving into an aggressive cytotoxic lymphoma.

Paired comparison of the analytical performance between the Oncomine™ Comprehensive Assay v3 and whole-exome sequencing of ovarian cancer tissue.

BACKGROUND: Next-generation sequencing (NGS) has been implemented in clinical oncology as a personalized medicine tool to identify targetable genetic alterations and to guide treatment decisions. However, the optimal NGS test strategy and target genes for clinical use are still being discussed. The aim was to compare the performance of the Oncomine™ Comprehensive Assay v3 (OCAv3) (targeted gene panel) and whole-exome sequencing (WES) to investigate somatic single and multiple nucleotide variants and small indels in ovarian cancer patients. METHODS AND RESULTS: Genomic DNA was isolated from fresh frozen samples of five high-grade serous (HGSC) and three clear cell ovarian (oCCC) cancer patients. Exome sequencing libraries were prepared by using the Ion AmpliSeq Exome RDY kit, whereas libraries for OCAv3 were prepared using by Ion AmpliSeq™ Library Kit Plus. Sequencing was performed using the Ion S5XL System (Thermo Fisher Scientific). When including only variants classified as pathogenic, likely pathogenic or unknown significance based on ClinVar database verdicts and comparing overlapping regions covered both by the OCAv3 assay and WES, 23 variants were detected by both assays. However, OCAv3 detected additionally two variants: ARID1A: p.Gln563Ter and TP53: p.Ser261ValfsTer84 that have not passed WES filtering criteria due to low coverage. CONCLUSIONS: With the present treatment possibilities, OCAv3 panel testing provided higher diagnostic yield due to better coverage. Our study emphasizes that WES, although offering the potential to identify novel findings in genes not covered by OCAv3, might overlook variants in genes relevant for OC.

Mutational landscape in Waldenström macroglobulinemia evaluated using a next-generation sequencing lymphoma panel in routine clinical practice.

Next-generation sequencing (NGS) affords comprehensive insights into the genomic landscape of lymphomas. We examined the mutational pattern in patients with Waldenström macroglobulinemia (WM) or lymphoplasmacytic lymphoma (LPL) as well as the diagnostic and clinical utility of a tailored NGS lymphoma panel. A consecutive series of 45 patients was reviewed and NGS analysis was performed as part of a routine diagnostic setup. The custom designed NGS panel assayed all coding sequences of 59 genes of known clinical significance in lymphoid neoplasms. The most frequently mutated genes were MYD88, CXCR4, BIRC3, CD79B, and ARID1A. Additional somatic mutations were detected in 17 genes with four mutations categorized as pathogenic or likely pathogenic. BIRC3 and TP53 mutations were associated with adverse clinical phenotypes. NGS performance for the MYD88L265P variant was 96% when compared to qPCR. In conclusion, targeted NGS provided important diagnostic and prognostic information in a routine clinical setting.

Next generation sequencing in routine diagnostics of mature non-Hodgkin's B-cell lymphomas.

INTRODUCTION: Integration of molecular characterization of lymphomas in clinical diagnostics may improve subclassification and risk-stratification, and we implemented a next generation sequencing (NGS) analysis as part of routine diagnostic work-up of all mature B-cell non-Hodgkin's lymphoma (B-NHL). Here, we present data of mutational profiles with potential complementary diagnostic, prognostic, and predictive value detected in our consecutive non-selected cohort of B-NHL patients. METHODS: NGS results from 298 patients with both newly diagnosed and relapsed/refractory disease were included as a single center study. NGS was performed as routine analysis together with standard diagnostic work-up using a custom-made amplicon PCR-based multiplex NGS panel covering all coding exons and consensus splice sites in 59 genes. RESULTS: Mutations were detected in 94% of the 298 samples. Most lymphomas could be classified definitively, but 24 cases were classified as small B-cell lymphomas without defining characteristics. Of these, 50% (12/24 cases) could retrospectively be assigned a likely diagnostic subtype according to mutational findings. CONCLUSION: Implementation of a 59 gene exome sequencing panel added diagnostic value to 50% of unclassified cases and provided in 94% of the cases possible biomarkers for disease monitoring as well as potential diagnostic, prognostic, and predictive markers for future studies.

Multi-site pre-therapeutic biopsies demonstrate genetic heterogeneity in patients with newly diagnosed diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease, both regarding clinical presentation, response to treatment and outcome. Recently, subclassification of DLBCL based on mutational profile has been suggested, and next generation sequencing (NGS) analysis may be relevant as part of the diagnostic workflow. This will, however, often be based on analysis of one tumor biopsy. Here, we present a prospective study where multi-site sampling was performed prior to treatment in patients with newly diagnosed DLBCL. Two spatially different biopsies from 16 patients were analyzed using NGS with an in-house 59-gene lymphoma panel. In 8/16 (50%) patients, mutational differences were found between the two biopsy sites, including differences in TP53 mutational status. Our data indicate that a biopsy from the extra-nodal site may represent the most advanced clone, and an extra-nodal biopsy should be preferred for analysis, if safely accessible. This will help ensure a standardized stratification and treatment decision.

Mismatch repair deficiency testing in Lynch syndrome-associated urothelial tumors.

Introduction: Lynch syndrome-associated cancer develops due to germline pathogenic variants in one of the mismatch repair (MMR) genes, MLH1, MSH2, MSH6 or PMS2. Somatic second hits in tumors cause MMR deficiency, testing for which is used to screen for Lynch syndrome in colorectal cancer and to guide selection for immunotherapy. Both MMR protein immunohistochemistry and microsatellite instability (MSI) analysis can be used. However, concordance between methods can vary for different tumor types. Therefore, we aimed to compare methods of MMR deficiency testing in Lynch syndrome-associated urothelial cancers. Methods: Ninety-seven urothelial (61 upper tract and 28 bladder) tumors diagnosed from 1980 to 2017 in carriers of Lynch syndrome-associated pathogenic MMR variants and their first-degree relatives (FDR) were analyzed by MMR protein immunohistochemistry, the MSI Analysis System v1.2 (Promega), and an amplicon sequencing-based MSI assay. Two sets of MSI markers were used in sequencing-based MSI analysis: a panel of 24 and 54 markers developed for colorectal cancer and blood MSI analysis, respectively. Results: Among the 97 urothelial tumors, 86 (88.7%) showed immunohistochemical MMR loss and 68 were successfully analyzed by the Promega MSI assay, of which 48 (70.6%) were MSI-high and 20 (29.4%) were MSI-low/microsatellite stable. Seventy-two samples had sufficient DNA for the sequencing-based MSI assay, of which 55 (76.4%) and 61 (84.7%) scored as MSI-high using the 24-marker and 54-marker panels, respectively. The concordance between the MSI assays and immunohistochemistry was 70.6% (p = 0.003), 87.5% (p = 0.039), and 90.3% (p = 1.00) for the Promega assay, the 24-marker assay, and the 54-marker assay, respectively. Of the 11 tumors with retained MMR protein expression, four were MSI-low/MSI-high or MSI-high by the Promega assay or one of the sequencing-based assays. Conclusion: Our results show that Lynch syndrome-associated urothelial cancers frequently had loss of MMR protein expression. The Promega MSI assay was significantly less sensitive, but the 54-marker sequencing-based MSI analysis showed no significant difference compared to immunohistochemistry. Data from this study alongside previous studies, suggest that universal MMR deficiency testing of newly diagnosed urothelial cancers, using immunohistochemistry and/or sequencing-based MSI analysis of sensitive markers, offer a potentially useful approach to identification of Lynch syndrome cases.

Test of the FlashFREEZE unit in tissue samples freezing for biobanking purposes.

Availability of molecularly intact biospecimens is essential in genetic diagnostics to obtain credible results. Integrity of nucleic acids (particularly RNA) may be compromised at various steps of tissue handling, and affected by factors such as time to freeze, freezing technique and storing temperature. At the same time, freezing and storing of the biological material should be feasible and safe for the operator. Here, we compared quality of DNA and RNA from biospecimens derived from different organs (breast, colon, adrenal glands, testes, rectum and uterus) frozen either using dry ice-cooled isopentane or with FlashFREEZE unit, in order to verify if the latter is suitable for routine use in biobanking. Implementing FlashFREEZE device would enable us to limit the use of isopentane, which is potentially toxic and environmentally harmful, whilst facilitate standardization of sample freezing time. We considered factors such RNA and DNA yield and purity. Furthermore, RNA integrity and RNA/DNA performance in routine analyses, such as qPCR, next generation sequencing or microarray, were also assessed. Our results indicate that freezing of tissue samples either with FlashFREEZE unit or isopentane ensures biological material with comparable expression profiles and DNA mutation status, indicating that RNA and DNA of similar quality can be extracted from both. Therefore, our findings support the use of the FlashFREEZE device in routine use for biobanking purposes.

Inactivating BTK mutations in large B-cell lymphoma in a real-world cohort: Strong correlation with BCL2 translocation.

Inactivating mutations in Bruton's tyrosine kinase (BTK) in patients with follicular lymphoma (FL) have recently been reported. These mutations were found in BTK inhibitor-treatment naïve patients. Here, we report the BTK mutation status in a real-world cohort of patients with non-Hodgkin lymphoma. We found primary BTK mutations in 7.7% of patients with large B-cell lymphoma (LBCL) and in 14.1% of patients with FL. All patients with BTK-mutated LBCL were BCL2 translocation positive, and the correlation between BCL2 translocation and BTK mutation persisted even when patients with known transformation from FL were excluded.

The value of next-generation sequencing in routine diagnostics and management of patients with cytopenia.

INTRODUCTION: We performed a single-center study of real-world health data to investigate the direct clinical consequence of targeted next-generation sequencing (NGS) results integrated in the clinicopathological evaluation of patients with cytopenia suspected of myelodysplastic syndrome (MDS). METHODS: The study included 87 newly referred patients, who had a bone marrow examination, which included targeted NGS analysis. NGS was requested at the discretion of either examining pathologist or hematologist. Data were collected retrospectively from patient files including pathology reports with integrated NGS results. RESULTS: The NGS results had a diagnostic impact in 67 cases (77%) when combining both histopathological and final clinical evaluation and provided prognostic value in 19 cases (22%). NGS supported a confident or tentative histopathological diagnosis in 52 cases (60%). Twenty cases (23%) had a final diagnosis of either Clonal Cytopenia of Undetermined Significance (CCUS) or Idiopathic Cytopenia of Undetermined Significance (ICUS). In 4 cases, NGS results affected the choice of principal treatment strategy, including considerations of allotransplantation. Twenty-one patients (24%) could be discharged to primary care physician. CONCLUSION: In a multidisciplinary clinicopathological real-world setting, NGS analysis of bone marrow samples from selected patients contributed substantially to the diagnostic evaluation and management of patients with cytopenia suspected of MDS. Consequently, we have now included NGS analysis in most routine bone marrow examinations from patients with MDS or unexplained cytopenia.