The E592K variant of SF3B1 creates unique RNA missplicing and associates with high-risk MDS without ring sideroblasts.

Among the most common genetic alterations in the myelodysplastic syndromes (MDS) are mutations in the spliceosome gene SF3B1. Such mutations induce specific RNA missplicing events, directly promote ring sideroblast (RS) formation, and generally associate with more favorable prognosis. However, not all SF3B1 mutations are the same, and little is known about how distinct hotspots influence disease. Here we report that the E592K variant of SF3B1 associates with high-risk disease features in MDS, including a lack of RS, increased myeloblasts, a distinct co-mutation pattern, and a lack of the favorable survival seen with other SF3B1 mutations. Moreover, compared to other hotspot SF3B1 mutations, E592K induces a unique RNA missplicing pattern, retains an interaction with the splicing factor SUGP1, and preserves normal RNA splicing of the sideroblastic anemia genes TMEM14C and ABCB7. These data have implications for our understanding of the functional diversity of spliceosome mutations, as well as the pathobiology, classification, prognosis, and management of SF3B1-mutant MDS.

Recipient clonal hematopoiesis in allogeneic bone marrow transplantation for lymphoid malignancies.

Allogeneic blood and marrow transplantation (alloBMT) is increasingly being used in older patients with blood cancer. Aging is associated with an increasing incidence of clonal hematopoiesis (CH). Although the effects of donor CH on alloBMT has been reported, the impact of recipient CH on alloBMT outcomes is unknown. In this retrospective study, alloBMT recipients age 60 and older with lymphoid malignancies were included. Among 97 consecutive patients who received alloBMT between 2017 and 2022, CH was detected in 60 (62%; 95% CI 51-72%). CH was found in 45% (95% CI 28-64%) of patients aged 60-64, 64% (95% CI 44-81%) of patients aged 65-69, and 73% (95% CI 59-87%) in those above 70. Pretransplant CH was associated with worse survival after alloBMT: 3-year overall survival (OS) was 78% (95% CI 65-94%) for patients without CH versus 47% (95% CI 35-63%) for those with CH, [unadjusted HR 3.1 (95%CI 1.4-6.8; P<0.001)]. Non-relapse mortality (NRM) was higher in patients with CH; cumulative incidence of NRM at one-year was 11% (95% CI 1-22%) versus 35% (95% CI 23-48%), [HR 3.4 (95% CI 1.4-8.5), p=0.009]. Among CH patients, worse OS and NRM was associated with CH burden and number of mutations. Recipient CH had no effect on relapse. In conclusion, older patients with CH experience worse outcomes after alloBMT, almost exclusively attributable to increased NRM. CH is a strong, independent predictor of outcomes. Novel strategies to ameliorate the adverse impacts of patient CH on transplant outcomes are being evaluated.

RET gene fusion and emergent Selpercatinib resistance in a calcitonin-rich neuroendocrine carcinoma: a case report.

Metastatic lung neuroendocrine carcinomas provide diagnostic challenges in identifying the cell of origin. High level calcitonin expression is not pathognomonic for medullary thyroid cancer. Tumor mutation analysis may provide essential clues regarding tissue origin and treatment targets. Oncogenic RET gene fusions have been identified in non-small cell lung cancer and non-medullary thyroid cancers, whereas RET point mutations are the key genetic finding in both inherited and sporadic MTC. Patients who receive radiation for the treatment of other cancers have an increased risk of developing a second malignancy, including a neuroendocrine carcinoma. Herein, we present a case of calcitonin-rich neuroendocrine carcinoma emerging on a background of prior radiation and chemotherapy for the treatment of Hodgkin's disease. Identification of a RET gene rearrangement (KIF5B-RET) led to initial successful treatment with selpercatinib, with eventual resistance associated with an activating mutation involving the MEK1 protein (MAP2K1 p. E102-I103 del) that led to relapse and progression of the disease.

Ring Chromosomes in Hematological Malignancies Are Associated with TP53 Gene Mutations and Characteristic Copy Number Variants.

Ring chromosomes (RC) are present in <10% of patients with hematological malignancies and are associated with poor prognosis. Until now, only small cohorts of patients with hematological neoplasms and concomitant RCs have been cytogenetically characterized. Here, we performed a conventional chromosome analysis on metaphase spreads from >13,000 patients diagnosed with hematological malignancies at the Johns Hopkins University Hospital and identified 98 patients with RCs-90 with myeloid malignancies and 8 with lymphoid malignancies. We also performed a targeted Next-Generation Sequencing (NGS) assay, using a panel of 642 cancer genes, to identify whether these patients harbor relevant pathogenic variants. Cytogenetic analyses revealed that RCs and marker chromosomes of unknown origin are concurrently present in most patients by karyotyping, and 93% of patients with NGS data have complex karyotypes. A total of 72% of these individuals have pathogenic mutations in TP53, most of whom also possess cytogenetic abnormalities resulting in the loss of 17p, including the loss of TP53. All patients with a detected RC and without complex karyotypes also lack TP53 mutations but have pathogenic mutations in TET2. Further, 70% of RCs that map to a known chromosome are detected in individuals without TP53 mutations. Our data suggest that RCs in hematological malignancies may arise through different mechanisms, but ultimately promote widespread chromosomal instability.

Recommendations for Cell-Free DNA Assay Validations: A Joint Consensus Recommendation of the Association for Molecular Pathology and College of American Pathologists.

Diagnosing, selecting therapy for, and monitoring cancer in patients using a minimally invasive blood test represents a significant advance in precision medicine. Wide variability exists in how circulating tumor DNA (ctDNA) assays are developed, validated, and reported in the literature, which hinders clinical adoption and may negatively impact patient care. Standardization is needed for factors affecting ctDNA assay performance and reporting, including pre-analytical variables, analytical considerations, and elements of laboratory assay reporting. The Association for Molecular Pathology Clinical Practice Committee's Liquid Biopsy Working Group (LBxWG), including organizational representation from the American Society of Clinical Oncology and the College of American Pathologists, has undertaken a full-text data extraction of 1228 ctDNA publications that describe assays performed in patients with lymphoma and solid tumor malignancies. With an emphasis on clinical assay validation, the LBxWG has developed a set of 13 best practice consensus recommendations for validating, reporting, and publishing clinical ctDNA assays. Recommendations include reporting key pre-analytical considerations and assay performance metrics; this analysis demonstrates these elements are inconsistently included in publications. The LBxWG recommendations are intended to assist clinical laboratories with validating and reporting ctDNA assays and to ensure high-quality data are included in publications. It is expected that these recommendations will need to be updated as the body of literature continues to mature.

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.

Novel PAX3::INO80D Fusion in Biphenotypic Sinonasal Sarcoma in an Adult.

This case report describes a novel gene fusion of PAX3::INO80D in a biphenotypic sinonasal carcinoma in a woman in her 40s.

Cell-free DNA measurable residual disease as a predictor of postallogeneic hematopoietic cell transplant outcomes.

The measurable residual disease (MRD) assessment provides an attractive predictor of allogeneic hematopoietic cell transplnat (alloHCT) outcomes. Cell-free DNA (cfDNA) has been applied to diagnosis, early detection, and disease burden monitoring in various tumors, but its utility as an MRD test in myeloid malignancies has not been systematically evaluated. We sought to determine the differential sensitivity between bone marrow (BM) and cfDNA MRD and to assess the effect of cfDNA MRD on alloHCT outcomes. The technical and clinical validation cohorts, including 82 patients participating in clinical trials (Bone Marrow Transplant Clinical Trials Network-0201 and 0402), were used. Ultradeep error-corrected targeted sequencing was performed on plasma and BM-derived DNA. We demonstrated that 94.6% (range, 93.9-95.3) of cfDNA was derived from hematopoietic tissue. The mutant allele fraction was congruent between BM and cfDNA (rho = 0.8; P < .0001); however, cfDNA seemed to be more sensitive in detecting clones with a variant allele frequency (VAF) of <0.26%. cfDNA-MRD clearance by day 90 after alloHCT (D90) was associated with improved relapse-free survival (RFS, median survival not reached vs 5.5 months; P < .0001) and overall survival (OS, median survival not reached vs 7.3 months; P < .0001) when compared with patients with persistent MRD. Irrespective of pre-alloHCT MRD, D90 cfDNA MRD was associated with inferior 2-year OS (16.7% vs 84.8%; P < .0001) and RFS (16.7% vs 80.7%; P < .0001). cfDNA seems to be an accurate, minimally invasive alternative to BM aspirates in MRD assessment and confers important prognostic implications in patients with myeloid malignancies undergoing alloHCT.

Complex/cryptic EWSR1::FLI1/ERG Gene Fusions and 1q Jumping Translocation in Pediatric Ewing Sarcomas.

Ewing sarcomas (ES) are rare small round cell sarcomas often affecting children and characterized by gene fusions involving one member of the FET family of genes (usually EWSR1) and a member of the ETS family of transcription factors (usually FLI1 or ERG). The detection of EWSR1 rearrangements has important diagnostic value. Here, we conducted a retrospective review of 218 consecutive pediatric ES at diagnosis and found eight patients having data from chromosome analysis, FISH/microarray, and gene-fusion assay. Three of these eight ES had novel complex/cryptic EWSR1 rearrangements/fusions by chromosome analysis. One case had a t(9;11;22)(q22;q24;q12) three-way translocation involving EWSR1::FLI1 fusion and 1q jumping translocation. Two cases had cryptic EWSR1 rearrangements/fusions, including one case with a cryptic t(4;11;22)(q35;q24;q12) three-way translocation involving EWSR1::FLI1 fusion, and the other had a cryptic EWSR1::ERG rearrangement/fusion on an abnormal chromosome 22. All patients in this study had various aneuploidies with a gain of chromosome 8 (75%), the most common, followed by a gain of chromosomes 20 (50%) and 4 (37.5%), respectively. Recognition of complex and/or cryptic EWSR1 gene rearrangements/fusions and other chromosome abnormalities (such as jumping translocation and aneuploidies) using a combination of various genetic methods is important for accurate diagnosis, prognosis, and treatment outcomes of pediatric ES.

Colorectal cancer in patients of advanced age is associated with increased incidence of BRAF p.V600E mutation and mismatch repair deficiency.

Introduction: The highest incidence of colorectal cancer (CRC) is in patients diagnosed at 80 years or older highlighting a need for understanding the clinical and molecular features of these tumors. Methods. In this retrospective cohort study, 544 CRCs underwent next generation sequencing and mismatch repair (MMR) evaluation. Molecular and clinical features were compared between 251 patients with traditional-onset CRC (50-69 years at diagnosis) and 60 with late-onset CRC (>80 years at diagnosis). Results: Late-onset CRC showed a significantly higher rate of right-sided tumors (82% vs 35%), MMR deficiency (35% vs. 8%) and BRAF p.V600E mutations (35% vs. 8%) and a significantly lower rate of stage IV disease (15% vs 28%) and APC mutations (52% vs. 78%). Association of these features with advanced age was supported by stratifying patients into 6 age groups (<40, 40-49, 50-59, 60-69, 70-79 and >80 years). However, the age-related rise in MMR deficient (dMMR) CRC was only seen in the female patients with an incidence of 48% (vs. 10% in the male patient) in the >80y group. In addition, BRAF p.V600E was significantly enriched in MMR deficient CRC of advanced age (67% in late-onset CRC). Categorizing CRC by mutational profiling, late-onset CRC revealed a significantly higher rate of dMMR/BRAF + APC - (18% vs. 2.0%), dMMR/BRAF - APC - (8.3% vs. 1.2%) and MMR proficient (pMMR)/BRAF + APC - (12% vs. 4.0%) as compared to traditional-onset CRC. Discussion: In summary, there was a higher rate of dMMR and BRAF p.V600E in late-onset CRC, independently or in combination. The higher incidence of dMMR in late-onset CRC in females is most likely predominantly driven by BRAF p.V600E induced hypermethylation. Prospective studies with treatment plans designed specifically for these older patients are warranted to improve their outcomes.

Co-occurring mutations in ASXL1, SRSF2, and SETBP1 define a subset of myelodysplastic/ myeloproliferative neoplasm with neutrophilia.

Identification of genomic signatures with consistent clinicopathological features in myelodysplastic/myeloproliferative neoplasm (MDS/MPN) is critical for improved diagnosis, elucidation of biology, inclusion in clinical trials, and development of therapies. We describe clinical and pathological features with co-existence of mutations in ASXL1 (missense or nonsense), SRSF2, and SKI homologous region of SETBP1, in 18 patients. Median age was 68 years with a male predominance (83%). Leukocytosis and neutrophilia were common at presentation. Marrow features included hypercellularity, granulocytic hyperplasia with megakaryocytic atypia, while the majority had myeloid hyperplasia and/or erythroid hypoplasia, myeloid dysplasia, and aberrant CD7 expression on blasts. Mutations in growth signaling pathways (RAS or JAK2) were noted at diagnosis or acquired during the disease course in 83% of patients. Two patients progressed upon acquisition of FLT3-TKD (acute myeloid leukemia) or KIT (aggressive systemic mastocytosis) mutations. The prognosis is poor with only two long-term survivors, thus far, who underwent blood or marrow transplantation. We propose that the presence of co-occurring ASXL1, SRSF2, and SETBP1 mutations can be diagnostic of a subtype of MDS/MPN with neutrophilia if clinical and morphological findings align. Our report underscores the association between genotype and phenotype within MDS/MPN and that genomic signatures should guide categorization of these entities.

Current clinical practices and challenges in molecular testing: a GOAL Consortium Hematopathology Working Group report.

While molecular testing of hematologic malignancies is now standard of care, there is variability in practice and testing capabilities between different academic laboratories, with common questions arising on how to best meet clinical expectations. A survey was sent to hematopathology subgroup members of the Genomics Organization for Academic Laboratories consortium to assess current and future practice and potentially establish a reference for peer institutions. Responses were received from 18 academic tertiary-care laboratories regarding next-generation sequencing (NGS) panel design, sequencing protocols and metrics, assay characteristics, laboratory operations, case reimbursement, and development plans. Differences in NGS panel size, use, and gene content were reported. Gene content for myeloid processes was reported to be generally excellent, while genes for lymphoid processes were less well covered. The turnaround time (TAT) for acute cases, including acute myeloid leukemia, was reported to range from 2 to 7 calendar days to 15 to 21 calendar days, with different approaches to achieving rapid TAT described. To help guide NGS panel design and standardize gene content, consensus gene lists based on current and future NGS panels in development were generated. Most survey respondents expected molecular testing at academic laboratories to continue to be viable in the future, with rapid TAT for acute cases likely to remain an important factor. Molecular testing reimbursement was reported to be a major concern. The results of this survey and subsequent discussions improve the shared understanding of differences in testing practices for hematologic malignancies between institutions and will help provide a more consistent level of patient care.

Familial Clonal Hematopoiesis in a Long Telomere Syndrome.

BACKGROUND: Telomere shortening is a well-characterized cellular aging mechanism, and short telomere syndromes cause age-related disease. However, whether long telomere length is advantageous is poorly understood. METHODS: We examined the clinical and molecular features of aging and cancer in persons carrying heterozygous loss-of-function mutations in the telomere-related gene POT1 and noncarrier relatives. RESULTS: A total of 17 POT1 mutation carriers and 21 noncarrier relatives were initially included in the study, and a validation cohort of 6 additional mutation carriers was subsequently recruited. A majority of the POT1 mutation carriers with telomere length evaluated (9 of 13) had long telomeres (>99th percentile). POT1 mutation carriers had a range of benign and malignant neoplasms involving epithelial, mesenchymal, and neuronal tissues in addition to B- and T-cell lymphoma and myeloid cancers. Five of 18 POT1 mutation carriers (28%) had T-cell clonality, and 8 of 12 (67%) had clonal hematopoiesis of indeterminate potential. A predisposition to clonal hematopoiesis had an autosomal dominant pattern of inheritance, as well as penetrance that increased with age; somatic DNMT3A and JAK2 hotspot mutations were common. These and other somatic driver mutations probably arose in the first decades of life, and their lineages secondarily accumulated a higher mutation burden characterized by a clocklike signature. Successive generations showed genetic anticipation (i.e., an increasingly early onset of disease). In contrast to noncarrier relatives, who had the typical telomere shortening with age, POT1 mutation carriers maintained telomere length over the course of 2 years. CONCLUSIONS: POT1 mutations associated with long telomere length conferred a predisposition to a familial clonal hematopoiesis syndrome that was associated with a range of benign and malignant solid neoplasms. The risk of these phenotypes was mediated by extended cellular longevity and by the capacity to maintain telomeres over time. (Funded by the National Institutes of Health and others.).

Clonal hematopoiesis in men living with HIV and association with subclinical atherosclerosis.

OBJECTIVES: People with HIV (PWH) are at increased risk for premature cardiovascular disease (CVD). Clonal hematopoiesis is a common age-related condition that may be associated with increased CVD risk. The goal of this study was to determine the prevalence of clonal hematopoiesis and its association with chronic inflammation and CVD in PWH. DESIGN: Cross-sectional study utilizing archived specimens and data from 118 men (86 PWH and 32 HIV-uninfected) from the Baltimore-Washington DC center of the Multicenter AIDS Cohort Study (MACS) who had had coronary computed tomography angiography (CTA) and measurement of 34 serologic inflammatory biomarkers. METHODS: Clonal hematopoiesis was assessed on peripheral blood mononuclear cells utilizing targeted error-corrected next generation sequencing (NGS) focused on 92 genes frequently mutated in hematologic malignancies. Clinical and laboratory data were obtained from the MACS database. RESULTS: Clonal hematopoiesis with a variant allele frequency (VAF) greater than 1% was significantly more common in PWH [20/86 (23.3%)] than in HIV-uninfected men [2/32 (6.3%)] ( P  = 0.035). PWH with clonal hematopoiesis (VAF > 1%) were more likely to have coronary artery stenosis of at least 50% than those without clonal hematopoiesis [6/20 (30%) vs. 6/64 (9%); P  = 0.021]. Presence of clonal hematopoiesis was not significantly associated with serological inflammatory markers, except for significantly lower serum leptin levels; this was not significant after adjustment for abdominal or thigh subcutaneous fat area. CONCLUSION: Clonal hematopoiesis was more common in PWH and among PWH was associated with the extent of coronary artery disease. Larger studies are needed to further examine the biological and clinical consequences of clonal hematopoiesis in PWH.

Utility of targeted next-generation sequencing assay to detect 1p/19q co-deletion in formalin-fixed paraffin-embedded glioma specimens.

Molecular classification of brain neoplasms is important for diagnosis, prognosis, and treatment outcome of histologically similar tumors. Oligodendroglioma is a glioma subtype characterized by 1p/19q co-deletion and IDH1/IDH2 mutations, which predict a good prognosis, responsiveness to therapy, and an improved overall survival compared to other adult gliomas. In a routine clinical setting, 1p/19q co-deletion is detected by interphase-FISH and SNP microarray, and somatic mutations are detected by targeted next-generation sequencing (NGS). The aim of this proof-of-principle study was to investigate the feasibility of using targeted NGS to simultaneously detect both 1p/19q co-deletion and somatic mutations. Among 247 consecutive patients with formalin-fixed paraffin-embedded brain tumors with various subtypes, NGS revealed 1p/19q co-deletion in 26 oligodendrogliomas and an IDH-wildtype astrocytoma, and partial loss across chromosomes 1p and 19q/whole-arm loss of 1p or 19q/copy neutral loss of heterozygosity in 11 nonoligodendrogliomas. For this 247 brain-tumor cohort, the overall sensitivity, specificity, and accuracy of detecting 1p/19q co-deletion by NGS in oligodendrogliomas were 96.2%, 99.6%, and 99.2%, respectively. The oligodendroglioma cohort had more mutations in IDH1/IDH2, CIC, FUBP1, and TERT, and fewer mutations in ATRX and TP53 than the nonoligodendroglioma cohort. This proof-of-concept study demonstrated that targeted NGS can simultaneously detect both 1p/19q co-deletion and somatic mutations, which can provide a more comprehensive genetic profiling for patients with gliomas using a single assay in a clinical setting.

Artificial Intelligence-Assisted Serial Analysis of Clinical Cancer Genomics Data Identifies Changing Treatment Recommendations and Therapeutic Targets.

PURPOSE: Given the pace of predictive biomarker and targeted therapy development, it is unknown whether repeat annotation of the same next-generation sequencing data can identify additional clinically actionable targets that could be therapeutically leveraged. In this study, we sought to determine the predictive yield of serial reanalysis of clinical tumor sequencing data. EXPERIMENTAL DESIGN: Using artificial intelligence (AI)-assisted variant annotation, we retrospectively reanalyzed sequencing data from 2,219 patients with cancer from a single academic medical center at 3-month intervals totaling 9 months in 2020. The yield of serial reanalysis was assessed by the proportion of patients with improved strength of therapeutic recommendations. RESULTS: A total of 1,775 patients (80%) had ≥1 potentially clinically actionable mutation at baseline, including 243 (11%) patients who had an alteration targeted by an FDA-approved drug for their cancer type. By month 9, the latter increased to 458 (21%) patients mainly due to a single pan-cancer agent directed against tumors with high tumor mutation burden. Within this timeframe, 67 new therapies became available and 45 were no longer available. Variant pathogenicity classifications also changed leading to changes in treatment recommendations for 124 patients (6%). CONCLUSIONS: Serial reannotation of tumor sequencing data improved the strength of treatment recommendations (based on level of evidence) in a mixed cancer cohort and showed substantial changes in available therapies and variant classifications. These results suggest a role for repeat analysis of tumor sequencing data in clinical practice, which can be streamlined with AI support.

Lynch syndrome caused by a novel deletion of the promoter and exons 1-13 of MLH1 gene.

Lynch syndrome (LS) is the most common hereditary cancer syndrome involving multiple organ systems. The mutation patterns of the involved major DNA mismatch repair (MMR) genes, namely MLH1, MSH2, MSH6, and PMS2, have not been fully elucidated. Herein, we report a case of LS caused by a novel large deletion in the promoter and exons 1-13 of MLH1 gene. A 30 year-old male was admitted for dull abdominal pain for 5 months with family history significant for dominant familial colon cancer. Abdominal computed tomography (CT) revealed masses in colon, lung and liver. His-plasma CA19-9 was 1250 units/ml and CEA 133 ng/ml. Targeted liver biopsy showed metastatic adenocarcinoma. Immunocytochemically, the tumor cells were positive for CK20 and CDX2, and displayed loss of MLH1 and PMS2 expression but with intact MSH2 and MSH6 proteins. Next-generation sequencing of the liver metastasis demonstrated copy loss of MLH1 gene spanning exons 1 to 13. Further SNP array detected copy neutral loss of heterozygosity (CN-LOH) expanding the short arm of chromosome 3p21.3 to 3pter regions and a 219 kb deletion involving the promoter and first 13 exons of MLH1 gene (arr[GRCh37] 3p22.2(36,856,328_37075457)x1). Germline sequencing using a blood sample confirmed the deletion of the MLH gene including the promoter and this first 13 exons (NG_007109.2(NM_000249.3:c.(?_-198)_(1558+1_1559-1)del). In summary, we identified a novel MLH1 mutation pattern of partial deletion and CN-LOH causing LS.