Citations

Abstract Myeloid neoplasms and acute leukemias derive from the clonal expansion of hematopoietic cells driven by somatic gene mutations. Although assessment of morphology plays a crucial role in the diagnostic evaluation of patients with these malignancies, genomic characterization has become increasingly important for accurate diagnosis, risk assessment, and therapeutic decision making. Conventional cytogenetics, a comprehensive and unbiased method for assessing chromosomal abnormalities, has been the mainstay of genomic testing over the past several decades and remains relevant today. However, more recent advances in sequencing technology have increased our ability to detect somatic mutations through the use of targeted gene panels, whole-exome sequencing, whole-genome sequencing, and whole-transcriptome sequencing or RNA sequencing. In patients with myeloid neoplasms, whole-genome sequencing represents a potential replacement for both conventional cytogenetic and sequencing approaches, providing rapid and accurate comprehensive genomic profiling. DNA sequencing methods are used not only for detecting somatically acquired gene mutations but also for identifying germline gene mutations associated with inherited predisposition to hematologic neoplasms. The 2022 International Consensus Classification of myeloid neoplasms and acute leukemias makes extensive use of genomic data. The aim of this report is to help physicians and laboratorians implement genomic testing for diagnosis, risk stratification, and clinical decision making and illustrates the potential of genomic profiling for enabling personalized medicine in patients with hematologic neoplasms.

Abstract In high-grade serous ovarian carcinoma (HGSC), deleterious mutations in DNA repair gene RAD51C are established drivers of defective homologous recombination and are emerging biomarkers of PARP inhibitor (PARPi) sensitivity. RAD51C promoter methylation (meRAD51C) is detected at similar frequencies to mutations, yet its effects on PARPi responses remain unresolved. In this study, three HGSC patient-derived xenograft (PDX) models with methylation at most or all examined CpG sites in the RAD51C promoter show responses to PARPi. Both complete and heterogeneous methylation patterns were associated with RAD51C gene silencing and homologous recombination deficiency (HRD). PDX models lost meRAD51C following treatment with PARPi rucaparib or niraparib, where a single unmethylated copy of RAD51C was sufficient to drive PARPi resistance. Genomic copy number profiling of one of the PDX models using SNP arrays revealed that this resistance was acquired independently in two genetically distinct lineages. In a cohort of 12 patients with RAD51C-methylated HGSC, various patterns of meRAD51C were associated with genomic “scarring,” indicative of HRD history, but exhibited no clear correlations with clinical outcome. Differences in methylation stability under treatment pressure were also observed between patients, where one HGSC was found to maintain meRAD51C after six lines of therapy (four platinum-based), whereas another HGSC sample was found to have heterozygous meRAD51C and elevated RAD51C gene expression (relative to homozygous meRAD51C controls) after only neoadjuvant chemotherapy. As meRAD51C loss in a single gene copy was sufficient to cause PARPi resistance in PDX, methylation zygosity should be carefully assessed in previously treated patients when considering PARPi therapy. Significance: Homozygous RAD51C methylation is a positive predictive biomarker for sensitivity to PARP inhibitors, whereas a single unmethylated gene copy is sufficient to confer resistance.

Abstract Acquired PARP inhibitor (PARPi) resistance in BRCA1- or BRCA2-mutant ovarian cancer often results from secondary mutations that restore expression of functional protein. RAD51C is a less commonly studied ovarian cancer susceptibility gene whose promoter is sometimes methylated, leading to homologous recombination (HR) deficiency and PARPi sensitivity. For this study, the PARPi-sensitive patient-derived ovarian cancer xenograft PH039, which lacks HR gene mutations but harbors RAD51C promoter methylation, was selected for PARPi resistance by cyclical niraparib treatment in vivo. PH039 acquired PARPi resistance by the third treatment cycle and grew through subsequent treatment with either niraparib or rucaparib. Transcriptional profiling throughout the course of resistance development showed widespread pathway level changes along with a marked increase in RAD51C mRNA, which reflected loss of RAD51C promoter methylation. Analysis of ovarian cancer samples from the ARIEL2 Part 1 clinical trial of rucaparib monotherapy likewise indicated an association between loss of RAD51C methylation prior to on-study biopsy and limited response. Interestingly, the PARPi resistant PH039 model remained platinum sensitive. Collectively, these results not only indicate that PARPi treatment pressure can reverse RAD51C methylation and restore RAD51C expression, but also provide a model for studying the clinical observation that PARPi and platinum sensitivity are sometimes dissociated.

Introduction: The NHLBI National MDS Study (NCT02775383) is a prospective cohort study conducted at 92 community hospitals and 29 academic centers. It enrolls patients undergoing work up for suspected MDS to understand the genetic, epigenetic, and biological factors associated with the initiation and progression of the disease. Previously untreated, cytopenic participants undergo both local and centralized pathology review and are assigned a diagnosis, including MDS, MDS/MPN, AML with blasts < 30%, and "Other". Emerging data suggests that Next Generation Sequencing (NGS), along with cytogenetics and clinical variables, may improve MDS diagnostic precision. Given that our study relies on central review (with additional tertiary pathology review used to adjudicate disagreements), we examined whether targeted gene sequencing data could be used to increase the agreement between local and central pathologic diagnosis of MDS vs. Other. Methods: Peripheral blood and bone marrow (BM) biopsy specimens from cytopenic patients, along with clinical history, CBC, and other results including karyotyping, FISH and pathology reports from local pathologists were reviewed by central pathologists. The updated 2016 WHO classifications were used to diagnose MDS. Targeted exon sequencing of 96 genes was performed using BM specimens. A subset of 648 individuals that were classified as MDS (n=212) or Other (n=436, including 90 CCUS and 89 individuals with other cancers) by pathology assessments were selected. A mean coverage of 1,317X was achieved and variants had a minimum variant allele frequency (VAF) of 2% (except FLT3). Variants for 596 subjects were manually reviewed to retain likely disease-causing variants to build a binary classifier (MDS vs. Other) using the maximum VAF per gene as input (Figure 1). Subjects diagnosed with MDS or Other by both central and local pathology were used for training, validation, and testing, and were considered "gold standard" (GS) cases (n=546). These subjects were split into 4 random groups with equal proportions of MDS cases. 75% of the GS cases were used to train and validate lasso-regularized logistic regression models using 3-fold cross validation. ROC curve analysis was carried out using the remaining 25% of GS cases (Test Set 1) on the best model to identify an optimal probability cut off point for classifying subjects as MDS. Model performance was then tested on 50 subjects for which the central and local pathology diagnosis disagreed (Test Set 2), as well as on 52 additional subjects irrespective of agreement (Test Set 3). Results : The best performing logistic regression model retained 7 genes as most informative in a discriminating diagnosis of MDS from Other based on their VAFs, in order of impact: TP53, SF3B1, U2AF1, ASXL1, TET2,STAG2, and SRSF2. We used this model to assign probabilities for each of the subjects in Test Set 1 and to estimate the performance using ROC analysis (Figure 1), resulting in a high area under the curve (AUC) of 0.89. We chose a probability cut-off of ≥0.17, being associated with a high percentage of correct classification of MDS with a sensitivity and specificity of 0.90 and 0.81, respectively. Among the cohort of 50 subjects with a discordant local and central pathology diagnosis (Test Set 2), the classifier accurately reassigned 37 subjects (accuracy = 74%) from the local to the central pathology. The blinded tertiary pathology reviewer agreed with central in all Test Set 2 cases. This included 24/34 MDS cases that had been labeled as Other by local pathology (positive predictive value [PPV]=0.89). 3/16 final pathology-classified Other cases were mis-classified as MDS by the local pathologist (negative predictive value [NPV] = 0.57). Next, we assessed the ability of the model to predict MDS vs. Other for 52 additional independent subjects using the third pathologist's diagnosis to break any ties (Test Set 3). The classifier correctly predicted 15/21 MDS cases (PPV=0.83) and misclassified 6/31 Others as MDS (NPV=0.82). The overall accuracy was 83%. Conclusions: We identified that VAFs for 7 genes can correctly re-classify subjects as either MDS or Other in 74% of cases that were misclassified between local and central pathology review. Further assessment on an independent cohort showed an accuracy of 83% of the model. Taken together, these data suggest that complementing pathology reviews with targeted sequencing of 7 genes could improve MDS diagnosis. Disclosures Lindsley: MedImmune: Research Funding; Jazz Pharmaceuticals: Consultancy, Research Funding; Bluebird Bio: Consultancy; Takeda Pharmaceuticals: Consultancy. Bejar:Aptose Biosciences: Current Employment; AbbVie/Genentech: Honoraria; Astex/Otsuka: Honoraria; Takeda: Honoraria, Research Funding; Celgene/BMS: Honoraria, Research Funding; Daiichi-Sankyo: Honoraria; Forty-Seven/Gilead: Honoraria; Genoptix/NeoGenomics: Honoraria. DeZern:MEI: Consultancy; Astex: Research Funding; Abbvie: Consultancy; Celgene: Consultancy, Honoraria. Foran:H3Biosciences: Research Funding; Aptose: Research Funding; Kura Oncology: Research Funding; Trillium: Research Funding; Takeda: Research Funding; Revolution Medicine: Consultancy; Xencor: Research Funding; Agios: Honoraria, Research Funding; Aprea: Research Funding; Actinium: Research Funding; Servier: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Research Funding. Gore:Abbvie: Consultancy, Honoraria, Research Funding. Komrokji:Acceleron: Honoraria; Incyte: Honoraria; Abbvie: Honoraria; Agios: Speakers Bureau; BMS: Honoraria, Speakers Bureau; Jazz: Honoraria, Speakers Bureau; Geron: Honoraria; Novartis: Honoraria. Maciejewski:Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria. Padron:Novartis: Honoraria; BMS: Research Funding; Incyte: Research Funding; Kura: Research Funding. Starczynowski:Captor Therapeutics: Consultancy; Tolero Therapeutics: Research Funding; Kurome Therapeutics: Consultancy, Current equity holder in private company, Research Funding. Sekeres:BMS: Consultancy; Takeda/Millenium: Consultancy; Pfizer: Consultancy.

Introduction: Myelodysplastic syndromes (MDS) are a heterogenous group of blood disorders defined by peripheral cytopenia(s), bone marrow failure, morphological dysplasia, and risk of progression. To understand the genetic, epigenetic and biological factors associated with the initiation and progression of MDS, NHLBI created the National MDS Study (NCT02775383). This is a prospective cohort study conducted at 92 community hospitals and 29 academic centers enrolling patients undergoing diagnostic work up for suspected MDS or MDS/myeloproliferative neoplasm (MPN) overlap syndrome. Eligible patients have yet to receive any therapy directed at their cytopenias. Previously untreated cytopenic participants underwent centralized histopathology and data review at the time of enrollment for assignment into distinct subcategories: MDS, MDS/MPN overlap, AML, and Other. Targeted exon sequencing of 96 genes was performed using marrow specimens from the first 300 consecutive individuals in the study. Here we report the genetic mutations for this cohort. Methods: NovaSeq 6000 was used for deep sequencing at a mean coverage of 1,286X and mean breadth (bases covered at ≥100X) of 99.8%. Reads were aligned against build GRCh38 using BWA-MEM, and VarScan2 was used to detect SNVs and INDELS. Variants were filtered for those with an allele base quality of >25 in combination with rule-based and manual review criteria. Subjects in the Other category without an identified malignancy were considered clonal cytopenias of undetermined significance (CCUS) when a mutation or a clonal cytogenetic change was present. Fisher's exact and Wilcoxon rank sum tests in combination with Bonferroni correction were applied to compare groups. Results : A total of 350 putative nonsynonymous pathogenic variants in 36 genes with an allele frequency of >.05 were identified across 150 patients (50%). At least one variant was noted in the following proportion of individuals: 61/72 (85%) with MDS, 13/13 (100%) with MDS/MPN, 15/17 (88%) with AML, and 61/198 (31%) in the Other category, of which 48 were CCUS and 13 were other cancers. Two CCUS patients only had a cytogenetic abnormality. Table 1 shows the distribution of variants in each subcategory of patients for the most commonly mutated genes in our cohort of 300 subjects. Mutations in these genes were enriched in specific groups: SF3B1, STAG2,TP53, and ASXL1 in MDS; TET2 in MDS/MPN; and IDH2 and TP53 in AML (one-sided p<0.0012). None were enriched in the Other group. Within the CCUS subset, 21 genes were mutated, with 37 of 50 (74%) patients having a mutation in TET2, ASXL1, SRSF2, SF3B1, or DNMT3A. The heatmap presented in Figure 1 summarizes variants by subject and allele frequency. Pair-wise comparisons of baseline characteristics of subjects between MDS, MDS/MPN, AML, or CCUS groups revealed no significant differences for age or sex. The CCUS group had significantly higher hemoglobins than the MDS group with median hemoglobin levels of 11.35 and 9.40 g/dL, respectively (p<0.0084). AML had significantly lower platelets compared to all other groups (median of 53 vs. > 110x109/L , respectively, p<0.0084). All groups significantly differed in their median ANC with AML having the lowest (0.8x109/L), followed by MDS (1.5x109/L), CCUS (2.45x109/L), and MDS/MPN overlap (5.95x109/L) (p<0.0084). There was no difference in the median number of variants per patient between groups or correlation with age (rs=0.11, p=0.18). The maximum variant allele frequency (maxVAF) per patient was highest in the MDS/MPN group (median = 0.42, range = 0.38-0.91) and lowest in the CCUS group (median = 0.37, range =0.06-0.98) with the MDS/MPN group having a significantly higher maxVAF compared to the MDS and the CCUS groups (p<0.0084). The proportions of subjects with mutations was similar for those who had abnormal (92% [34/37]) and normal (91% [80/88]) cytogenetics. Conclusions: Incorporation of gene-panel sequencing in the comprehensive evaluation of 300 adult cytopenic patients identified half of the cohort with potentially pathogenic variants. Ultimately, a diagnosis of CCUS was possible in 48 of 183 subjects (26%) not diagnosed with MDS, MDS/MPN overlap syndrome, AML, other cancers or clonal cytogenetics. This study continues to serially bank samples from patients with CCUS, in addition to MDS, MDS/MPN, and ICUS, with the goal to better understand the natural history of these diseases and their progression. Disclosures Lindsley: Jazz Pharmaceuticals: Research Funding; Takeda Pharmaceuticals: Consultancy; Medlmmune: Research Funding. Bejar:Celgene: Consultancy; Takeda Pharmaceuticals: Research Funding; AbbVie/Genentech: Consultancy, Honoraria; Astex/Otsuka: Consultancy; Modus Outcomes: Consultancy; Daiichi-Sankyo: Consultancy. Al Baghdadi:Celgene: Consultancy, Honoraria; Heron: Consultancy, Honoraria; Tracon: Equity Ownership; Epizyme: Equity Ownership; Bristol Myer Squibb: Consultancy, Honoraria; Sunesis: Equity Ownership; Portola: Equity Ownership; Heron therapeutics: Equity Ownership; Cardinal health: Consultancy, Honoraria; Bristol Myer Squibb: Equity Ownership; Celgene: Equity Ownership; Spectrum pharmaceutical: Equity Ownership; Astrazeneca: Equity Ownership; Seattle genetics: Equity Ownership; Roche: Consultancy, Honoraria. DeZern:Astex Pharmaceuticals, Inc.: Consultancy; Celgene: Consultancy. Foran:Agios: Honoraria, Research Funding. Gore:Celgene Corporation: Consultancy, Research Funding. Komrokji:DSI: Consultancy; Agios: Consultancy; Novartis: Speakers Bureau; JAZZ: Speakers Bureau; pfizer: Consultancy; celgene: Consultancy; Incyte: Consultancy; JAZZ: Consultancy. Maciejewski:Alexion: Consultancy; Novartis: Consultancy. Starczynowski:Kurome Therapeutics: Consultancy. Sekeres:Millenium: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Purpose This study compared orofacial strength between adults with dysarthria and neurologically normal (NN) matched controls. In addition, orofacial muscle weakness was examined for potential relationships to speech impairments in adults with dysarthria. Method Matched groups of 55 adults with dysarthria and 55 NN adults generated maximum pressure (P max ) against an air-filled bulb during lingual elevation, protrusion and lateralization, and buccodental and labial compressions. These orofacial strength measures were compared with speech intelligibility, perceptual ratings of speech, articulation rate, and fast syllable-repetition rate. Results The dysarthria group demonstrated significantly lower orofacial strength than the NN group on all tasks. Lingual strength correlated moderately and buccal strength correlated weakly with most ratings of speech deficits. Speech intelligibility was not sensitive to dysarthria severity. Individuals with severely reduced anterior lingual elevation P max (< 18 kPa) had normal to profoundly impaired sentence intelligibility (99%–6%) and moderately to severely impaired speech (26%–94% articulatory imprecision; 33%–94% overall severity). Conclusions Results support the presence of orofacial muscle weakness in adults with dysarthrias of varying etiologies but reinforce tenuous links between orofacial strength and speech production disorders. By examining individual data, preliminary evidence emerges to suggest that speech, but not necessarily intelligibility, is likely to be impaired when lingual weakness is severe.

Abstract High-grade epithelial ovarian carcinomas containing mutated BRCA1 or BRCA2 (BRCA1/2) homologous recombination (HR) genes are sensitive to platinum-based chemotherapy and PARP inhibitors (PARPi), while restoration of HR function due to secondary mutations in BRCA1/2 has been recognized as an important resistance mechanism. We sequenced core HR pathway genes in 12 pairs of pretreatment and postprogression tumor biopsy samples collected from patients in ARIEL2 Part 1, a phase II study of the PARPi rucaparib as treatment for platinum-sensitive, relapsed ovarian carcinoma. In 6 of 12 pretreatment biopsies, a truncation mutation in BRCA1, RAD51C, or RAD51D was identified. In five of six paired postprogression biopsies, one or more secondary mutations restored the open reading frame. Four distinct secondary mutations and spatial heterogeneity were observed for RAD51C. In vitro complementation assays and a patient-derived xenograft, as well as predictive molecular modeling, confirmed that resistance to rucaparib was associated with secondary mutations. Significance: Analyses of primary and secondary mutations in RAD51C and RAD51D provide evidence for these primary mutations in conferring PARPi sensitivity and secondary mutations as a mechanism of acquired PARPi resistance. PARPi resistance due to secondary mutations underpins the need for early delivery of PARPi therapy and for combination strategies. Cancer Discov; 7(9); 984–98. ©2017 AACR. See related commentary by Domchek, p. 937. See related article by Quigley et al., p. 999. See related article by Goodall et al., p. 1006. This article is highlighted in the In This Issue feature, p. 920