Panel Of Mutations Research Articles

Simultaneous analysis of mutations and methylations in circulating cell-free DNA for hepatocellular carcinoma detection.

Cell-free DNA (cfDNA)-based liquid biopsy is a promising approach for the early detection of cancer. A major hurdle is the limited yield of cfDNA from one blood draw, limiting the use of most samples to one test of either mutation or methylation. Here, we develop a technology, Mutation Capsule Plus (MCP), which enables multiplex profiling of one cfDNA sample, including simultaneous detection of genetic and epigenetic alterations and genome-wide discovery of methylation markers. With this technology, we performed de novo screening of methylation markers on cfDNA samples from 30 hepatocellular carcinoma (HCC) cases and 30 non-HCC controls. The methylation markers enriched in HCC cfDNA were further profiled in parallel with a panel of mutations on a training cohort of 60 HCC and 60 non-HCC cases, resulting in an HCC detection model. We validated the model in an independent retrospective cohort with 58 HCC and 198 non-HCC cases and got 90% sensitivity with 94% specificity. Furthermore, we applied the model to a prospective cohort of 311 asymptomatic hepatitis B virus carriers with normal liver ultrasonography and serum AFP concentration. The model detected four of the five HCC cases in the cohort, showing 80% sensitivity and 94% specificity. These findings demonstrate that the MCP technology has potential for the discovery and validation of multiomics biomarkers for the noninvasive detection of cancer. This study also provides a comprehensive database of genetic and epigenetic alterations in the cfDNA of a large cohort of HCC cases and high-risk non-HCC individuals.

Towards a single-assay approach: a combined DNA/RNA sequencing panel eliminates diagnostic redundancy and detects clinically-relevant fusions in neuropathology

Since the introduction of integrated histological and molecular diagnoses by the 2016 World Health Organization (WHO) Classification of Tumors of the Nervous System, an increasing number of molecular markers have been found to have prognostic significance in infiltrating gliomas, many of which have now become incorporated as diagnostic criteria in the 2021 WHO Classification. This has increased the applicability of targeted-next generation sequencing in the diagnostic work-up of neuropathology specimens and in addition, raises the question of whether targeted sequencing can, in practice, reliably replace older, more traditional diagnostic methods such as immunohistochemistry and fluorescence in-situ hybridization. Here, we demonstrate that the Oncomine Cancer Gene Mutation Panel v2 assay targeted-next generation sequencing panel for solid tumors is not only superior to IHC in detecting mutation in IDH1/2 and TP53 but can also predict 1p/19q co-deletion with high sensitivity and specificity relative to fluorescence in-situ hybridization by looking at average copy number of genes sequenced on 1p, 1q, 19p, and 19q. Along with detecting the same molecular data obtained from older methods, targeted-next generation sequencing with an RNA sequencing component provides additional information regarding the presence of RNA based alterations that have diagnostic significance and possible therapeutic implications. From this work, we advocate for expanded use of targeted-next generation sequencing over more traditional methods for the detection of important molecular alterations as a part of the standard diagnostic work up for CNS neoplasms.

Genetic Mutation Signature for Relapse Prediction in Normal Karyotype Acute Myeloid Leukemia

Background: Risk stratification for normal karyotype acute myeloid leukemia (NK-AML) remains unsatisfactory owing to the high incidence of leukemia relapse. This study aimed to evaluate the role of gene mutations and clinical characterization in predicting the relapse of patients with NK-AML. Methods: A prognostic system for NK-AML was constructed based on gene mutations, measurable residual disease (MRD), and clinical characteristics. A panel of gene mutations was explored using next-generation sequencing. The least absolute shrinkage and selection operator, and nomogram algorithm were used to build a genomic mutation signature (GMS) and nomogram (GMSN) model that combines GMS, MRD, and clinical factors to predict relapse in 347 patients with NK-AML from four centers. Results: Patients with GMS-high had a higher 5-year incidence of relapse than those with GMS-low (P < 0.001). The 5-year incidence of relapse was also higher in patients with GMSN-high than those with GMSN-intermediate and -low (P < 0.001). The 5-year disease-free survival and overall survival rates were lower in patients with GMSN-high than in those with GMSN-intermediate and -low (P < 0.001) as confirmed by training and validation cohorts. Conclusions: This study illustrated the potential of GMSN as a predictor of NK-AML relapse.

Thrombophilia Gene Mutations for the Prediction of Venous Thromboembolism in Ambulatory Cancer Patients Receiving Chemotherapy

Inherited thrombophilia and cancer are both independently known to increase the risk of venous thromboembolism (VTE) due to genetic procoagulant predisposition, tissue damage and inflammatory mediators, respectively. The role of inherited thrombophilia on the risk of VTE in cancer patients is unclear and determination of thrombophilic status is not part of clinical practice. Our objective was to determine the prevalence of common thrombophilia gene mutations and their influence on the risk of developing VTE in a cohort of moderate to high-risk ambulatory cancer patients receiving chemotherapy. We used buffy coat blood samples at the baseline visit from patients enrolled in the AVERT trial (a randomized, placebo-controlled, double-blind trial to assess the efficacy and safety of apixaban as primary thromboprophylaxis in ambulatory cancer patients with intermediate to high risk for VTE) to determine if a panel of thrombophilia gene mutations (including Prothrombin G20210A mutation, Factor XI, Fibrinogen gamma chain (FGG), SERPINA10, Factor V K858R, Factor XIII, Factor V Leiden and, ABO blood gene mutations) is associated with a diagnosis of VTE during the 7-months follow-up after randomization (which in all cases coincided with the start of chemotherapy). Bivariate tests of association were performed using the Fisher exact test. Logistic regression analysis was used to calculate unadjusted and adjusted odds ratios (OR) with 95% confidence intervals comparing heterozygous and homozygous mutations (combined) to wild type. When a gene mutation was rare or absent in the cohort, we used exact logistic regression to obtain OR estimates. We adjusted for age, sex, anticoagulation treatment group, and concomitant antiplatelet therapy. 438 patients (260 females) (76.3% of the entire cohort of 574) with available samples were included in this study. The patients' median age was 62. During the 7-months of follow-up, there were 39 VTE events including 25 deep vein thrombosis (DVT), 13 pulmonary embolisms (PE), and 1 DVT with PE. Among the 39 (8.90%) patients who suffered a VTE event, the prevalence of mutations was Prothrombin G20210A mutation: 2.56%, Factor XI: 79.5%, FGG: 31.6%, SERPINA10: 0%, Factor V K858R: 35.9%, Factor XIII: 44.7%, Factor V Leiden: 15.4% and ABO: 79.5%, compared to Prothrombin G20210A mutation: 2.01%, Factor XI: 75.2%, FGG: 42.0%, SERPINE10: 0.75%, Factor V K858R: 45.6%, Factor XIII: 43.7%, Factor V Leiden: 3.52% and ABO: 58.9% in those who did not develop a VTE (n=399, Table 1). The odds of VTE were significantly increased in patients with Factor V Leiden mutation compared to those without the mutation, in both unadjusted and adjusted models [OR: 4.99 (p=0.0020) and adjusted OR: 5.84 (p=0.0011)] (Table 2). Patients with non-O blood type also had increased odds of VTE compared to those with O blood type before and after adjustment [OR: 2.70 (p=0.0153) and adjusted OR: 2.71 (p=0.0156)] (Table 2). No other thrombophilia genes were significantly associated with the occurrence of VTE (Table 2). In conclusion, our data indicate that some thrombophilia gene mutations may be important predictors for VTE in cancer patients. The Factor V Leiden and ABO gene mutations independently predicted VTE in this cohort of moderate to high-risk ambulatory cancer patients receiving chemotherapy. Whether testing for thrombophilia gene mutations can help risk stratify or influence outcomes should be further explored in prospective studies before clinical application. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Impaired Clearance of DNMT3A/TET2/ASXL1-Mutant Clones with Hypomethylating Agent or Intensive Induction Chemotherapy for Myelodysplastic Syndrome and Acute Myeloid Leukemia

Background: Mutations in the epigenetic regulators or chromatin modifiers DNMT3A, TET2, and ASXL1, collectively termed DTA mutations, are common early hits in myeloid neoplasms, including myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Our group has recently shown the impact of pre-transplant mutation status on survival for patients with DTA-mutant clones, in addition to IDH1/2- and EZH2-mutant clones (Selove et al., eJHaem. 2022; 2:514-19). In principle, hypomethylating agent (HMA)-based therapy might specifically eliminate TET2-mutant clones. Wild-type TET2 is a demethylase, and TET2 mutation imparts a hypermethylated phenotype that could potentially be reversed by HMAs. However, this is rarely observed in the clinical setting. Methods: Identification of patients with DTA mutations from the UMass Chan Leukemia Registry was performed via UMass Clarity, a Microsoft SQL Server database comprised of 23,000 tables, in conjunction with the main reporting environment in EPIC. The Registry identified patients harboring one or more aberrations in the DTA cluster and with ICD-10 codes of either D46.9 (MDS, including its subentities) or C92.00 (AML, including its subentities) between 2011-2021. We evaluated the frequency of DTA mutations in 495 patients with confirmed diagnoses of myeloid malignancies, of whom 36 (7.3%) harbored DNMT3A mutation(s), 54 (10.9%) harbored TET2 mutation(s), and 32 (6.5%) harbored ASXL1 mutation(s) (Panel A). Responses to treatment were assessed through serial bone marrow examinations, which were available for 21 patients with DNMT3A mutations, 22 patients with TET2 mutations, and 14 patients with ASXL1 mutations. We investigated the effects of HMA- and cytarabine-based therapies on DTA-mutant clones to discern the effectiveness of first-line treatment. Results:DNMT3A mutations persisted in 21 (100%) of 21 patients assessed who received first-line HMA-based therapy (n = 14) or cytarabine-based therapy (n = 7) (Panel B). In 20 (95.2%) of the 21 patients assessed with DNMT3A mutations, HMA-based (13 of 14 patients) and cytarabine-based (7 of 7 patients) therapy resulted in relative clonal enrichment of DNMT3A mutations, despite eliminating clones harboring mutations in other biological clusters. Of note, targeted therapy often extinguished the susceptible mutation but did not reduce the burden of the DNMT3A-mutant clone. In contrast, the DNMT3A-mutant clone was eradicated in all 10 (100%) of the patients who underwent allogeneic hematopoietic cell transplant (allo-HCT). TET2 mutations persisted in 19 (100%) of 19 patients who received first-line HMA-based (n = 14) or cytarabine-based (n = 5) therapy (Panel B). The relative TET2 mutational burden increased in 16 (84.2%) of the 19 patients treated with HMA-based or cytarabine-based therapy. In 2 patients, the original TET2 mutation disappeared, but a new TET2 mutation emerged. The TET2 mutation(s) were eradicated in 6 (85.7%) of 7 patients who underwent allo-HCT. ASXL1 mutations persisted in 9 (81.8%) of 11 patients treated with HMA and 2 (66.6%) of 3 patients treated with cytarabine-based chemotherapy (Panel B). Of the 3 patients who had clearance of the ASXL1-mutant clone, clearance was transient in 2 patients, and 1 patient had expansion of a TP53-mutant clone. In 12 (85.7%) of 14 patients treated with HMA-based therapy or cytarabine-based therapy, the relative ASXL1-mutant clonal size increased. ASXL1 mutations were eradicated in 2 (66.7%) of 3 patients who underwent allo-HCT. For ASXL1 with TP53 co-mutation, the TP53-mutant clone expanded on HMA-based therapy. Conclusions: Neither HMA-based nor cytarabine-based therapy reliably eradicated DTA-mutant clones. In fact, the relative clonal size of DTA mutations increased after such therapy, despite morphologic remission in many cases, highlighting the resistance of DTA-mutant cells. However, subsequent allo-HCT was effective at eliminating DTA-mutant clones (including 100% of DNMT3A-mutant clones). A successful therapeutic strategy for patients with DTA mutations may therefore be initial treatment with either HMA- or cytarabine-based therapy for disease control, followed by allo-HCT for curative intent, when feasible. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Rapid Genotyping Assay for CSF Accelerates Diagnosis and Treatment Initiation for CNS Malignancies

The overlapping clinical and radiographic features of CNS neoplasms, combined with low yields of conventional CSF studies, complicate diagnostic workflows and delay treatment initiation. Neurosurgical biopsies of high-risk lesions are frequently required, and decisions regarding extent of surgical resection are challenging. In order to address these challenges, we previously developed the Targeted Rapid Sequencing (TetRS) assay to detect highly recurrent point mutations characteristic of CNS neoplasms from CSF, within 80 minutes of sample collection. This qPCR-based panel offers parallel detection of MYD88, TERT promoter, IDH1/2, BRAF and H3F3A point mutations. In this study, we describe the clinical performance and real-time utilization of this assay in a large, single-center prospective patient sample. We asked whether rapid point mutation detection from CSF liquid biopsies can (1) accelerate times to diagnosis and (2) provide a basis for safely initiating disease-directed therapies in the absence of solid tissue sampling. We collected CSF from 98 patients with new CNS lesions suspicious for neoplasms over an 18-month period. Patient enrollment, selection of specific assay variants, and utilization of results in clinical decision-making were at the discretion of providing physicians. IRB approval was obtained. In this diverse cohort, 40 of 98 patients ultimately established a diagnosis of a CNS neoplasm. A point mutation was detected by TetRS in 15 out of 40 patients with a neoplasm (37.5%); this included cases of primary and secondary CNS lymphoma (PCNSL), glioblastoma, IDH-mutant brainstem glioma and H3K27M-mutant diffuse midline glioma. In time-to-event analyses, we found that detection of a mutation by TetRS accelerated the return of diagnostic information from CSF sampling when compared to contemporaneous and historical control cases in which a mutation was not detected (median 0.5 vs 10.5 days, P<0.01). We found multiple cases in which mutation detection by TetRS helped to avoid neurosurgical biopsy. In one representative case, detection of the MYD88 L265P mutation established the diagnosis of PCNSL; methotrexate and rituximab were initiated within 3 days of CSF sampling. In this patient, TetRS results over serial CSF samplings correlated with treatment response and detecting disease recurrence. In another representative case, a lesion initially thought to be an H3K27M-mutant diffuse midline glioma on clinical and radiographic grounds was instead diagnosed as an IDH-mutant brainstem glioma based on TetRS detection of the IDH1 R132G mutation in CSF. This enabled rapid initiation of fractionated radiation, as well as targeted IDH inhibitor therapy; the patient experienced clinical and radiographic response over 10 months of follow-up. TetRS thus represents a novel, targeted point mutation panel that can rapidly and specifically diagnose a broad array of CNS neoplasms. We demonstrate the potential of this novel tool to accelerate diagnosis, follow treatment response and detect recurrence in selected patients without solid tissue sampling. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Mutation Spectrum, Characteristics and Impact of Mutation Profiling on Prognosis, Outcome and Treatment Responses in Patients (pts) with Mantle Cell Lymphoma

Background: Genomic profiles of mantle cell lymphoma (MCL) are associated with clinical outcomes. BTKi Resistance in MCL can be predicted with mutation profile. Identifying these patients (pts) early can help guide therapy decisions and predict outcomes. We describe results from a clinical NGS based assay with a customized 162-gene panel in routine work-ups of MCL pts. Materials and Methods: We reviewed data from MCL patients who underwent CLIA approved lymphoma gene panel testing at MDACC from October 2020 until the end of June 2022. Data on treatment response, outcomes, and patient characteristics were collected. Mutation panel testing was conducted on various patient samples. Genomic DNA was isolated from fresh bone marrow (BM) aspirate, formalin-fixed paraffin-embedded (FFPE) tissues, fine needle aspirate (FNA) and peripheral blood (PB) specimens. Sequencing was performed using the Illumina NextSeq platform (Illumina, San Diego, CA). Variant calling was performed using Agilent SureCall software v4.1 and post-variant call analysis and annotation were performed using an in-house bioinformatics pipeline (OncoSeek v 1.1). Analytical sensitivity of the assay was established at 2% using a per base coverage cut-off of 200x. The assay was performed in cases with >10% lymphoma cells in the sample. Results: We included 227 MCL pts in this study: 129 treatment naïve and 97 with prior therapies. The distribution of patients according to the sample type included: BM (n=107), FFPE (n=50; 24 lymph node, 20 GI tract, and 6 miscellaneous tissues), FNA from involved nodal and non-nodal tissues (n=50) and PB (n=20). Baseline pt features included a median age of 67 years (range, 38-88 years). There were 164 men and 63 women. Histomorphology was classic in 165 pts, blastoid in 31, pleomorphic in 19 and blastoid/pleomorphic in 2. Ten pts had unclear histomorphology. Ki-67% in involved tissues was evaluable in 180 pts (in 47 pts Ki-67% was not available). Ki-67% was high (>=30%) in 98 and low in 82 biopsy specimens. Among the 97 previously treated pts, 47 had BTKi refractory disease. Twenty-three pts were treated with standard of care brexucabtagene autoleucel (CART). The spectrum of gene mutations is depicted by both previously untreated and pts with prior therapy in figure-1A. The most common mutated genes were: ATM (51.5%), TP53 (29.5%), KMT2D (21.1%), CCND1 (19.3%), BIRC3 (16.2%), NSD2 (11.4%), SMARCA4 (10.1%), UBR5 (9%), NOTCH1 (8.3%), CARD11 (7%), SAMHD1 (7%), NFKB1E (6.6%), SP140 (6.6%), S1PR1 (6.6%), DNMT3A (6%), NOTCH2 (6%), IGLL5 (6%), TRAF2 (5%), TET2 (5%). Unlike chronic lymphocytic leukemia, mutations of BTK (n=5; 2%) and PLCg (n=4; 2%) were rare. BTK mutations included missense mutations: C481Y, C481R, T474I and 2 with L528W. All patients with BTK mutations were resistant to prior BTKi and 4/5 died with refractory disease. Additional mutations of prognostic interest in other lymphomas were detected at very low frequency (<3%) and included CDKN2A, CD79b, MYD88, MEF2B, MAP3K14, Rb1, PAX5, SPEN, SF3B1, DIS3. We also evaluated pt survival from the date of gene panel testing and noted that TP53 mutant MCL pts had significantly inferior survival. We further divided the pts based on >=3 mutations (n=77) vs < 3 mutations (n=150) and identified that pts with a higher number of mutations had inferior survival, P <0.0001 (Figure 1C). Pts with SMARCA4 and TRAF2 mutations also demonstrated shorter survival compared to those which were wild type (not shown). Twenty-three pts received CART therapy and 7 had progressed and 16 have not. The distribution of mutations before CART in the two groups was not significantly different. Among the 47 BTKi refractory MCL, the frequency of certain mutations was higher: TP53 (49%), ATM (55%), KMT2D (31%), CCND1 (23%), 10% each for SMARCA4, NSD2 and SP140. Currently, we are utilizing this test to evaluate and include pts with high risk MCL on our clinical trials in MCL and treat with risk stratified treatments. Conclusion: The clinical NGS-based assay for MCL pts at our institution has proven to have prognostic significance. Resistant MCL pts exhibit a preponderance of mutations involving TP53, epigenetic modifier and chromatin regulator genes. Comprehensive risk stratification of pts early on and during therapy is very useful and improves patient care, helps design next generation clinical trials and improves our understanding of the biology of resistant MCL in the BTKi and CART era. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Survival and Outcomes after Relapse in Newly Diagnosed Acute Myeloid Leukemia (AML) Patients Treated with Venetoclax-Based Chemotherapy

Background Venetoclax (bcl-2 inhibitor) was first approved in 2018 for use in combination with hypomethylating agents (HMA) or low-dose cytarabine (LDAC) for treatment of newly diagnosed acute myeloid leukemia (AML) in older patients, or those who have comorbidities that preclude use of intensive induction chemotherapy. However, AML is also known to evolve and undergo clonal selection when treated with venetoclax, leading to treatment failure. Mechanisms for adaptive resistance include kinase activation (e.g. FLT3-ITD), clones that acquire biallelic silencing of TP53, and monocytic AML cells that upregulate MCL1 to evade BCL2 blockade. Other issues with venetoclax-based regimens include prolonged cytopenias and increased risk of infection. Our aim was to investigate survival after relapse, subsequent responses to second- line agents, and pattern of infections in patients treated with first-line venetoclax-based combination therapies. Methods This was a retrospective study of patients who received venetoclax-based regimens for untreated AML or myelodysplastic syndrome with excess blasts (MDS-EB) at LAC+USC Medical Center and Norris Cancer Center in Los Angeles, CA from January 1, 2018 to June 30, 2022. The electronic medical record was reviewed for demographics, disease characteristics, clinical history, transplant status, and survival. Results 61 patients received frontline venetoclax-based regimens for untreated AML or MDS-EB with a mean age of 65 (range 22-84); 36% were female, 44% non-Hispanic white and 31% were Hispanic/Latino. 41% had AML-MRC (myelodysplasia-related changes), 36% had De Novo AML, 16% had therapy-related AML, and 7% had MDS-EB. The majority (70%) were adverse risk group per ELN 2017 and 46% had complex cytogenetics. 58 patients had available myeloid mutation panel at time of diagnosis. The most common mutations (median allele frequencies) were: TP53=33% (0.45), ASXL1=17% (0.37), NPM1=16% (0.39), IDH2=9% (0.42), IDH1=7% (0.29), FLT3-ITD=5% (0.51). 70% received venetoclax with decitabine, 21% received venetoclax with azacitidine, and 2% received venetoclax with LDAC. Median number of cycles was 3 (1-16). 10 patients underwent allogeneic stem cell transplant. Out of 61 patients, 12 (20%) had primary refractory disease and 23 (38%) relapsed on frontline venetoclax. 49% of these relapsed/refractory patients received salvage chemotherapy. The median time to relapse and number of cycles prior to relapse was 6.3 months (1.6-17.4) and 4 (1-12) respectively. There was no correlation between cytogenetics and genetic variables and risk of relapse. Only 3 (19%) out of 16 patients and 3 (27%) out of 11 patients who received second- and third-line treatment achieved CRi respectively. Myeloid mutation panel was available for 12 patients at time of relapse. Most common mutations were FLT3-ITD/TKD (n=5), TP53 (n=3) and NPM1 (n=3). Six patients acquired new mutations at the time of relapse: FLT3-ITD (n=2), TP53 (n=1), WT1 (n=2) and ETV6 (n=1). Median time from relapse to death was 3 months (1-23). Median overall survival was 10.2 months. Median survival in responder compared to non-responders after cycle 1 and cycle 2 was 16.8 vs. 5.3 months (p <0.001) and 19.4 months vs. 7.4 months (p=0.031) respectively. Median survival in patients with relapse compared to non-relapse was 12.1 vs 6 months (p=0.187). 40 (66%) patients died; major causes of death were disease progression (50%), bleeding (10%) and infection (25%). 28% patients died in intensive care unit. 9 patients had fungal infections; most common were aspergillosis (n=5) and mucormycosis (n=2). Conclusions Venetoclax-based regimens are effective options for untreated AML in elderly patients. Response to first and second cycle of combination treatment predict survival. Although we observed clonal evolution in relapsed patients, it did not affect their survival. FLT3-ITD was a commonly acquired mutation at time of relapse, thus patients received salvage with targeted agents. Also, as patients relapse, the chance of attaining CR/CRi with subsequent lines of treatment is less than 30% and risk of invasive fungal infection was approximately 15%. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Characterizing the Development of Secondary Myelodysplastic Syndrome (MDS) with Antecedent or Concurrent Chronic Lymphocytic Leukemia (CLL) in the Era of Small Molecule Inhibitors for the Treatment of CLL

Introduction Secondary MDS (sMDS) is defined by MDS arising from another bone marrow disorder or related to previous therapy [chemotherapy (CT), radiation, or immunosuppression], and comprises about 10% of MDS cases. The rate of sMDS following CLL (including treatment-naïve CLL) has been reported as high as 3.5%. Developing in elderly populations, both CLL and MDS are associated with clonal hematopoiesis (CH), and chemoimmunotherapy (CIT) treatment for CLL likely contributes to the occurrence of MDS. There is little published data on the characteristics of patients (pts) who develop sMDS with antecedent/concurrent CLL, particularly in the era where kinase inhibitor therapy has displaced CIT. Therefore, we sought to characterize a cohort of CLL pts who develop sMDS in the era of modern treatments. Methods We performed a retrospective chart review at The Ohio State University between 01/01/2016 and 02/10/2021 of pts with a diagnosis of CLL who subsequently or concurrently were diagnosed with MDS. Clinical, next generation sequencing (NGS), and outcome data were collected. NGS test refers to a 50 gene hematologic neoplasm mutation panel. Overall survival (OS) was calculated from MDS diagnosis and estimated using Kaplan-Meier method. Results We identified 27 sMDS pts with antecedent/concurrent CLL. At CLL diagnosis, median age was 58 years (range 40-86). 5 pts (18.5%) had a TP53 mutation and/or del(17p) noted during their CLL disease course prior to sMDS diagnosis. Median number of therapies for CLL prior to sMDS was 1 (range 0-10), with 4 pts diagnosed with CLL at the same time as MDS. CLL treatments, as any line of therapy, included CT/CIT in 21 pts (77.8%) and ibrutinib in 10 pts (37.0%). Of the pts that received CLL therapy, only 1 pt did not receive CT/CIT. 5 pts (18.5%) were not treated for CLL. A median of 6.9 years (range 0-31.8) elapsed between the diagnosis of CLL and sMDS. At sMDS diagnosis median age was 70 years (range 50-86), 17 pts (65.4%) were MDS IPSS-R category high/very high. 5 pts transformed to AML after a median of 9.2 months (mos) from MDS diagnosis (range 4.1-21.3). After a median follow-up of 6.3 mos (range 0.8-109.2), the median OS from MDS diagnosis was 18.8 mos (95% CI, 10.2-not reached). (Table 1) In pts who received CT/CIT for CLL (n=21) and those who did not (n=6), a TP53 mutation developed following CLL or MDS diagnosis in 10 pts (47.6%) and 1 pt, respectively. All but 1 pt who developed TP53 mutations (n=11) received prior CT. 15 pts (71.4%) who received CT/CIT for CLL therapy had MDS with complex karyotype; this was seen in 2 pts who never received CT/CIT. Twenty-six (96.3%) pts had NGS testing. 5 pts (18.5%) had 1 or more tests completed for CLL prior to sMDS: 4 pts had TP53 mutations, 2 pts each had DNMT3A and SF3B1 mutations, and 1 pt each had an ASXL1, KMT2A, BTK, or PLCG2 mutation. Of these 5 pts, 1 pt had 5 NGS tests performed on serial B-cell selected samples (as opposed to total leukocytes): mutations included 2 TP53, 1 SF3B1 and 1 BTK mutation. 25 (92.6%) pts had 1 or more tests completed at or after sMDS diagnosis (all assessed total leukocytes): 8 pts (36.4%) had TP53, 6 pts (27.3%) had DNMT3A, 5 pts (22.7%) had RUNX1, and 3 pts (13.6%) each had ASXL1, SF3B1, and TET2 mutations. Finally, 4 pts (14.8%) had NGS testing done both pre and post sMDS diagnosis (all assessed total leukocytes). Two pts had the same mutation present pre and post sMDS diagnosis: 1 ASXL1, 1 DNMT3A. A third pt had 3 identical mutations pre and post sMDS diagnosis: 1 TP53 p.G244D, 1 TP53 p.N235_Y236del, and 1 SF3B1. (Table 2) Conclusions Here we have characterized a cohort of CLL pts who developed sMDS in the modern era, where small molecule inhibitors are commonly used for CLL, and NGS is widely available for both CLL and MDS. While the majority of pts with CLL received CT, 5 pts were treatment-naive, and 1 pt only received a BTK inhibitor and an anti-CD20 antibody. The OS of this cohort following MDS diagnosis is similar to historical data of pts with comparable IPSS-R scores, despite the fact that this is a sMDS population; this may be due to lead time bias as pts likely received frequent blood work for CLL. Prior to sMDS diagnosis, complex karyotype and TP53, SF3B1 and CH-associated mutations DNMT3A and ASXL1 were prevalent. TP53, SF3B1, and CH-associated mutations DNMT3A, ASXL1, and TET2 mutations were prevalent at the time of or following a sMDS diagnosis. Further investigation into the interplay between CLL and CH in MDS development in the modern therapy era is needed. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Identification of Boronate-Containing Diarylpyrimidine Derivatives as Novel HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitors.

In this study, privileged boronic acid ester was introduced into the right wing of etravirine (ETR) to obtain a series of novel boronate-containing derivatives. These newly synthesized derivatives were evaluated for their anti-HIV potency in MT-4 cells using the MTT method, and their inhibitory activity to HIV-1 reverse transcriptase (RT) was assayed by the ELISA method. Most of the synthesized compounds displayed promising antiviral activity against the wild-type and a wide range of HIV-1 mutant strains. In particular, 4a exhibited the most potent activity against the wild-type and a panel of single mutations (L100I, K103N, Y181C, and E138K) with EC50 values ranging from 0.005 to 0.648 μM, which were much superior to those of nevirapine (EC50 = 0.151 μM). Moreover, 4b turned out to be an effective inhibitor against the double-mutant strains F227L + V106A and RES056 with EC50 values of 3.21 and 2.30 μM, respectively. RT inhibition activity and molecular docking were also investigated.

RASopathy mutations provide functional insight into the BRAF cysteine-rich domain and reveal the importance of autoinhibition in BRAF regulation

BRAF is frequently mutated in human cancer and the RASopathy syndromes, with RASopathy mutations often observed in the cysteine-rich domain (CRD). Although the CRD participates in phosphatidylserine (PS) binding, the RAS-RAF interaction, and RAF autoinhibition, the impact of these activities on RAF function in normal and disease states is not well characterized. Here, we analyze a panel of CRD mutations and show that they increase BRAF activity by relieving autoinhibition and/or enhancing PS binding, with relief of autoinhibition being the major factor determining mutation severity. Further, we show that CRD-mediated autoinhibition prevents the constitutive plasma membrane localization of BRAF that causes increased RAS-dependent and RAS-independent function. Comparison of the BRAF- and CRAF-CRDs also indicates that the BRAF-CRD is a stronger mediator of autoinhibition and PS binding, and given the increased catalytic activity of BRAF, our studies reveal a more critical role for CRD-mediated autoinhibition in BRAF regulation.

PSUN249 Starvation Ketoacidosis as the Presenting Finding of Glycogen Storage Disorder Type III

Abstract A 30-year woman with a history of heart transplantation and a history of post-transplant diabetes mellitus presents with severe euglycemic ketoacidosis. Initial concern was for diabetes related ketoacidosis given history of diabetes however blood glucose was within goal range in the absence of pregnancy and use of sodium glucose transporter type 2 inhibitor use. Initial laboratory evaluation showed a serum glucose of 177 mg/dL, a serum beta hydroxybutyrate of greater than 12.66 mmol/L, serum lactate of 0.6 mmol/L, with a calculated anion gap of greater than 30 mmol/L. C-peptide levels were repeatedly normal and appropriate for blood glucose level ranging between 2.8 ng/mL and 7.5 ng/mL ruling out insulin deficiency as causative. It was discovered that the patient had intentionally fasted for greater than 18 hours and thus had not used the premixed insulin regimen prescribed. Metabolic disturbance corrected with a dextrose and insulin infusion. Evaluation for inborn error of metabolism showed: acylcarnitine panel returned normal making an inborn error of fatty acid metabolism unlikely. The mitochondrial DNA mutation panel was negative for point mutations or deletions. Further testing for urinary organic acids did not reveal elevation of 3-hydroxybutyric acid suggesting SCOT enzyme levels were sufficient in our patient. Review of pathology records for heart transplantation completed for idiopathic cardiomyopathy 20 years prior to this presentation showed high levels of glycogen deposition prompting evaluation for glycogen storage disorders. Results revealed a heterozygous abnormality at exon 9 of the AGL gene; AGL gene c.1139A&amp;gt;G (p.Glu380Gly). This is not a reported pathologic mutation, and given the heterozygosity of the mutation in this autosomal recessive condition, it is felt that either environmental factors or the presence of other unrecognized genetic mutations allowed for the phenotypic expression of GSDIII with euglycemic starvation ketoacidosis. The patient was treated with dietary counseling regarding ketone suppression with basal insulin during periods of fasting and preference to avoid periods of prolonged fasting with frequent carbohydrate-containing meals. Conclusion This case represents a novel, potentially pathologic defect in the AGL gene. This finding should prompt further investigations into genetic variabilities that may lead to this phenotypic expression. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

A Subset of Pancreatoblastomas May Arise From an Adenomatous Precursor: An Ampullary Pancreatoblastoma and Adjacent Adenoma With a Shared Molecular Phenotype in an Adult Patient.

Pancreatoblastomas are rare pediatric tumors. In adults, they are exceedingly rare and seem to have a worse prognosis. Most are sporadic, though rare, cases occur in patients with familial adenomatous polyposis. Unlike pancreatic ductal adenocarcinomas, pancreatoblastomas are not believed to arise from dysplastic precursor lesions. Clinical history, along with endoscopic, pathological, and molecular findings, was reviewed for a 57-year-old male patient with an ampullary mass who presented with obstructive jaundice. Microscopic examination showed a pancreatoblastoma subjacent to an adenomatous polyp with intestinal differentiation and low-grade dysplasia. Both tumors had abnormal p53 (complete loss) and nuclear β-catenin immunostaining. Mutational panel analysis showed an identical CTNNB1 (p.S45P) mutation in both. This case adds to our understanding of the pathogenesis of these rare tumors and suggests that a subset may arise from an adenomatous precursor. In addition, this case is just the second pancreatoblastoma to originate in the duodenal ampulla, and the preceding case suggests that an ampullary location leads to earlier diagnosis. Moreover, this case highlights the difficulty in diagnosing pancreatoblastoma on limited tissue specimens and illustrates the need to include pancreatoblastoma in the differential diagnosis in all tumors in and around the pancreas, including those in adult patients.

Spatially aware graph neural networks and cross-level molecular profile prediction in colon cancer histopathology: a retrospective multi-cohort study

Digital whole-slide images are a unique way to assess the spatial context of the cancer microenvironment. Exploring these spatial characteristics will enable us to better identify cross-level molecular markers that could deepen our understanding of cancer biology and related patient outcomes. We proposed a graph neural network approach that emphasises spatialisation of tumour tiles towards a comprehensive evaluation of predicting cross-level molecular profiles of genetic mutations, copy number alterations, and functional protein expressions from whole-slide images. We introduced a transformation strategy that converts whole-slide image scans into graph-structured data to address the spatial heterogeneity of colon cancer. We developed and assessed the performance of the model on The Cancer Genome Atlas colon adenocarcinoma (TCGA-COAD) and validated it on two external datasets (ie, The Cancer Genome Atlas rectum adenocarcinoma [TCGA-READ] and Clinical Proteomic Tumor Analysis Consortium colon adenocarcinoma [CPTAC-COAD]). We also predicted microsatellite instability and result interpretability. The model was developed on 459 colon tumour whole-slide images from TCGA-COAD, and externally validated on 165 rectum tumour whole-slide images from TCGA-READ and 161 colon tumour whole-slide images from CPTAC-COAD. For TCGA cohorts, our method accurately predicted the molecular classes of the gene mutations (area under the curve [AUCs] from 82·54 [95% CI 77·41-87·14] to 87·08 [83·28-90·82] on TCGA-COAD, and AUCs from 70·46 [61·37-79·61] to 81·80 [72·20-89·70] on TCGA-READ), along with genes with copy number alterations (AUCs from 81·98 [73·34-89·68] to 90·55 [86·02-94·89] on TCGA-COAD, and AUCs from 62·05 [48·94-73·46] to 76·48 [64·78-86·71] on TCGA-READ), microsatellite instability (MSI) status classification (AUC 83·92 [77·41-87·59] on TCGA-COAD, and AUC 61·28 [53·28-67·93] on TCGA-READ), and protein expressions (AUCs from 85·57 [81·16-89·44] to 89·64 [86·29-93·19] on TCGA-COAD, and AUCs from 51·77 [42·53-61·83] to 59·79 [50·79-68·57] on TCGA-READ). For the CPTAC-COAD cohort, our model predicted a panel of gene mutations with AUC values from 63·74 (95% CI 52·92-75·37) to 82·90 (73·69-90·71), genes with copy number alterations with AUC values from 62·39 (51·37-73·76) to 86·08 (79·67-91·74), and MSI status prediction with AUC value of 73·15 (63·21-83·13). We showed that spatially connected graph models enable molecular profile predictions in colon cancer and are generalised to rectum cancer. After further validation, our method could be used to infer the prognostic value of multiscale molecular biomarkers and identify targeted therapies for patients with colon cancer. This research has been partially funded by ARO MURI 805491, NSF IIS-1793883, NSF CNS-1747778, NSF IIS 1763523, DOD-ARO ACC-W911NF, and NSF OIA-2040638 to Dimitri N Metaxas.

An assessment of patient-provider communication surrounding tumor genetic profiling in thoracic oncology.

285 Background: Thoracic oncologists regularly order mutational testing for lung cancer, called the Stanford Actionable Mutation Panel (STAMP) at the Stanford Cancer Center, that assesses for somatic tumor based mutational testing. This project surveyed patients who had this testing and assessed their understanding of the test’s purpose and results in thoracic oncology. We also gathered information about which providers on the care team are conducting this counseling and time spent on patient education. Methods: This quality assurance project was deemed exempt from IRB review. We obtained an EHR query of patients who had the STAMP testing between July 2020 and July 2021, which was kept in a password protected file in a secured shared folder. Patients were contacted by phone using Doximity Dialer and answers were recorded in Redcap as the questionnaire was administered over the phone. Patient responses were labeled using a random number generator and no protected patient information was recorded. Results: The EHR query of patients who had the STAMP test performed in the last year resulted in 77 unique patients and 34 patients agreed to complete the survey. The majority of respondents (73.5%) knew that their physician or oncologist had ordered the STAMP test for them. Of those patients, the majority (84%) stated that their provider, most commonly their oncologist, explained the purpose of the test. The majority of respondents (64%) stated that their provider, most commonly their oncologist, had discussed the test results with them, which usually took approximately 5-10 minutes. Almost all respondents (93.8%) felt that they had time to ask the questions they had regarding their results. 43.8% of respondents stated that their test results changed their treatment options and 18.8% were unsure. Similarly, 43.8% of respondents stated that they had a targetable mutation, while 25% were unsure. Overall, 42.9% of respondents felt confident in their understanding of their test results, 33.3% were very confident, 4.8% felt neutral, and 19% were not confident in their understanding. Conclusions: In this single center survey, thoracic oncology teams appear to be communicating effectively with their patients about the purpose and results of molecular testing. However, patients were more uncertain about how the test influenced their treatment, identifying a potential area of improvement in patient education.

A Retrospective Evaluation of the Diagnostic Performance of an Interdependent Pairwise MicroRNA Expression Analysis with a Mutation Panel in Indeterminate Thyroid Nodules.

Background: The addition of genetic analysis to the evaluation of thyroid nodule fine-needle aspiration biopsy samples improves diagnostic accuracy of cytologically indeterminate thyroid nodules (ITNs) with Bethesda III or IV cytopathology. We previously reported the performance of a multiplatform molecular test, referred to in this study as MPTXv1, that includes a mutation panel (ThyGeNEXT®) plus an algorithmic microRNA (miRNA) risk classifier (ThyraMIR®). Complex interactions of growth-promoting and -suppressing miRNAs affect the phenotype. We previously demonstrated that accounting for these interactions with pairwise miRNA expression analysis improves the diagnosis of medullary thyroid carcinoma. In this study, we assess the impact of pairwise miRNA expression analysis on risk stratification of ITNs. Methods: Pairwise expression analysis of 11 miRNAs was performed on a training cohort of histopathology-proven benign nodules (n = 50) to define the mean and standard deviation of each pairwise analysis and create a Benign/Malignant Profiler (MPTXv2), deviations from which predicted the malignancy risk. Clinical validation of MPTXv2 was assessed using a cohort of 178 ITN (Bethesda III and IV) samples from a multicentered, blinded retrospective study, previously evaluated by MPTXv1. Results: Compared with MPTXv1, MPTXv2 significantly improved the test performance. The receiver operating characteristic (ROC) areas under the curve (AUC) increased from 0.85 to 0.97 (p < 0.001), and the diagnostic accuracy at the positive threshold increased significantly (p < 0.05) from 83% [95% confidence interval (CI) = 76-88] to 93% [CI = 89-96]. The significant improvement in the ROC AUC and the diagnostic accuracy was due to a strong statistical trend for improvement in specificity at the positive threshold. At the positive threshold, the specificity for MPTXv1 was 90% [CI = 84-95] and improved to 98% [CI = 94-99] for MPTXv2. Using the MPTXv2, the Moderate-Risk cohort decreased from 50 samples (28% of the cohort) to 24 samples (13% of the cohort). This 52% decrease is statistically significant (p < 0.001) and clinically meaningful. Conclusion: As compared with MPTXv1, pairwise miRNA expression analysis used in MPTXv2 significantly improved the diagnostic accuracy of ITN risk stratification and reduced the size of the Moderate-Risk group. Prospective trials are indicated to confirm these findings in a clinical practice setting.

Heritable cancer predisposition testing in pediatric cancer patients excluding retinoblastoma in a middle-income country.

Resource-limited settings often have financial barriers to genetic testing for heritable cancer. This retrospective study investigated the pattern of heritable cancer predisposition testing in a middle-income country over the period 2014-2021, excluding retinoblastoma. After establishing a specific fund in 2019, rate of tests increased from 1.1% to 10.9% of new diagnoses. Most common testing was for constitutional mismatch repair deficiency (CMMRD), rhabdoid predisposition syndrome, TP53 (tumor protein 53) mutation, and hereditary cancer panel. Of 33 patients, 13 (39%) tested positive, 12 (36%) negative, and eight (24%) had variants of unknown significance. Positivity rate was 43% for a clinical phenotype and 44% for a tumor type indication.

Plexiform fibrohistiocytic tumor: a clinicopathological and immunohistochemical study of 39 tumors, with evidence for a CSF1-producing "null cell" population.

Plexiform fibrohistiocytic tumor (PFHT) is a mesenchymal tumor of intermediate malignancy, typically occurring in the superficial soft tissues of young patients and displaying a biphasic pattern, with nodules of histiocytoid cells surrounded by fascicles of myofibroblastic spindled cells. The pathogenesis of PHFT is unknown. We comprehensively studied 39 PFHT, occurring in 25 females (66%) and 13 males (34%), ranging from 2 to 55years of age (median 21years). The tumors most often occurred in the upper extremity (n = 16, 41%) and ranged from 0.4 to 6.1cm in size (median 1.5cm). One patient with known neurofibromatosis type 1 presented with metachronous tumors of the finger and back. Clinical follow-up (29 patients; range 5-168months; median 60months) showed 3 tumors to have recurred locally; none was metastasized. One patient died of an unrelated cause; all others were alive without disease at the time of last follow-up. Immunohistochemistry showed the histiocytoid nodules of all cases to contain CD163/CD11c-positive histiocytes and cells negative for both markers ("null cells"). CSF1 expression was present in "null cells" in 7/10 cases (RNAscope chromogenic in situ hybridization). The Ki-67 labeling index was very low (< 5%); Ki-67-positive cells within histiocytoid nodules appeared to represent "null cells." All tested cases were negative for significant mutations or fusion events (TruSight Mutation Panel, TruSight Fusion Panel, Mayo Clinic Melanoma Targeted Gene Panel). We conclude that PHFT may be even more indolent than has been appreciated, although classification as an "intermediate" tumor is correct. We hypothesize that the CSF1-producing "null cells" of PHFT may represent the neoplastic element, with the bulk of the tumor masses comprising recruited and reactive cell populations.

EP04.01-008 Factors Impacting Time from Biopsy to Initiation of Treatment for Advanced NSCLC at an Academic Hospital and Affiliate Hospitals

Delays in initiation of treatment for advanced cancers are associated with poorer outcomes. In advanced NSCLC, one factor impacting time of treatment initiation is next-generation sequencing (NGS) testing. In our hospital system, Northwestern Medicine, the standard for NGS testing at Northwestern Memorial, the 894-bed academic hospital, is in-house reflex testing on all histologies and stages for a 50-gene panel of mutations and fusions. At affiliate hospitals, there is no set protocol so testing is sent to private vendors.

Spectrum of Cystic Fibrosis Conductance Regulator Gene Mutations Reported in Pakistani Descent Cystic Fibrosis Patients

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. This study aims to determine the genotypic and phenotypic spectrum of the CFTR gene mutations reported in the literature for Pakistani-origin CF patients. Databases were searched for such studies from 1947-2019 for sample size, method of diagnosis, and CFTR gene mutations. The authors identified 12 studies reporting 33 CFTR gene mutations, both intronic as well as exonic in Pakistani origin patients. The most widely tested mutation was D508 with a frequency of 17%-60%. No hotspot zone was identified and not all reported mutations were causing disease. There is a need to identify common mutations in the Pakistani population to develop population-specific CFTR mutations panel. This will enable the researchers to perform phenotype-genotype correlation studies to improve the CF detection rate. Key Words: Cystic fibrosis, Pakistan, Mutations, CFTR.