Case Report: Effectiveness of Targeted Treatment in a Patient With Pancreatic Cancer Harboring PALB2 Germline Mutation and KRAS Somatic Mutation

Pancreatic cancer is one of the most leading causes of cancer death worldwide. The rapid development of next-generation sequencing (NGS) and precision medicine promote us to seek potential targets for the treatment of pancreatic cancer. Here, we report a female pancreatic cancer patient who underwent radical surgical excision after neoadjuvant chemotherapy. After the surgery, the patient underwent gemcitabine + S-1 therapy, capecitabine + albumin paclitaxel therapy and irinotecan therapy successively, however, MRI review revealed tumor progression. The surgical tissue sample was subjected to next-generation sequencing (NGS), and PALB2 germline mutation and KRAS somatic mutation were identified. The patient then received olaparib (a PARP inhibitor) + irinotecan and the disease stabilized for one year. Due to the increased CA19-9, treatment of the patient with a combination of trametinib (a MEK inhibitor) and hydroxychloroquine resulted in stable disease (SD) with a significant decrease of CA19-9. This case demonstrated that the NGS may be a reliable method for finding potential therapeutic targets for pancreatic cancer.

The mutationathon highlights the importance of reaching standardization in estimates of pedigree-based germline mutation rates

In the past decade, several studies have estimated the human per-generation germline mutation rate using large pedigrees. More recently, estimates for various non-human species have been published. However, methodological differences among studies in detecting germline mutations and estimating mutation rates make direct comparisons difficult. Here, we describe the many different steps involved in estimating pedigree-based mutation rates, including sampling, sequencing, mapping, variant calling, filtering, and how to appropriately account for false-positive and false-negative rates. For each step, we review the different methods and parameter choices that have been used in the recent literature. Additionally, we present the results from a 'Mutationathon', a competition organized among five research labs to compare germline mutation rate estimates for a single pedigree of rhesus macaques. We report almost a two-fold variation in the final estimated rate among groups using different post-alignment processing, calling, and filtering criteria and provide details into the sources of variation across studies. Though the difference among estimates is not statistically significant, this discrepancy emphasizes the need for standardized methods in mutation rate estimations and the difficulty in comparing rates from different studies. Finally, this work aims to provide guidelines for computational and statistical benchmarks for future studies interested in identifying germline mutations from pedigrees.

Germline Mutation Landscape and Associated Clinical Characteristics in Chinese Patients With Renal Cell Carcinoma

BackgroundRenal cell carcinoma (RCC) is a disease of genomic alterations, of which the complete panorama helps in facilitating molecular-guided therapy. Germline mutation profiles and associated somatic and clinical characteristics remains unexplored in Chinese RCC patients.MethodsWe retrospectively profiled the germline and somatic mutations of 322 unselected RCC patients using a panel consisting of 808 cancer-related genes. We categorized patients into three groups based on germline mutation status and compared the somatic mutation spectrum among different groups.ResultsApproximately one out of ten (9.9%) RCC patients were identified to carry pathogenic/likely pathogenic (P/LP) germline variants (PGVs), of which 3.7% were variants in syndromic RCC-associated genes and 6.2% were other cancer-predisposition genes. The most common PGV was found in VHL (2.2%), followed by FH, TSC2, ATM, BRCA1, NBN, and BLM (0.6% each). Young patients (≤46 years) were more likely to harbor PGVs. Variants in syndromic RCC-associated genes were predominant identified in young patients, while variants in other cancer-predisposition genes were found in patients >46 years more frequently. Furthermore, 39.3% (11/28) of patients carrying PGVs were detected to have somatic “second hit” events. Germline and somatic sequencing, including microsatellite instability (MSI) status analysis, provided potentially actionable therapeutic targets in 17.1% of patients in the whole cohort.ConclusionsOur results revealed that approximately 10% of RCC patients carried clinically significant germline mutations. Current guidelines recommendation for genetic testing seemed not sensitive enough to identify patients with hereditary RCC susceptibility. It is rational to promote genetic testing in RCC population.

Clonal hematopoiesis in individuals with ANKRD26 or ETV6 germline mutations

Currently, there are at least a dozen recognized hereditary hematopoietic malignancies (HHMs), some of which phenocopy others. Among these, three HHMs driven by germline mutations in ANKRD26, ETV6, or RUNX1 share a phenotype of thrombocytopenia, qualitative platelet defects, and an increased lifetime risk of hematopoietic malignancies (HMs). Prior work has demonstrated that RUNX1 germline mutation carriers experience an elevated lifetime risk (66%) for developing clonal hematopoiesis (CH) prior to age 50. Germline mutations in ANKRD26 or ETV6 phenocopy RUNX1 germline mutations, but no studies have focused on the risk of CH in individuals with germline mutations in ANKRD26 or ETV6. To determine the prevalence of CH in individuals with germline mutations in ANKRD26 or ETV6, we performed next generation sequencing on hematopoietic tissue from twelve individuals with either germline ANKRD26 or germline ETV6 mutations. Each patient had thrombocytopenia but had not developed HMs. Among the seven individuals with germline ANKRD26 mutations, one patient had a CH clone driven by a somatic SF3B1 mutation (p.Lys700Glu). This mutation increased from a variant allele frequency (VAF) of 9.4% at age 56 to 17.4% at age 60. None of the germline ETV6 mutation carriers had evidence of CH at the limits of detection of the NGS assay (5% VAF). Unlike individuals with germline mutations in RUNX1, no individuals under the age of 50 with germline mutations in ANKRD26 or ETV6 had detectable CH. This work demonstrates that ANKRD26 germline mutation carriers, but not ETV6 mutation carriers, experience elevated risk for CH.

Trametinib for Refractory Chylous Effusions in Children with Noonan Syndrome

BACKGROUND Noonan syndrome (NS) is one of several autosomal dominant multisystem disorders known as RASopathies. Common manifestations of NS include congenital heart defects and cardiomyopathy, lymphatic malformations, and predisposition to myeloproliferative disorders. Chylous fluid accumulation secondary to lymphatic malformations are seen in NS and are a major cause of morbidity and mortality often refractory to conventional medical management. There has been increasing interest in the use of pharmacologic MEK inhibition in the management of these patients given that activating RAS pathway mutations lead to downstream MEK activation that is causative of this pathology. DESIGN/METHODS Three patients with a confirmed diagnosis of NS are described. Each patient developed complications from chylous effusions refractory to conventional management and were subsequently enrolled on-study to treat with compassionate use oral trametinib from Novartis Pharmaceuticals on a single patient Investigational New Drug from the Food and Drug Administration (FDA). All patients were consented to be monitored for one year of therapy following a local protocol approved by the Colorado Institutional Review Board (COMIRB). Patient 1: a 4-year-old female with NS due to a pathogenic germline mutation of the RIT1 gene [c.246T>G, p.Phe82Leu] born with severe hypertrophic cardiomyopathy, mitral valve dysplasia, and pulmonary valve stenosis. She developed bilateral chylous pleural effusions that were refractory to dietary modification, diuretics, octreotide, and sirolimus. Patient 2: a 3-month-old female with NS due to a pathogenic germline mutation of the SOS1 gene [c.1322G>A; p.Cys441Tyr] born with esophageal atresia/tracheoesophageal fistula and moderate pulmonary valve stenosis. She developed bilateral chylous pleural effusions and ascites that were refractory to dietary modification and octreotide therapy. Patient 3: a 4-month-old male with NS due to a gain-of-function mutation of PTPN11 [c.854T>C; p.Phe285Ser] with hypertrophic cardiomyopathy, pulmonary valve stenosis, respiratory insufficiency with suspected pulmonary lymphangiectasia, and persistent chylous pleural effusions in addition to Noonan syndrome-associated myeloproliferative disorder (NS-MPD) that had been refractory to traditional management. RESULTS MEK inhibition with trametinib was used in three patients with NS and life-threatening complications with no medical or surgical treatment options. All three patients had dynamic contrast magnetic resonance lymphangiography (DCMRL) evidence of primary, central lymphatic dysplasia that manifested in lymphatic accumulation affecting cardiorespiratory function, nutrition, and the immune system. DCMRL imaging for patient 2 are highlighted in Figure 1 A and B. Within one month of initiating trametinib oral therapy, all three patients demonstrated response adequate to wean from mechanical ventilation and other supportive care modalities. Serum albumin levels improved as lymphatic leak resolved (Figure 1C). Patient 3 showed improvement in hypertrophic cardiomyopathy as evidenced by a decrease in both NT-proBNP and left ventricular mass by echocardiogram. Patients 1 and 2 demonstrated notable improvements in growth after one year of therapy, with increase in both weight and height percentiles. Patient 3 also presented with NS-MPD that responded with marked improvements in total WBC count as well as absolute monocyte count (Figure 1D). DISCUSSION Our experience adds to the growing body of evidence demonstrating the effectiveness of MEK inhibition on disease processes that are common in patients with NS and other RASopathies. None of the patients in our series experienced significant adverse effects from the medication aside from patient 2 who developed mild dermatitis. The efficacy of this therapy does not appear to be based on the underlying genotype, as each of the three patients we describe had different underlying molecular alterations (SOS1, RIT1, PTPN11). Substantial improvements in a variety of parameters including lymphatic malformations, cardiomyopathy, pulmonary valve stenosis, growth, and NS-MPD highlight the potential utility of trametinib in this patient population. Larger, prospective studies are necessary to confirm the efficacy of MEK inhibition and to assess the long-term safety of its use in this population. Figure 1 Figure 1. Disclosures Nakano: Novartis: Consultancy. OffLabel Disclosure: Trametinib is a MEK1/2 inhibitor that has been approved for the use in certain malignancies. Its off label use in children with Noonan Syndrome with significant lymphatic anomalies is based on the up regulation of the MAPK pathway in these patients.

Clonal Hematopoiesis in Telomere Biology Disorders Associates with the Underlying Germline Defect and Somatic Mutations in POT1, PPM1D, and TERT promoter

Introduction: Telomere biology (TBD) disorders are caused by pathogenic germline variants in genes related to telomere maintenance. In TBD, clonal hematopoiesis (CH) has been hypothesized to compensate for restricted cell fitness and to lead to development of myelodysplastic syndromes and acute myeloid leukemia (MDS/AML). We sought to characterize the clonal landscape and dynamics by deep sequencing of a large cohort of TBD patients with a broad spectrum of phenotypes and ages. Methods: We screened 120 TBD patients (median age=29) from the National Institutes of Health and the University of Sao Paulo for somatic mutations in genes related to myeloid malignancies and telomere diseases using an error-correcting DNA sequencing panel (minimum allele frequency [VAF] of 0.5%). Patients had either a pathogenic germline variant in telomere-related genes or short telomeres in blood and a strong clinical suspicion for TBD. Relatives were included if they harbored the proband's germline mutation. Single-cell DNA sequencing was performed in marrow samples from two TBD patients with MDS (TBD-MDS) to elucidate clonal trajectories Results: Fifty-eight TBD patients (48%) had somatic mutations in peripheral blood (median age and range, 42 years; 9-57), most frequently in PPM1D (all exon 6 truncated; n=18) , TERTp (-57, -124, and -146; n=14), POT1 (n=12), U2AF1 (n=12), and other MDS-associated genes. Clinically, these patients had dyskeratosis congenita (DC; n=12/27), aplastic anemia (AA; n=11/27), isolated cytopenias (n=7/10), MDS/AML (n=7/8), pulmonary or liver fibrosis (n=4/8), and multi-organ disease (n=19/26). In this series, no relatives had somatic mutations (n=14). CH frequency increased with age and was significantly more frequently observed than in healthy controls, regardless of age (p<0.001). POT1, PPM1D, and TERTp clones size was lower than the size of MDS-associated clones (VAF of 1% vs 8%). These mutations often co-occurred, except for POT1 and TERTp mutation. Patients' clonal profiles correlated with the underlying germline defect. Somatic P OT1 mutations strongly associated with TINF2 germline variants, and consequently DC: 5/9 TINF2 patients had one (n=2) or >2 POT1 clones (n=3). In contrast, both TERTp and PPM1D clones were mostly detected in TERT/TERC patients with multi-organ disease, especially pulmonary fibrosis and marrow failure. No telomere elongation or improved blood counts were seen in serial samples. TINF2 patients with somatically mutated POT1 clones were older despite their DC diagnosis (median age=19 vs 5 years in POT1 mutated and wild type, respectively). A single patient with a germline TINF2 R282C and somatically mutated POT1 clone at VAF=29%, which was stable for 5 years, had MAA. The median ages (range) of TERT/TERC patients with TERTp and PPM1D mutations were 41 (25-64) and 43 (12-72), respectively, whereas TERT/TERC patients without TERTp and PPM1D mutations were at a median age of 27 (8-58). Most clones were stable regardless of clinical phenotype, even after danazol treatment. PPM1D clones were stable for 2-9 years of follow-up. TERTp and POT1 clones' size decreased while on androgens but consistently increased after the drug was discontinued. In single-cell DNA analysis of two TBD-MDS patients, the U2AF1 S34F and Q157R were driver mutations and occurred with mutations in RUNX1, ETV6, ASXL1; these clones were stable for 3-6 years. In the first case, the U2AF1 clone subsequently acquired a RUNX1 mutation; this clone was coincident with an independent clone containing PPM1D and POT1 mutations. In the second patient, a U2AF1 clone acquired successive mutations in SETBP1 and AXSL1; a second clone with U2AF1 and additional mutations in GATA2 and KRAS arose at evolution to AML. Conclusion: In TBD, the somatic landscape differed from age-related CH, with recurrent TERTp, POT1, and truncated PPM1D mutations. Mutations' frequency increased with age but was related to the underlying germline mutation. It is uncertain whether clonal selection is a probabilistic consequence of older age or the cause of mild phenotypes and prolonged lifespan. Despite the association of POT1 and PPM1D with malignancies, no patients in our cohort had POT1-related cancers or had received chemotherapy. POT1 and PPM1D like TERTp mutations may arise to compensate for cell fitness. Clinically, this distinct clonal landscape, not found in immune BMF, could serve as a molecular marker of underlying TBD. Disclosures Calado: Instituto Butantan: Consultancy; AA&MDS International Foundation: Research Funding; Agios: Membership on an entity's Board of Directors or advisory committees; Novartis Brasil: Honoraria; Alexion Brasil: Consultancy; Team Telomere, Inc.: Membership on an entity's Board of Directors or advisory committees. Young: Novartis: Research Funding.

A deep learning-based framework for estimating fine-scale germline mutation rates

Germline mutation rates are essential for genetic and evolutionary analyses. Yet, estimating accurate fine-scale mutation rates across the genome is a great challenge, due to relatively few observed mutations and intricate relationships between predictors and mutation rates. Here we present MuRaL (Mutation Rate Learner), a deep learning-based framework to predict fine-scale mutation rates using only genomic sequences as input. Harnessing human germline variants for comprehensive assessment, we show that MuRaL achieves better predictive performance than current state-of-the-art methods. Moreover, MuRaL can build models with relatively few training mutations and a moderate number of sequenced individuals. It can leverage transfer learning to build models with further less training data and time. We apply MuRaL to produce genome-wide mutation rate profiles for four species - Homo sapiens, Macaca mulatta, Arabidopsis thaliana and Drosophila melanogaster, demonstrating its high applicability. The generated mutation rate profiles and open source software can greatly facilitate related research.