Donor Clonal Hematopoiesis and Recipient Outcomes After Transplantation

PURPOSE Clonal hematopoiesis (CH) can be transmitted from a donor to a recipient during allogeneic hematopoietic cell transplantation. Exclusion of candidate donors with CH is controversial since its impact on recipient outcomes and graft alloimmune function is uncertain. PATIENTS AND METHODS We performed targeted error-corrected sequencing on samples from 1,727 donors age 40 years or older and assessed the effect of donor CH on recipient clinical outcomes. We measured long-term engraftment of 102 donor clones and cytokine levels in 256 recipients at 3 and 12 months after transplant. RESULTS CH was present in 22.5% of donors, with DNMT3A (14.6%) and TET2 (5.2%) mutations being most common; 85% of donor clones showed long-term engraftment in recipients after transplantation, including clones with a variant allele fraction < 0.01. DNMT3A-CH with a variant allele fraction ≥ 0.01, but not smaller clones, was associated with improved recipient overall (hazard ratio [HR], 0.79; P = .042) and progression-free survival (HR, 0.72; P = .003) after adjustment for significant clinical variables. In patients who received calcineurin-based graft-versus-host disease prophylaxis, donor DNMT3A-CH was associated with reduced relapse (subdistribution HR, 0.59; P = .014), increased chronic graft-versus-host disease (subdistribution HR, 1.36; P = .042), and higher interleukin-12p70 levels in recipients. No recipient of sole DNMT3A or TET2-CH developed donor cell leukemia (DCL). In seven of eight cases, DCL evolved from donor CH with rare TP53 or splicing factor mutations or from donors carrying germline DDX41 mutations. CONCLUSION Donor CH is closely associated with clinical outcomes in transplant recipients, with differential impact on graft alloimmune function and potential for leukemic transformation related to mutated gene and somatic clonal abundance. Donor DNMT3A-CH is associated with improved recipient survival because of reduced relapse risk and with an augmented network of inflammatory cytokines in recipients. Risk of DCL in allogeneic hematopoietic cell transplantation is driven by somatic myelodysplastic syndrome–associated mutations or germline predisposition in donors.

Bockenheimer Disease is Associated With a TEK Variant

Bockenheimer disease is a venous malformation involving all tissues of an extremity. Patients have significant morbidity and treatment is palliative. The purpose of this study was to identify the cause of Bockenheimer disease to develop pharmacotherapy for the condition. Paraffin-embedded tissue from 9 individuals with Bockenheimer disease obtained during a clinically-indicated operation underwent DNA extraction. Droplet digital PCR (ddPCR) was used to screen for variants most commonly associated with sporadic venous malformations [TEK (NM_000459.5:c.2740C>T; p.Leu914Phe), PIK3CA (NM_006218.4:c.1624G>A; p.Glu542Lys and NM_006218.4:c.3140A>G; p.His1047Arg)]. ddPCR detected a TEK L914F variant in all 9 patients (variant allele fraction 2%-13%). PIK3CA E542K and H1047R variants were not identified in the specimens. Sanger sequencing and restriction enzyme digestion confirmed variants identified by ddPCR. A pathogenic variant in the endothelial cell tyrosine kinase receptor TEK is associated with Bockenheimer disease. Pharmacotherapy targeting the TEK signaling pathway might benefit patients with the condition.

SCReadCounts: estimation of cell-level SNVs expression from scRNA-seq data

Background Recent studies have demonstrated the utility of scRNA-seq SNVs to distinguish tumor from normal cells, characterize intra-tumoral heterogeneity, and define mutation-associated expression signatures. In addition to cancer studies, SNVs from single cells have been useful in studies of transcriptional burst kinetics, allelic expression, chromosome X inactivation, ploidy estimations, and haplotype inference. Results To aid these types of studies, we have developed a tool, SCReadCounts, for cell-level tabulation of the sequencing read counts bearing SNV reference and variant alleles from barcoded scRNA-seq alignments. Provided genomic loci and expected alleles, SCReadCounts generates cell-SNV matrices with the absolute variant- and reference-harboring read counts, as well as cell-SNV matrices of expressed Variant Allele Fraction (VAFRNA) suitable for a variety of downstream applications. We demonstrate three different SCReadCounts applications on 59,884 cells from seven neuroblastoma samples: (1) estimation of cell-level expression of known somatic mutations and RNA-editing sites, (2) estimation of cell- level allele expression of biallelic SNVs, and (3) a discovery mode assessment of the reference and each of the three alternative nucleotides at genomic positions of interest that does not require prior SNV information. For the later, we applied SCReadCounts on the coding regions of KRAS, where it identified known and novel somatic mutations in a low-to-moderate proportion of cells. The SCReadCounts read counts module is benchmarked against the analogous modules of GATK and Samtools. SCReadCounts is freely available (https://github.com/HorvathLab/NGS) as 64-bit self-contained binary distributions for Linux and MacOS, in addition to Python source. Conclusions SCReadCounts supplies a fast and efficient solution for estimation of cell-level SNV expression from scRNA-seq data. SCReadCounts enables distinguishing cells with monoallelic reference expression from those with no gene expression and is applicable to assess SNVs present in only a small proportion of the cells, such as somatic mutations in cancer.

APOBEC Mutagenesis Is Concordant between Tumor and Viral Genomes in HPV-Positive Head and Neck Squamous Cell Carcinoma

APOBEC is a mutagenic source in human papillomavirus (HPV)-mediated malignancies, including HPV+ oropharyngeal squamous cell carcinoma (HPV + OPSCC), and in HPV genomes. It is unknown why APOBEC mutations predominate in HPV + OPSCC, or if the APOBEC-induced mutations observed in both human cancers and HPV genomes are directly linked. We performed sequencing of host somatic exomes, transcriptomes, and HPV16 genomes from 79 HPV + OPSCC samples, quantifying APOBEC mutational burden and activity in both host and virus. APOBEC was the dominant mutational signature in somatic exomes. In viral genomes, there was a mean of five (range 0–29) mutations per genome. The mean of APOBEC mutations in viral genomes was one (range 0–5). Viral APOBEC mutations, compared to non-APOBEC mutations, were more likely to be low-variant allele fraction mutations, suggesting that APOBEC mutagenesis actively occurrs in viral genomes during infection. HPV16 APOBEC-induced mutation patterns in OPSCC were similar to those previously observed in cervical samples. Paired host and viral analyses revealed that APOBEC-enriched tumor samples had higher viral APOBEC mutation rates (p = 0.028), and APOBEC-associated RNA editing (p = 0.008), supporting the concept that APOBEC mutagenesis in host and viral genomes is directly linked and occurrs during infection. Using paired sequencing of host somatic exomes, transcriptomes, and viral genomes, we demonstrated for the first-time definitive evidence of concordance between tumor and viral APOBEC mutagenesis. This finding provides a missing link connecting APOBEC mutagenesis in host and virus and supports a common mechanism driving APOBEC dysregulation.

Venetoclax and Azacitidine Therapy in Elder Acute Myeloid Leukemia: A Retrospective Evaluation of Real-World Experience

Background：To investigate the efficacy of venetoclax combined with azacytidine in the treatment of elderly patients with relapsed and refractory (R/R) acute myeloid leukemia（AML). Methods： The clinical data of 9 elderly AML patients over 65 years old, including 5 with R/R AML, using venetoclax and azacytidine were retrospectively analyzed. Results： Six males and 3 females with a median age of 71 years were included in this study, of which four patients had at least one relapse, and one patient did not get go into remission after 4 cycles of azacytidine monotherapy, deeming it refractory. Four patients had AML with myelodysplasia-related changes (AML-MRC). After 1 to 13 cycles of treatment using venetoclax and azacytidine, one of the 9 patients died early due to long duration of neutropenia and severe pulmonary infection caused by drugs. and six of the remaining 8 patients obtained complete response or complete response with incomplete hematologic recovery (CR/ CRi) , including five R/R patients. One patient did not respond to treatment after two cycles. For the side effects of the treatment, granulocytopenia occurred in all patients, and neutropenia occurred in 8 patients, lasting for an average of 10.5 (6-15) days and was most obvious in the second to third week of treatment. Three patients with TP53 gene mutation positive had following different outcomes. One relapsed patient achieved progression free remission (PFS) for 16 months up to date, and a second patient achieved complete remission but relapsed two months thereafter. Another patient had complete remission in myelology for 4months, but the variant allele fraction value (VAF) gradually increased, indicative that the disease was about to progress. Conclusion：Venetoclax combined with azacytidine regimen in elderly patients is an effective and well tolerated rescue scheme for R/R AML.The patients with TP53 mutation with lower VAF may be benifit from Venetoclax and azacytidine. Severe infection caused by neutropenia is an adverse reaction worthy of attention in the treatment process of the regimen.

Serial Monitoring of Circulating Tumor DNA by Next-Generation Gene Sequencing as a Biomarker of Response and Survival in Patients With Advanced NSCLC Receiving Pembrolizumab-Based Therapy

PURPOSE Although the majority of patients with metastatic non–small-cell lung cancer (mNSCLC) lacking a detectable targetable mutation will receive pembrolizumab-based therapy in the frontline setting, predicting which patients will experience a durable clinical benefit (DCB) remains challenging. MATERIALS AND METHODS Patients with mNSCLC receiving pembrolizumab monotherapy or in combination with chemotherapy underwent a 74-gene next-generation sequencing panel on blood samples obtained at baseline and at 9 weeks. The change in circulating tumor DNA levels on-therapy (molecular response) was quantified using a ratio calculation with response defined by a > 50% decrease in mean variant allele fraction. Patient response was assessed using RECIST 1.1; DCB was defined as complete or partial response or stable disease that lasted > 6 months. Progression-free survival and overall survival were recorded. RESULTS Among 67 patients, 51 (76.1%) had > 1 variant detected at a variant allele fraction > 0.3% and thus were eligible for calculation of molecular response from paired baseline and 9-week samples. Molecular response values were significantly lower in patients with an objective radiologic response (log mean 1.25% v 27.7%, P < .001). Patients achieving a DCB had significantly lower molecular response values compared to patients with no durable benefit (log mean 3.5% v 49.4%, P < .001). Molecular responders had significantly longer progression-free survival (hazard ratio, 0.25; 95% CI, 0.13 to 0.50) and overall survival (hazard ratio, 0.27; 95% CI, 0.12 to 0.64) compared with molecular nonresponders. CONCLUSION Molecular response assessment using circulating tumor DNA may serve as a noninvasive, on-therapy predictor of response to pembrolizumab-based therapy in addition to standard of care imaging in mNSCLC. This strategy requires validation in independent prospective studies.

Circulating tumor DNA in identifying resistant subclones post EGFR blockade: Implications for EGFR rechallenge.

131 Background: For patients with metastatic RAS/RAF wild-type refractory colorectal cancer anti-EGFR therapy can be reused in subsequent lines of therapy. However, it is not entirely clear if all patients derive benefit. Increasingly it is been recognized that these patients acquire mechanisms of resistance which can be detected on circulating tumor DNA-based testing. We present a series of patients who had serial testing post EGFR therapy showing its feasibility and value. This would have implications for EGFR rechallenge. Methods: We reviewed records for patients who initially were noted to have tissue RAS/RAF Wild type who received prior anti-EGFR therapy and then subsequently had at least 1 circulating tumor DNA-based testing. Included a result of patients who had comprehensive NGS based profiling for both tissue as well as ctDNA. Results: Median duration of initial prior anti EGFR therapy was around 10 months. Table shows results of patient's tissue based genomic testing in parallel with the serial circulating tumor DNA-based testing that was done later when subsequent lines of therapy were being decided. As noted, known acquired mechanisms of resistance were noted in 100% of the cases. These included KRAS, NRAS, extracellular domain mutations in EGFR and BRAF mutations. Interestingly the levels of the sub-clones expressed as variant allele fraction percentage varied and decreased over time in relation to timing of the prior EGFR exposure. Additionally, these were noted to be polyclonal and the number of clones also varied including some disappearing over time during non-EGFR based therapy (EGFR holiday). Conclusions: Patient's post EGFR blockade may have multiple mechanisms of acquired resistance that can be easily detected on noninvasive liquid biopsies. These patients likely will not benefit from EGFR rechallenge based on the results of the recently reported CRICKET (NCT02296203) and CAVE (EudraCT number: 2017-004392-32) clinical trials. Rechecking liquid biopsy plasma RAS/RAF status is one thing that may be incorporated into practice with EGFR rechallenge only if acquired mechanisms of resistance are absent. [Table: see text]

SCReadCounts: Estimation of cell-level SNVs from scRNA-seq data

SummarySCReadCounts is a method for a cell-level estimation of the sequencing read counts bearing a particular nucleotide at genomic positions of interest from barcoded scRNA-seq alignments. SCReadCounts generates an array of outputs, including cell-SNV matrices with the absolute variant-harboring read counts, as well as cell-SNV matrices with expressed Variant Allele Fraction (VAFRNA); we demonstrate its application to estimate cell level expression of somatic mutations and RNA-editing on cancer datasets. SCReadCounts is benchmarked against GATK and Samtools and is freely available as a 64-bit self-contained binary distribution (Linux), along with MacOS and Python installation.Availabilityhttps://github.com/HorvathLab/NGS/tree/master/SCReadCountsSupplementary InformationSCReadCounts_Supplementary_Data.zip