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Supplementary Data from Bone Marrow Surveillance of Pediatric Cancer Survivors Identifies Clones that Predict Therapy-Related Leukemia

Supplementary Figure from Bone Marrow Surveillance of Pediatric Cancer Survivors Identifies Clones that Predict Therapy-Related Leukemia

High-risk neuroblastoma is generally metastatic and often lethal. Using genomic profiling of 470 sequential and spatially separated samples from 283 patients, we characterize subtype-specific genetic evolutionary trajectories from diagnosis, through progression and end-stage metastatic disease. Clonal tracing timed disease initiation to embryogenesis. Continuous acquisition of structural variants at disease defining loci (MYCN, TERT, MDM2-CDK4) followed by convergent evolution of mutations targeting shared pathways emerged as the predominant feature of progression. At diagnosis metastatic clones were already established at distant sites where they could stay dormant, only to cause relapses years later and spread via metastasis-to-metastasis and polyclonal seeding after therapy.

Homologous recombination deficiency (HRd) is a DNA repair defect prevalent in but not exclusive to breast and ovarian cancer most commonly associated with BRCA1 or BRCA2 alterations. HRd results in accumulation of small and large scale genetic alterations across the genome, including allele specific copy number alterations (aCNAs), small nucleotide variants (SNVs), deletions, and structural variants (SVs). Detection of HRd in tumors predicts response to genotoxic drugs such as PARP inhibitors and platinum. Genome wide aCNAs such as large state transitions (LST), loss of heterozygosity (LOH), and telomeric allelic imbalances (TAI) in conjunction with BRCA1/2 mutation detection have been implemented in routine diagnostic testing to identify HRd in tumors. However, these features represent a subset of the genetic signatures predictive of HRd, and we hypothesize that a significant portion of tumors with HRd are missed using these existing assays. Whole genome sequencing (WGS) enables the detection of the full spectrum of genetic lesions that arise in an HRd tumor in a single assay. To demonstrate the added value of WGS to identify HRd, we trained and validated a pan-cancer classifier of HRd. A tumor/normal matched cohort of 321 cancer patients sequenced by WGS was assembled and analyzed as part of a retrospective study, representing 62 tumor types. An unbiased analysis of HRd associated SV signatures revealed the top quartile of samples harboring tandem duplications (Dups) and deletions (Dels) in the size range of 1-10kbp were enriched with BRCA1, BRCA2, and RAD51C/D alterations. Through curating Dels, Dups, HRd SNV/InDel signatures, and alteration of HRd associated genes, 37 unique patients were found to have high confidence HRd, out of which 13% had no alterations in BRCA1, BRCA2, or other HRd genes. We then trained a random forest classifier to identify HRd tumors. The most important predictive features were WGS-specific, namely small deletions with microhomology, SV Dels, and SV Dups. The HRd classifier was validated using an independent cohort of 556 samples from the Pan-Cancer Analysis of Whole Genomes (PCAWG) study. Of 46 samples with biallelic BRCA1/2 alterations, the classifier achieved high areas under receiver-operator characteristic (AUROC, 0.99) and precision recall curves (AUPRC, 0.96). The aCNA score, the number of segments harboring LST, LOH, and TAI, had similar AUROC (0.96) but lower AUPRC (0.87). There were 11 BRCA1/2 non-altered cases predicted to be HRd with the classifier which were not identified by CNA scores, in which 10 had at least 1 alteration in an HRd gene, including RAD51C, CHEK2 biallelic alterations and SVs in PALB2, Fanconi pathway genes, and ATM/ATR. We conclude that a classifier incorporating the additional mutational features which can only be detected using WGS can achieve superior precision in identifying HRd tumors and, in the future, uncover additional patients for therapeutic options. Citation Format: Kevin Hadi, Gunes Gundem, Max F. Levine, Aditya Deshpande, Minal Patel, Stan Skzrypczak, Majd Al Assaad, Juan Miguel Mosquera, Olivier Elemento, Andrew L. Kung, Juan S. Medina-Martínez, Elli Papaemmanuil. A whole genome sequencing classifier of homologous recombination deficiency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2149.

Whole genome sequencing (WGS) has emerged as a tool to characterize the entire genomic landscape of cancer and capture potential new targets and predictive biomarkers. Using state-of-the-art whole-genome and transcriptome sequencing (WGTS) analysis we interrogated a pan cancer cohort to nominate new treatment options. WGS was performed on 414 tumor/germline pairs from our pan cancer cohort of 355 patients, of which 103 had results of other commercial NGS assays. RNAseq data was available in 96 samples. We employed the Isabl GxT analytic platform and manually curated single base substitution (COSMIC v3) mutational signatures and structural variants (SVs). WGTS results were correlated with available clinico-pathologic data. High throughput drug screening that included PARP inhibitors and functional validation were performed in 61 matched patient-derived tumor organoids (PDTO). We found 5,129 SVs affecting at least one cancer gene in 88% of cases. High-confidence homologous recombination deficiency (HRD), determined by curation of mutational signatures and mutations/SVs affecting oncogenes and tumor suppressor genes, was detected in 8.3% of solid tumors (e.g., prostate, pancreas, ovarian, uterine, breast, gallbladder, and stomach carcinomas). HRD status included tumors with BRCA1/2 wildtype and SVs impacting BRCA1/2, RAD51, ATM and other HRD genes. CDK12-loss associated genomic instability was detected in 7 cases including 3 cases of prostate adenocarcinoma. 93 gene fusions involving cancer genes were detected including known and novel fusions of potential significance (e.g., SLC45A3::ALK in metastatic prostate adenocarcinoma, TMEM106A::BRCA1 in serious ovarian carcinoma, ESR1::EP300 in metastatic breast cancer). Some tumor cohorts demonstrated enrichment of drivers e.g., FGFR1 amplification in male breast cancer, rearrangements at the TERT locus in adrenocortical carcinoma, enrichment for repeat-mediated deletion attributed to mismatch repair (MMR) in gynecological carcinosarcoma, and SVs affecting FHIT in gastric adenocarcinoma. Drug screening of PDTO and functional testing with RAD51 immunofluorescence assay validated high-confidence HRD annotation. One example is a BRCA1/2 wildtype pancreatic adenocarcinoma with increased tandem duplications and SV (inversion) impacting BRCA2, and high-confidence HRD by our novel WGS classifier. Drug screening of the corresponding PDTO showed an enhanced response to Olaparib, in contrast to cases without HRD that did not. We have used state-of-the-art analysis of WGTS to improve the detection of targets and significant biomarkers in a pan cancer cohort, including HRD, CDK12-type genomic instability, pathogenic germline mutations and potential targetable novel fusions. A robust and clinically relevant ex vivo preclinical model allowed us to validate a WGS-based classifier that detects HRD even in cancers with wildtype BRCA1/2. Citation Format: Majd Al Assaad, Hui-Hsuan Kuo, Jones Nauseef, Max F. Levine, Gunes Gundem, Juan S. Medina-Martínez, Jyothi Manohar, Michael Sigouros, Ahmed Elsaeed, David Wilkes, Elli Papaemmanuil, Eloise Chapman, Scott T. Tagawa, Allyson Ocean, Cora N. Sternberg, Eleni Andreopoulou, Manish Shah, Andrea Sboner, Kevin Holcomb, David Nanus, Laura Martin, Olivier Elemento, Juan Miguel Mosquera. Whole genome and transcriptome sequencing of a pan cancer cohort unearths novel therapeutic avenues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3437.

Purpose: Therapy-related myelodysplastic syndrome and acute leukemias (t-MDS/AL) are a major cause of nonrelapse mortality among pediatric cancer survivors. Although the presence of clonal hematopoiesis (CH) in adult patients at cancer diagnosis has been implicated in t-MDS/AL, there is limited published literature describing t-MDS/AL development in children. Experimental Design: We performed molecular characterization of 199 serial bone marrow samples from 52 patients treated for high-risk neuroblastoma, including 17 with t-MDS/AL (transformation), 14 with transient cytogenetic abnormalities (transient), and 21 without t-MDS/AL or cytogenetic alterations (neuroblastoma-treated control). We also evaluated for CH in a cohort of 657 pediatric patients with solid tumor. Results: We detected at least one disease-defining alteration in all cases at t-MDS/AL diagnosis, most commonly TP53 mutations and KMT2A rearrangements, including involving two novel partner genes (PRDM10 and DDX6). Backtracking studies identified at least one t-MDS/AL-associated mutation in 13 of 17 patients at a median of 15 months before t-MDS/AL diagnosis (range, 1.3–32.4). In comparison, acquired mutations were infrequent in the transient and control groups (4/14 and 1/21, respectively). The relative risk for development of t-MDS/AL in the presence of an oncogenic mutation was 8.8 for transformation patients compared with transient. Unlike CH in adult oncology patients, TP53 mutations were only detectable after initiation of cancer therapy. Last, only 1% of pediatric patients with solid tumor evaluated had CH involving myeloid genes. Conclusions: These findings demonstrate the clinical relevance of identifying molecular abnormalities in predicting development of t-MDS/AL and should guide the formation of intervention protocols to prevent this complication in high-risk pediatric patients.

Introduction Sarcopenia likely comprises muscle fiber denervation and re-innervation, resulting in clustering of muscle fibers of the same type (classified by myosin heavy chain isoform composition). Development of methodology to quantitatively evaluate clustering of muscle fibers according to fiber type is necessary. Methods Fiber type specific immunofluorescence histology was used to quantify fiber clustering in murine diaphragm muscle (n = 15) at ages 6 and 24 months. Results With age, fiber type clustering is evidenced by fiber type specific changes in distances between fibers, specifically a 14% decrease to the closest fiber for type I and 24% increase for type IIx and/or IIb fibers (P

We developed and tested a semi-automated algorithm to generate large data sets of ventilatory information (amplitude, premotor drive and timing) across a range of motor behaviors. Adult spontaneously breathing, anesthetized mice (n = 27) underwent measurements of transdiaphragmatic pressure (Pdi) during eupnea, hypoxia-hypercapnia, and tracheal occlusion with values ranging from 8 ± 1 to 9 ± 2 to 44 ± 3 cmH2O, respectively. Premotor drive to phrenic motor neurons (estimated by the rate of rise during initial 60 ms) was ∼ 5-fold greater during tracheal occlusion compared to other behaviors. Variability in Pdi amplitude (normalized to spontaneously occurring sighs for each animal) displayed minimal evidence of complex temporal structure or dynamic clustering across the entire period of examination. Using a deterministic model to evaluate predictor variables for Pdi amplitude between successive inspiratory events, there was a large correlation for premotor drive and preceding Pdi amplitude vs. Pdi amplitude (r = 0.52). These findings highlight substantial variability in Pdi amplitude that primarily reflects linear components rather than complex, dynamic effects over time.

To perform a range of ventilatory and nonventilatory behaviors, the diaphragm muscle (DIAm) must be able to generate sufficient forces throughout the lifespan. We hypothesized that sarcopenia impacts DIAm force generation and thus limits performance of expulsive, higher force, nonventilatory behaviors. Male and female mice ( n = 79) at 6 and 24 mo of age (100 vs. 70–75% survival, respectively) were used to examine transdiaphragmatic pressure (Pdi) generation across motor behaviors in vivo and in vitro DIAm specific force. We found a significant effect of age on maximum Pdi (20–41% decline during tracheal occlusion and bilateral phrenic nerve stimulation), maximum DIAm specific force (30% decline), and DIAm fatigue resistance (15% increase). There were no differences between sexes in these age effects on DIAm performance. These results support our hypothesis that sarcopenia primarily impacts higher force, nonventilatory motor behaviors of the DIAm. Such functional limitations may have negative implications in the ability of the DIAm to generate forces needed for airway clearance in old age and thereby contribute to age-related respiratory complications.