Development and validation of a targeted gene sequencing panel for application to disparate cancers

Next generation sequencing has revolutionised genomic studies of cancer, having facilitated the development of precision oncology treatments based on a tumour’s molecular profile. We aimed to develop a targeted gene sequencing panel for application to disparate cancer types with particular focus on tumours of the head and neck, plus test for utility in liquid biopsy. The final panel designed through Roche/Nimblegen combined 451 cancer-associated genes (2.01 Mb target region). 136 patient DNA samples were collected for performance and application testing. Panel sensitivity and precision were measured using well-characterised DNA controls (n = 47), and specificity by Sanger sequencing of the Aryl Hydrocarbon Receptor Interacting Protein (AIP) gene in 89 patients. Assessment of liquid biopsy application employed a pool of synthetic circulating tumour DNA (ctDNA). Library preparation and sequencing were conducted on Illumina-based platforms prior to analysis with our accredited (ISO15189) bioinformatics pipeline. We achieved a mean coverage of 395x, with sensitivity and specificity of >99% and precision of >97%. Liquid biopsy revealed detection to 1.25% variant allele frequency. Application to head and neck tumours/cancers resulted in detection of mutations aligned to published databases. In conclusion, we have developed an analytically-validated panel for application to cancers of disparate types with utility in liquid biopsy

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Peak study: A phase 3, randomized, open-label multicenter clinical study of bezuclastinib (CGT9486) and sunitinib in combination versus sunitinib in patients with gastrointestinal stromal tumors (GIST)

TPS766 Background: Imatinib is the worldwide standard for first-line therapy of advanced KIT-mutant GIST. However, secondary resistance mutations in the KIT ATP-binding domain (exons 13, 14), activation loop (exons 17, 18), or both develop and result in loss of imatinib-sensitivity. While no single tyrosine kinase inhibitor (TKI) inhibits all KIT mutations, the combination of bezuclastinib + sunitinib targets commonly occurring primary (exons 9, 11) and secondary (exons 13, 14, 17, and 18) KIT mutations. In a Phase 1/2 clinical study, pts with relapsed and/or refractory GIST and a median of 3 prior lines of therapy treated with bezuclastinib + sunitinib (n=15) experienced clinical benefit and an acceptable safety profile, warranting further evaluation in a randomized trial. Methods: Peak (NCT05208047) is a global, randomized, open-label, multi-part Phase 3 study evaluating the efficacy and safety of bezuclastinib + sunitinib versus sunitinib as second-line treatment in adult pts who were intolerant to imatinib or whose tumors had imatinib-resistance. The lead-in portion, to test a new formulation of bezuclastinib, (Part 1) has completed enrollment. Based upon PK and safety, a dose of bezuclastinib 600 mg QD + sunitinib 37.5 mg QD has been determined for Part 2 of the Peak study. Part 2 will enroll ~388 pts to be randomized (1:1) to bezuclastinib 600 mg QD + sunitinib 37.5 mg QD or sunitinib 37.5 mg QD alone. Key inclusion: >1 measurable lesion per modified Response Evaluation Criteria in Solid Tumors (mRECIST) v1.1, Eastern Cooperative Oncology Group Performance Status 0-2, adequate organ function, and prior imatinib therapy (no other prior therapy). Key exclusion: PDGFR mutations or succinate dehydrogenase deficiency, clinically significant cardiac disease, and use of strong CYP3A4 inhibitors or inducers. The primary endpoint is progression-free survival (PFS) confirmed by blinded independent central review per mRECIST v1.1. Additional efficacy (including overall survival and objective response rate) and safety endpoints will be evaluated. Clinical trial information: NCT05208047

ETCTN/NCI 10330: A phase 2 study of belinostat with SGI-110 (guadecitabine) or ASTX727 (decitabine/cedazuridine) for the treatment of unresectable and metastatic conventional chondrosarcoma

11531 Background: Conventional chondrosarcoma (cCS) is the 2nd most common primary bone tumor and is resistant to chemotherapy and radiation. IDH1/2 mutations (m) occur in 50% of cCS. Both IDHm and wild-type (wt) cCS harbor epigenetic dysregulation. In preclinical models of IDHm and wt cCS, combination treatment with HDAC and DNMT inhibitors (i) suppressed growth in vitro and in vivo by reversing the hypermethylated state and inducing tumor suppressors, interferon response genes and apoptosis (Sheikh T, Schwartz G. Mol Cancer Ther 2021;20). Methods: NCI 10330 is a single-arm, multicenter, phase 2 study evaluating the HDACi belinostat (B) with the DNMTi SGI-110 (S) or ASTX727 (A). A replaced S due to drug availability (pts were replaced). Pts had advanced cCS, ECOG PS ≤ 2 and could be treatment naïve. Progression was required for grade 1 cCS. Pts received B 1000mg/m2 IV + S 45mg/m2 SC both days 1-5 or B (same dosing) + A (cedazuridine 100mg/decitabine 35mg) PO both days 1-5, in 28-day cycles. 1° endpoint was objective response. A Simon 2-stage design was used. If ≥ 2/13 responses occurred in stage 1, the study would proceed to full accrual. Design had 85% power with α = 0.05 to test ORR 8% vs 28%. 2° endpoints included safety, PFS and OS. A safety lead-in was performed. Paired biopsies were collected. Results: Stage 1 is complete. 19 pts were treated: 6 on B+S and 13 on B+A. Median age was 50 and 67 years, respectively. All pts had prior surgery. 17% (B+S) and 38% (B+A) had prior radiation. 33% (B+S) and 55% (B+A) were IDHm. 67% (B+S) and 75% (B+A) were histologic grade ≥ 2. There were no objective responses. Best response (at 8 weeks) was stable disease (SD) in 4/6 pts (67%) on B+S and 6/10 pts (60%) on B+A. mPFS was 4.2 mos (95% CI 1.97-NR) for B+S and 3.8 mos (95% CI 2.17-NR) for B+A. mOS has not been reached. For B+A, mPFS for IDHm vs wt pts was 4.7 and 3.1 mos, respectively (p=0.21). One pt with IDHm grade 2 cCS who progressed on FT-2102 (IDH1i) remains on B+A > 1 year. There were no DLTs during either safety lead-in. Grade 3/4 treatment-related adverse events (TRAEs) occurred in 17% (B+S) and 69% (B+A). For B+A, the most common grade 3/4 TRAE was neutropenia (54%) and the most common all-grade TRAEs were nausea (69%), leukopenia (61%), neutropenia (54%), anemia (46%) and fatigue (46%). Paired tumor biopsies are being evaluated with whole exome sequencing, RNAseq, methylation array and multiplex IHC with results forthcoming. Conclusions: Combination HDACi + DNMTi was well-tolerated in advanced cCS. There were no objective responses; however, a subset of pts experienced prolonged SD with a trend towards improved mPFS in IDHm pts. Correlative work is ongoing with a focus on differential effects on IDHm tumors and whether modulation of the immune microenvironment might support combinations with immunotherapy. Support: UM1CA186689. Clinical trial information: NCT04340843

Correlative results from NCI CTEP/ETCTN 10330: A phase 2 study of belinostat with SGI-110 (guadecitabine) or ASTX727 (decitabine/cedazuridine) for advanced conventional chondrosarcoma (cCS)

11526 Background: There are no FDA-approved treatments for advanced cCS. Chemotherapy provides limited benefit. IDH1/2 mutations (m) occur in 50% of cCS. Both IDHm and wild-type cCS harbor epigenetic dysregulation. In preclinical models, HDAC + DNMT inhibition (i) suppressed growth by inducing apoptosis, reversing the hypermethylated state, and upregulating expression of interferon (IFN) response genes including PDL1(1). This prompted a phase 2 study of HDACi + DNMTi in cCS, which failed to meet the 1° endpoint of ORR. The majority of pts had a best response of stable disease, with a trend towards improved mPFS in IDHm (2). Here we report correlative analysis of NCI 10330. Methods: NCI 10330 is a single-arm, multicenter, phase 2 study of belinostat with SGI-110 or ASTX727 in advanced cCS. All pts were required to have pre- and on-treatment (tx) biopsies (Cycle 2, Day 3-5). Tissue was evaluated with whole exome sequencing (WES) and RNA sequencing (RNAseq). From RNAseq, Tumor Inflammation Signature Scores (TISS) and Tumor Microenvironment functional Gene Set Enrichment Analysis scores (TME GSEA) were calculated as the mean of the log2 normalized counts of 18 signature genes (Danaher et al. J Imm Can 2018) and pre-defined gene sets (3), respectively. Wilcoxon rank sum test was used to analyze TME GSEA between pre- and on-tx samples. Differential expression was significant if |FC| > 1.33, adjusted P <0.01. Results: 19 pts were treated; all received paired biopsies. WES was adequate in 7/19 (37%) pts, with IDHm identified in 3/7 (43%) pts. All pts were MSI-stable, TMB: 1.01-3.57 mut/Mb. RNAseq was adequate in 7/19 (37%) pts at pre-tx and 5/19 (26%) pts at on-tx. TISS ranges trended higher for pre-tx (8.77-10.14) vs on-tx (7.36-9.51) samples. TME GSEA identified significantly enriched tumor immune infiltration (p < 0.05) in pre- vs on-tx samples. Differential analysis identified several gene sets related to inflammation overexpressed in pre-tx samples only, including IFN response. On-tx samples were enriched in myogenesis and coagulation gene sets. Conclusions: This is the first study to describe transcriptomic changes following epigenetic tx in cCS. Contradictory to our preclinical data, HDACi + DNTMi resulted in low expression of inflammation. Prior studies reported that the immune infiltrate of CS at progression is immunosuppressive; higher immune infiltrate is correlated with worse outcomes (4). Loss of inflammation may have implications for disease stability experienced by most pts on NCI 10330. Further analyses are planned to correlate TME (pro- vs anti-inflammatory infiltrates) and outcomes. Analyses are limited by small sample size, highlighting the challenges in collecting adequate CS specimens. 1. Sheikh, Schwartz et al. Mol Cancer Ther 2021. 2. Lacuna et al. ASCO 2023: #11531. 3. Bagaev et al. Can Cell 2021. 4. Richert et al. J Bone Onc 2020. Clinical trial information: NCT04340843