Sotorasib in KRAS p.G12C-Mutated Advanced Pancreatic Cancer.

BACKGROUND: KRAS p.G12C mutation occurs in approximately 1 to 2% of pancreatic cancers. The safety and efficacy of sotorasib, a KRAS G12C inhibitor, in previously treated patients with KRAS p.G12C-mutated pancreatic cancer are unknown. METHODS: We conducted a single-group, phase 1-2 trial to assess the safety and efficacy of sotorasib treatment in patients with KRAS p.G12C-mutated pancreatic cancer who had received at least one previous systemic therapy. The primary objective of phase 1 was to assess safety and to identify the recommended dose for phase 2. In phase 2, patients received sotorasib at a dose of 960 mg orally once daily. The primary end point for phase 2 was a centrally confirmed objective response (defined as a complete or partial response). Efficacy end points were assessed in the pooled population from both phases and included objective response, duration of response, time to objective response, disease control (defined as an objective response or stable disease), progression-free survival, and overall survival. Safety was also assessed. RESULTS: The pooled population from phases 1 and 2 consisted of 38 patients, all of whom had metastatic disease at enrollment and had previously received chemotherapy. At baseline, patients had received a median of 2 lines (range, 1 to 8) of therapy previously. All 38 patients received sotorasib in the trial. A total of 8 patients had a centrally confirmed objective response (21%; 95% confidence interval [CI], 10 to 37). The median progression-free survival was 4.0 months (95% CI, 2.8 to 5.6), and the median overall survival was 6.9 months (95% CI, 5.0 to 9.1). Treatment-related adverse events of any grade were reported in 16 patients (42%); 6 patients (16%) had grade 3 adverse events. No treatment-related adverse events were fatal or led to treatment discontinuation. CONCLUSIONS: Sotorasib showed anticancer activity and had an acceptable safety profile in patients with KRAS p.G12C-mutated advanced pancreatic cancer who had received previous treatment. (Funded by Amgen and others; CodeBreaK 100 ClinicalTrials.gov number, NCT03600883.).

Trastuzumab deruxtecan in patients with solid tumours harbouring specific activating HER2 mutations (DESTINY-PanTumor01): an international, phase 2 study.

BACKGROUND: Trastuzumab deruxtecan is a HER2-directed antibody-drug conjugate approved by the US Food and Drug Administration and the European Medicines Agency for HER2-mutant non-small-cell lung cancer. Few treatment options exist for patients with HER2-mutant solid tumours beyond lung cancers. We investigated trastuzumab deruxtecan in metastatic solid tumours with specific activating HER2 mutations. METHODS: In this open-label, phase 2, basket study done in 29 centres in Asia, Europe, and North America, we investigated trastuzumab deruxtecan (5·4 mg/kg every 3 weeks by intravenous infusion) in patients aged 18 years or older with unresectable or metastatic solid tumours with specific activating HER2 mutations, an Eastern Cooperative Oncology Group performance status of 0 or 1, and disease progression following previous treatment (previous HER2-targeted therapy was permitted) or with no satisfactory alternative treatment options. The primary endpoint was confirmed objective response rate by independent central review. Anti-tumour activity and safety were analysed in all patients who received at least one dose of trastuzumab deruxtecan. This trial is registered with ClinicalTrials.gov, NCT04639219, and is active but no longer recruiting. FINDINGS: Between Dec 30, 2020, and Jan 25, 2023, 102 patients (62 [61%] female and 40 [39%] male; median age 66·5 years [IQR 58-72]; 51 [50%] White, two [2%] Black or African American, 38 [37%] Asian, and 11 [11%] did not have race information reported) with solid tumours with activating HER2 mutations received trastuzumab deruxtecan and were included in the anti-tumour activity and safety analyses sets. Patients had a median of three (IQR 2-4) previous treatment regimens. The median duration of follow-up was 8·61 months (IQR 3·71-12·68). The objective response rate by independent central review was 29·4% (95% CI 20·8-39·3; 30 of 102 patients). 52 (51%) patients had a treatment-emergent adverse event of grade 3 or worse; the most common events (in ≥5% of patients) were anaemia (16 [16%]) and neutrophil count decreased (eight [8%]). Drug-related treatment-emergent serious adverse events occurred in ten (10%) patients. Adjudicated drug-related interstitial lung disease or pneumonitis of any grade occurred in 11 patients (11%; three grade 1, five grade 2, one grade 3, and two grade 5); there were two (2%) cases of fatal adjudicated drug-related interstitial lung disease or pneumonitis. INTERPRETATION: Trastuzumab deruxtecan showed anti-tumour activity and durable responses in heavily pretreated patients across multiple tumour types with activating HER2 mutations, with no new safety signals. Prespecified HER2 mutations might be targeted by HER2-directed antibody-drug conjugates and our findings support further investigation of trastuzumab deruxtecan in the pan-tumour setting. FUNDING: AstraZeneca and Daiichi Sankyo.

Neoadjuvant and adjuvant pembrolizumab plus chemotherapy in locally advanced gastric or gastro-oesophageal cancer (KEYNOTE-585): an interim analysis of the multicentre, double-blind, randomised phase 3 study.

BACKGROUND: The benefit of combination neoadjuvant and adjuvant chemotherapy and immune checkpoint inhibition in patients with locally advanced, resectable gastric or gastro-oesophageal adenocarcinoma is unknown. We assess the antitumor activity of neoadjuvant and adjuvant pembrolizumab plus chemotherapy in patients with locally advanced resectable gastric or gastro-oesophageal adenocarcinoma. METHODS: The KEYNOTE-585 study is a multicentre, randomised, placebo-controlled, double-blind, phase 3 study done at 143 medical centres in 24 countries. Eligible patients were aged 18 years or older with untreated, locally advanced, resectable gastric or gastro-oesophageal adenocarcinoma, and an Eastern Cooperative Oncology Group performance status 0-1. Patients were randomly assigned (1:1) by an interactive voice response system and integrated web response system to neoadjuvant pembrolizumab 200 mg intravenously or placebo (saline) plus cisplatin-based doublet chemotherapy (main cohort) every 3 weeks for 3 cycles, followed by surgery, adjuvant pembrolizumab or placebo plus chemotherapy for 3 cycles, then adjuvant pembrolizumab or placebo for 11 cycles. A small cohort was also randomly assigned (1:1) to pembrolizumab or placebo plus fluorouracil, docetaxel, and oxaliplatin (FLOT)-based chemotherapy (FLOT cohort) every 2 weeks for four cycles, followed by surgery, adjuvant pembrolizumab, or placebo plus FLOT for four cycles, then adjuvant pembrolizumab or placebo for 11 cycles. Patients were stratified by geographic region, tumour stage, and chemotherapy backbone. Primary endpoints were pathological complete response (reviewed centrally), event-free survival (reviewed by the investigator), and overall survival in the intention-to-treat population, and safety assessed in all patients who received at least one dose of study treatment. The study is registered at ClinicalTrials.gov, NCT03221426, and is closed to accrual. FINDINGS: Between Oct 9, 2017, and Jan 25, 2021, of 1254 patients screened, 804 were randomly assigned to the main cohort, of whom 402 were assigned to the pembrolizumab plus cisplatin-based chemotherapy group and 402 to the placebo plus cisplatin-based chemotherapy group, and 203 to the FLOT cohort, of whom 100 were assigned to the pembrolizumab plus FLOT group and 103 to placebo plus FLOT group. In the main cohort of 804 participants, 575 (72%) were male and 229 (28%) were female. In the main cohort, after median follow-up of 47·7 months (IQR 38·0-54·8), pembrolizumab was superior to placebo for pathological complete response (52 [12·9%; 95% CI 9·8-16·6] of 402 vs eight [2·0%; 0·9-3·9] of 402; difference 10·9%, 95% CI 7·5 to 14·8; p<0·00001). Median event-free survival was longer with pembrolizumab versus placebo (44·4 months, 95% CI 33·0 to not reached vs 25·3 months, 20·6 to 33·9; hazard ratio [HR] 0·81, 95% CI 0·67 to 0·99; p=0·0198) but did not meet the threshold for statistical significance (p=0·0178). Median overall survival was 60·7 months (95% CI 51·5 to not reached) in the pembrolizumab group versus 58·0 months (41·5 to not reached) in the placebo group (HR 0·90, 95% CI 0·73 to 1·12; p=0·174). Grade 3 or worse adverse events of any cause occurred in 312 (78%) of 399 patients in the pembrolizumab group and 297 (74%) of 400 patients in the placebo group; the most common were nausea (240 [60%] vs 247 [62%]), anaemia (168 [42%] vs 158 [40%]), and decreased appetite (163 [41%] vs 172 [43%]). Treatment-related serious adverse events were reported in 102 (26%) and 97 (24%) patients. Treatment-related adverse events that led to death occurred in four (1%) patients in the pembrolizumab group (interstitial ischaemia, pneumonia, decreased appetite, and acute kidney injury [n=1 each]) and two (<1%) patients in the placebo group (neutropenic sepsis and neutropenic colitis [n=1 each]). INTERPRETATION: Although neoadjuvant and adjuvant pembrolizumab versus placebo improved the pathological complete response, it did not translate to significant improvement in event-free survival in patients with untreated, locally advanced resectable gastric or gastro-oesophageal cancer. FUNDING: Merck Sharp & Dohme.

Pembrolizumab plus trastuzumab and chemotherapy for HER2-positive gastric or gastro-oesophageal junction adenocarcinoma: interim analyses from the phase 3 KEYNOTE-811 randomised placebo-controlled trial.

BACKGROUND: Evidence for the efficacy of combined PD-1 and HER2 blockade with chemotherapy on progression-free and overall survival in HER2-positive gastro-oesophageal cancer is scarce. The first interim analysis of the randomised, phase 3 KEYNOTE-811 study showed a superior objective response with pembrolizumab compared with placebo when added to trastuzumab plus fluoropyrimidine and platinum-based chemotherapy. Here, we report results from protocol-specified subsequent interim analyses of KEYNOTE-811. METHODS: The randomised, phase 3 KEYNOTE-811 trial involved 168 medical centres in 20 countries worldwide. Patients aged 18 years or older with locally advanced or metastatic HER2-positive gastro-oesophageal junction adenocarcinoma, without previous first-line treatment, were randomly assigned (1:1) by an integrated interactive voice-response and web-response system to intravenous pembrolizumab 200 mg or placebo, both to be combined with standard chemotherapy (fluoropyrimidine and platinum-based therapy) plus trastuzumab every 3 weeks for up to 35 cycles or until disease progression, unacceptable toxic effects, or investigator or participant-initiated withdrawal. Randomisation used a block size of four and was stratified by region, PD-L1 status, and chemotherapy. Dual primary endpoints were progression-free and overall survival, analysed by intention to treat. Safety was assessed in all randomly assigned patients who received at least one dose of study treatment according to the treatment received. KEYNOTE-811 is registered with ClinicalTrials.gov (NCT03615326) and is active but not recruiting. FINDINGS: Between Oct 5, 2018, and Aug 6, 2021, 698 patients were assigned to pembrolizumab (n=350) or placebo (n=348). 564 (81%) were male and 134 (19%) were female. At the third interim analysis, 286 (82%) of 350 patients in the pembrolizumab group and 304 (88%) of 346 in the placebo group who received treatment had discontinued treatment, mostly due to disease progression. At the second interim analysis (median follow-up 28·3 months [IQR 19·4-34·3] in the pembrolizumab group and 28·5 months [20·1-34·3] in the placebo group), median progression-free survival was 10·0 months (95% CI 8·6-11·7) in the pembrolizumab group versus 8·1 months (7·0-8·5) in the placebo group (hazard ratio [HR] 0·72, 95% CI 0·60-0·87; p=0·0002). Median overall survival was 20·0 months (17·8-23·2) versus 16·9 months (15·0-19·8; HR 0·87 [0·72-1·06]; p=0·084). At the third interim analysis (median follow-up 38·4 months [IQR 29·5-44·4] in the pembrolizumab group and 38·6 months [30·2-44·4] in the placebo group), median progression-free survival was 10·0 months (8·6-12·2) versus 8·1 months (7·1-8·6; HR 0·73 [0·61-0·87]), and median overall survival was 20·0 months (17·8-22·1) versus 16·8 months (15·0-18·7; HR 0·84 [0·70-1·01]), but did not meet prespecified criteria for significance and will continue to final analysis. Grade 3 or worse treatment-related adverse events occurred in 204 (58%) of 350 patients in the pembrolizumab group versus 176 (51%) of 346 patients in the placebo group. Treatment-related adverse events that led to death occurred in four (1%) patients in the pembrolizumab group and three (1%) in the placebo group. The most common treatment-related adverse events of any grade were diarrhoea (165 [47%] in the pembrolizumab group vs 145 [42%] in the placebo group), nausea (154 [44%] vs 152 [44%]), and anaemia (109 [31%] vs 113 [33%]). INTERPRETATION: Compared with placebo, pembrolizumab significantly improved progression-free survival when combined with first-line trastuzumab and chemotherapy for metastatic HER2-positive gastro-oesophageal cancer, specifically in patients with tumours with a PD-L1 combined positive score of 1 or more. Overall survival follow-up is ongoing and will be reported at the final analysis. FUNDING: Merck Sharp & Dohme.

Lenvatinib plus Pembrolizumab or Everolimus for Advanced Renal Cell Carcinoma.

BACKGROUND: Lenvatinib in combination with pembrolizumab or everolimus has activity against advanced renal cell carcinoma. The efficacy of these regimens as compared with that of sunitinib is unclear. METHODS: In this phase 3 trial, we randomly assigned (in a 1:1:1 ratio) patients with advanced renal cell carcinoma and no previous systemic therapy to receive lenvatinib (20 mg orally once daily) plus pembrolizumab (200 mg intravenously once every 3 weeks), lenvatinib (18 mg orally once daily) plus everolimus (5 mg orally once daily), or sunitinib (50 mg orally once daily, alternating 4 weeks receiving treatment and 2 weeks without treatment). The primary end point was progression-free survival, as assessed by an independent review committee in accordance with Response Evaluation Criteria in Solid Tumors, version 1.1. Overall survival and safety were also evaluated. RESULTS: A total of 1069 patients were randomly assigned to receive lenvatinib plus pembrolizumab (355 patients), lenvatinib plus everolimus (357), or sunitinib (357). Progression-free survival was longer with lenvatinib plus pembrolizumab than with sunitinib (median, 23.9 vs. 9.2 months; hazard ratio for disease progression or death, 0.39; 95% confidence interval [CI], 0.32 to 0.49; P<0.001) and was longer with lenvatinib plus everolimus than with sunitinib (median, 14.7 vs. 9.2 months; hazard ratio, 0.65; 95% CI, 0.53 to 0.80; P<0.001). Overall survival was longer with lenvatinib plus pembrolizumab than with sunitinib (hazard ratio for death, 0.66; 95% CI, 0.49 to 0.88; P = 0.005) but was not longer with lenvatinib plus everolimus than with sunitinib (hazard ratio, 1.15; 95% CI, 0.88 to 1.50; P = 0.30). Grade 3 or higher adverse events emerged or worsened during treatment in 82.4% of the patients who received lenvatinib plus pembrolizumab, 83.1% of those who received lenvatinib plus everolimus, and 71.8% of those who received sunitinib. Grade 3 or higher adverse events occurring in at least 10% of the patients in any group included hypertension, diarrhea, and elevated lipase levels. CONCLUSIONS: Lenvatinib plus pembrolizumab was associated with significantly longer progression-free survival and overall survival than sunitinib. (Funded by Eisai and Merck Sharp and Dohme; CLEAR ClinicalTrials.gov number, NCT02811861.).

Silmitasertib plus gemcitabine and cisplatin first-line therapy in locally advanced/metastatic cholangiocarcinoma: A Phase 1b/2 study.

BACKGROUND AND AIMS: This study aimed to investigate safety and efficacy of silmitasertib, an oral small molecule casein kinase 2 inhibitor, plus gemcitabine and cisplatin (G+C) versus G+C in locally advanced/metastatic cholangiocarcinoma. APPROACH AND RESULTS: This work is a Phase 1b/2 study (S4-13-001). In Phase 2, patients received silmitasertib 1000 mg twice daily for 10 days with G+C on Days 1 and 8 of a 21-day cycle. Primary efficacy endpoint was progression-free survival (PFS) in the modified intent-to-treat population (defined as patients who completed at least one cycle of silmitasertib without dose interruption/reduction) from both phases (silmitasertib/G+C n = 55, G+C n = 29). The response was assessed by Response Evaluation Criteria in Solid Tumors v1.1. The median PFS was 11.2 months (95% confidence interval [CI], 7.6, 14.7) versus 5.8 months (95% CI, 3.1, not evaluable [NE]) ( p  = 0.0496); 10-month PFS was 56.1% (95% CI, 38.8%, 70.2%) versus 22.2% (95% CI, 1.8%, 56.7%); and median overall survival was 17.4 months (95% CI, 13.4, 25.7) versus 14.9 months (95% CI, 9.9, NE) with silmitasertib/G+C versus G+C. Overall response rate was 34.0% versus 30.8%; the disease control rate was 86.0% versus 88.5% with silmitasertib/G+C versus G+C. Almost all silmitasertib/G+C (99%) and G+C (93%) patients reported at least one treatment emergent adverse event (TEAE). The most common TEAEs (all grades) with silmitasertib/G+C versus G+C were diarrhea (70% versus 13%), nausea (59% vs. 30%), fatigue (47% vs. 47%), vomiting (39% vs. 7%), and anemia (39% vs. 30%). Twelve patients (10%) discontinued treatment because of TEAEs during the study. CONCLUSIONS: Silmitasertib/G+C demonstrated promising preliminary evidence of efficacy for the first-line treatment of patients with locally advanced/metastatic cholangiocarcinoma.

Real-world outcomes of third-line immune checkpoint inhibitors versus irinotecan-based chemotherapy in patients with advanced gastric cancer: a Korean, multicenter study (KCSG ST22-06).

BACKGROUND: Immune checkpoint inhibitor (ICI) or irinotecan-based chemotherapy is frequently used after failure of second-line paclitaxel plus ramucirumab treatment for patients with locally advanced unresectable or metastatic advanced gastric cancer (AGC). This study aimed to compare the efficacy between ICI and irinotecan-based chemotherapy as third-line treatment in patients with AGC. METHODS: We retrospectively reviewed patients with AGC, whose third-line treatment started between July 2019 and June 2021 at 17 institutions in Korea. The ICI group included patients who received nivolumab or pembrolizumab, and the irinotecan-based chemotherapy group included patients who received irinotecan or FOLFIRI (5-fluorouracil, leucovorin and irinotecan). RESULTS: A total of 363 patients [n = 129 (ICI) and n = 234 (irinotecan-based chemotherapy)] were analyzed. The median progression-free survival was 2.3 and 2.9 months in ICI and irinotecan-based chemotherapy groups, respectively (p = 0.802). The median overall survival (OS) was 5.5 and 6.0 months in ICI and irinotecan-based chemotherapy groups, respectively (p = 0.786). For all patients included in this study, multivariable analysis showed that weight loss, peritoneal metastasis, low serum sodium or albumin, and short duration of second-line treatment were associated with inferior OS (p < 0.05). ICI showed significantly longer OS than irinotecan-based chemotherapy in patients without peritoneal metastasis. Whereas ICI showed significantly shorter OS in patients without PD-L1 expression than irinotecan-based chemotherapy. CONCLUSIONS: No significant difference in survival outcome was observed between ICI and irinotecan-based chemotherapy as third-line treatment for AGC patients. ICI might be preferred for patients without peritoneal metastasis and irinotecan-based chemotherapy for patients with tumors without PD-L1 expression. TRIAL REGISTRATION: This study was registered in the Clinical Trial Registry of Korea ( https://cris.nih.go.kr : KCT 0007732).

Second-line chemoimmunotherapy with nivolumab and paclitaxel in immune-related biomarker-enriched advanced gastric cancer: a multicenter phase Ib/II study.

BACKGROUND: We conducted a trial to evaluate the efficacy and safety of nivolumab and paclitaxel as second-line therapy for immune-related biomarker-enriched advanced gastric cancer (AGC). METHODS: This open-label, single-arm, phase Ib/II study was a part of multi-institutional, biomarker-integrated umbrella study conducted in Korea. In phase Ib, patients received nivolumab (3 mg/kg) on Days 1 and 15 and paclitaxel (dose level 1, 70 mg/m2 or dose level 2, 80 mg/m2) on Days 1, 8, 15 every four weeks. In phase II, patients with Epstein-Barr virus-related, deficient mismatch repair or programmed cell death-ligand-1-positive AGC were enrolled. The primary endpoints were recommended phase II dose (RP2D, phase Ib) and progression-free survival (PFS, phase II). Secondary endpoints included objective response rate (ORR), overall survival (OS), safety, and exploratory biomarker analysis. RESULTS: Dose level 2 was selected as RP2D. In phase II, 48 patients were enrolled. The median PFS and OS were 3.9 and 11.2 months, respectively. The ORR was 23.3%, and the median response duration was 16.7 months. Grade 3 or higher treatment-related adverse events, mainly neutropenia, occurred in 20 patients (41.7%). Targeted sequencing revealed that patients with RTK/RAS pathway alterations or the HLA-A02 supertype had better survival. Patients with elevated baseline interleukin-1 receptor antagonist levels had worse survival. CONCLUSIONS: Although the study did not meet its primary end point, nivolumab and paclitaxel for AGC demonstrated a durable response with manageable toxicity profiles. Genomic analysis or plasma cytokine analysis may provide information for the selection of patients who would benefit more from immunotherapy combined with chemotherapy.

The phase I/II eNRGy trial: Zenocutuzumab in patients with cancers harboring NRG1 gene fusions.

Neuregulin 1 (NRG1) fusions are oncogenic drivers that have been detected in non-small-cell lung cancer (NSCLC), pancreatic ductal adenocarcinoma (PDAC) and other solid tumors. NRG1 fusions are rare, occurring in less than 1% of solid tumors. Patients with NRG1 fusion positive (NRG1+) cancer have limited therapeutic options. Zenocutuzumab is a novel, bispecific IgG1 antibody that targets both HER2 and HER3 proteins and inhibits NRG1 binding through a 'Dock & Block®' mechanism of action. Here, we describe the rationale and design of the phase II component of the eNRGy trial, part of the overall, open-label phase I/II, multicenter trial exploring the safety, tolerability, pharmacokinetics, pharmacodynamics, immunogenicity and antitumor activity of zenocutuzumab in patients with NRG1+ NSCLC, PDAC or other solid tumors.

eNRGy: a clinical trial of zenocutuzumab for cancer caused by NRG1 gene fusionsNRG1 gene fusions are rare mutations that cause cancer cells to grow. These fusions are found in many different types of cancer. Tumors with NRG1 gene fusions do not respond well to standard treatment options. Zenocutuzumab, or Zeno, is a treatment that is being tested to see if it can stop cancer that is growing because of NRG1 gene fusions. Here, we describe the reasoning for and design of an ongoing clinical trial (eNRGy) designed to study the efficacy (how well it works) and safety of Zeno in patients with cancer that has NRG1 gene fusions. The eNRGy trial is recruiting patients with cancer that has NRG1 gene fusions, including non-small-cell lung cancer, pancreatic cancer and others. Patients who join this trial will receive Zeno once every 2 weeks until their cancer grows. The main goal (primary end point) of this trial is to determine the percentage of patients whose tumors decrease in size by 30% or more. The eNRGy trial is currently enrolling patients. For more information, refer to ClinicalTrials.gov (Identifier: NCT02912949), visit https://nrg1.com/, or call 1-833-NRG-1234.

Discovery of novel 1H-benzo[d]imidazole-4,7-dione based transglutaminase 2 inhibitors as p53 stabilizing anticancer agents in renal cell carcinoma.

Overexpression of transglutaminase 2 (TGase 2; TG2) has been implicated in the progression of renal cell carcinoma (RCC) through the inactivation of p53 by forming a protein complex. Because most p53 in RCC has no mutations, apoptosis can be increased by inhibiting the binding between TG2 and p53 to increase the stability of p53. In the present study, a novel TG2 inhibitor was discovered by investigating the structure of 1H-benzo[d]imidazole-4,7-dione as a simpler chemotype based on the amino-1,4-benzoquinone moiety of streptonigrin, a previously reported inhibitor. Through structure-activity relationship (SAR) studies, compound 8j (MD102) was discovered as a potent TG2 inhibitor with an IC50 value of 0.35 µM, p53 stabilization effect and anticancer effects in the ACHN and Caki-1 RCC cell lines with sulforhodamine B (SRB) GI50 values of 2.15 µM and 1.98 µM, respectively. The binding property of compound 8j (MD102) with TG2 was confirmed to be reversible in a competitive enzyme assay, and the binding interaction was expected to be formed at the ß-sandwich domain, a p53 binding site, in the SPR binding assay with mutant proteins. The mode of binding of compound 8j (MD102) to the ß-sandwich domain of TG2 was analyzed by molecular docking using the crystal structure of the active conformation of human TG2. Compound 8j (MD102) induced a decrease in the downstream signaling of p-AKT and p-mTOR through the stabilization of p53 by TG2 inhibition, resulting in tumor cell apoptosis. In a xenograft animal model using ACHN cancer cells, oral administration and intraperitoneal injection of compound 8j (MD102) showed an inhibitory effect on tumor growth, confirming increased levels of p53 and decreased levels of Ki-67 in tumor tissues through immunohistochemical (IHC) tissue staining. These results indicated that the inhibition of TG2 by compound 8j (MD102) could enhance p53 stabilization, thereby ultimately showing anticancer effects in RCC. Compound 8j (MD102), a novel TG2 inhibitor, can be further applied for the development of an anticancer candidate drug targeting RCC.

Pembrolizumab plus chemotherapy versus placebo plus chemotherapy for HER2-negative advanced gastric cancer (KEYNOTE-859): a multicentre, randomised, double-blind, phase 3 trial.

BACKGROUND: PD-1 inhibitors combined with chemotherapy have shown efficacy in gastric or gastro-esophageal junction cancer. We compared the efficacy and safety of pembrolizumab plus chemotherapy with placebo plus chemotherapy in participants with locally advanced or metastatic HER2-negative gastric or gastro-esophageal junction adenocarcinoma. METHODS: KEYNOTE-859 is a multicentre, double-blind, placebo-controlled, randomised, phase 3 trial, done at 207 medical centres across 33 countries. Eligible participants were aged 18 years and older with previously untreated histologically or cytologically confirmed locally advanced or metastatic HER2-negative gastric or gastro-esophageal junction adenocarcinoma and an Eastern Cooperative Oncology Group performance status of 0 or 1. Patients were randomly assigned (1:1) to receive pembrolizumab or placebo 200 mg, administered intravenously every 3 weeks for up to 35 cycles. All participants received investigator's choice of fluorouracil (intravenous, 800 mg/m2 per day) administered continuously on days 1-5 of each 3-week cycle plus cisplatin (intravenous, 80 mg/m2) administered on day 1 of each 3-week cycle or capecitabine (oral, 1000 mg/m2) administered twice daily on days 1-14 of each 3-week cycle plus oxaliplatin (intravenous, 130 mg/m2) administered on day 1 of each 3-week cycle. Randomisation was done using a central interactive voice-response system and stratified by geographical region, PD-L1 status, and chemotherapy in permuted block sizes of four. The primary endpoint was overall survival, assessed in the intention-to-treat (ITT) population, and the populations with a PD-L1 combined positive score (CPS) of 1 or higher, and PD-L1 CPS of 10 or higher. Safety was assessed in the as-treated population, which included all randomly assigned participants who received at least one dose of study intervention. Here, we report the results of the interim analysis. This study is registered with ClinicalTrials.gov, NCT03675737, and recruitment is complete. FINDINGS: Between Nov 8, 2018, and June 11, 2021, 1579 (66%) of 2409 screened participants were randomly assigned to receive pembrolizumab plus chemotherapy (pembrolizumab group; n=790) or placebo plus chemotherapy (placebo group; n=789). Most participants were male (527 [67%] of 790 participants in the pembrolizumab plus chemotherapy group; 544 [69%] of 789 participants in the placebo plus chemotherapy group) and White (426 [54%]; 435 [55%]). Median follow-up at the data cutoff was 31·0 months (IQR 23·0-38·3). Median overall survival was longer in the pembrolizumab group than in the placebo group in the ITT population (12·9 months [95% CI 11·9-14·0] vs 11·5 months [10·6-12·1]; hazard ratio [HR] 0·78 [95% CI 0·70-0·87]; p<0·0001), in participants with a PD-L1 CPS of 1 or higher (13·0 months [11·6-14·2] vs 11·4 months [10·5-12·0]; 0·74 [0·65-0·84]; p<0·0001), and in participants with a PD-L1 CPS of 10 or higher (15·7 months [13·8-19·3] vs 11·8 months [10·3-12·7]; 0·65 [0·53-0·79]; p<0·0001). The most common grade 3-5 adverse events of any cause were anaemia (95 [12%] of 785 participants in the pembrolizumab group vs 76 [10%] of 787 participants in the placebo group) and decreased neutrophil count (77 [10%] vs 64 [8%]). Serious treatment-related adverse events occurred in 184 (23%) participants in the pembrolizumab group and 146 (19%) participants in the placebo group. Treatment-related deaths occurred in eight (1%) participants in the pembrolizumab group and 16 (2%) participants in the placebo group. No new safety signals were identified. INTERPRETATION: Participants in the pembrolizumab plus chemotherapy group had a significant and clinically meaningful improvement in overall survival with manageable toxicity compared with participants in the placebo plus chemotherapy group. Therefore, pembrolizumab with chemotherapy might be a first-line treatment option for patients with locally advanced or metastatic HER2-negative gastric or gastro-esophageal junction adenocarcinoma. FUNDING: Merck Sharp and Dohme.

Lenvatinib plus pembrolizumab versus sunitinib as first-line treatment of patients with advanced renal cell carcinoma (CLEAR): extended follow-up from the phase 3, randomised, open-label study.

BACKGROUND: In the primary analysis of the CLEAR study, lenvatinib plus pembrolizumab significantly improved progression-free survival and overall survival versus sunitinib in patients with advanced renal cell carcinoma (data cutoff Aug 28, 2020). We aimed to assess overall survival based on 7 months of additional follow-up. METHODS: This is a protocol-prespecified updated overall survival analysis (data cutoff March 31, 2021) of the open-label, phase 3, randomised CLEAR trial. Patients with clear-cell advanced renal cell carcinoma who had not received any systemic anticancer therapy for renal cell carcinoma, including anti-vascular endothelial growth factor therapy, or any systemic investigational anticancer drug, were eligible for inclusion from 200 sites (hospitals and cancer centres) across 20 countries. Patients were randomly assigned (1:1:1) to receive lenvatinib (20 mg per day orally in 21-day cycles) plus pembrolizumab (200 mg intravenously every 21 days; lenvatinib plus pembrolizumab group), lenvatinib (18 mg per day orally) plus everolimus (5 mg per day orally; lenvatinib plus everolimus group [not reported in this updated analysis]) in 21-day cycles, or sunitinib (50 mg per day orally, 4 weeks on and 2 weeks off; sunitinib group). Eligible patients were at least 18 years old with a Karnofsky performance status of 70 or higher. A computer-generated randomisation scheme was used, and stratification factors were geographical region and Memorial Sloan Kettering Cancer Center prognostic groups. The primary endpoint was progression-free survival assessed by independent imaging review according to Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST v1.1). In this Article, extended follow-up analyses for progression-free survival and protocol-specified updated overall survival data are reported for the intention-to-treat population. No safety analyses were done at this follow-up. This study is closed to new participants and is registered with ClinicalTrials.gov, NCT02811861. FINDINGS: Between Oct 13, 2016, and July 24, 2019, 1417 patients were screened for inclusion in the CLEAR trial, of whom 1069 (75%; 273 [26%] female, 796 [74%] male; median age 62 years [IQR 55-69]) were randomly assigned: 355 (33%) patients (255 [72%] male and 100 [28%] female) to the lenvatinib plus pembrolizumab group, 357 (33%) patients (275 [77%] male and 82 [23%] female) to the sunitinib group, and 357 (33%) patients to the lenvatinib plus everolimus group (not reported in this updated analysis). Median follow-up for progression-free survival was 27·8 months (IQR 20·3-33·8) in the lenvatinib plus pembrolizumab group and 19·4 months (5·5-32·5) in the sunitinib group. Median progression-free survival was 23·3 months (95% CI 20·8-27·7) in the lenvatinib plus pembrolizumab group and 9·2 months (6·0-11·0) in the sunitinib group (stratified hazard ratio [HR] 0·42 [95% CI 0·34-0·52]). Median overall survival follow-up was 33·7 months (IQR 27·4-36·9) in the lenvatinib plus pembrolizumab group and 33·4 months (26·7-36·8) in the sunitinib group. Overall survival was improved with lenvatinib plus pembrolizumab (median not reached [95% CI 41·5-not estimable]) versus sunitinib (median not reached [38·4-not estimable]; HR 0·72 [95% CI 0·55-0·93]). INTERPRETATION: Efficacy benefits of lenvatinib plus pembrolizumab over sunitinib were durable and clinically meaningful with extended follow-up. These results support the use of lenvatinib plus pembrolizumab as a first-line therapy for patients with advanced renal cell carcinoma. FUNDING: Eisai and Merck Sharp & Dohme.

Efficacy and safety of lenvatinib plus pembrolizumab vs sunitinib in the East Asian subset of patients with advanced renal cell carcinoma from the CLEAR trial.

In the CLEAR trial, lenvatinib plus pembrolizumab met study endpoints of superiority vs sunitinib in the first-line treatment of patients with advanced renal cell carcinoma. We report the efficacy and safety results of the East Asian subset (ie, patients in Japan and the Republic of Korea) from the CLEAR trial. Of 1069 patients randomly assigned to receive either lenvatinib plus pembrolizumab, lenvatinib plus everolimus or sunitinib, 213 (20.0%) were from East Asia. Baseline characteristics of patients in the East Asian subset were generally comparable with those of the global trial population. In the East Asian subset, progression-free survival was considerably longer with lenvatinib plus pembrolizumab vs sunitinib (median 22.1 vs 11.1 months; HR 0.38; 95% CI: 0.23-0.62). The HR for overall survival comparing lenvatinib plus pembrolizumab vs sunitinib was 0.71; 95% CI: 0.30-1.71. The objective response rate was higher with lenvatinib plus pembrolizumab vs sunitinib (65.3% vs 49.2%; odds ratio 2.14; 95% CI: 1.07-4.28). Dose reductions due to treatment-emergent adverse events (TEAEs) commonly associated with tyrosine kinase inhibitors occurred more frequently than in the global population. Hand-foot syndrome was the most frequent any-grade TEAE with lenvatinib plus pembrolizumab (66.7%) and sunitinib (57.8%), a higher incidence compared to the global population (28.7% and 37.4%, respectively). The most common grade 3 to 5 TEAEs were hypertension with lenvatinib plus pembrolizumab (20%) and decreased platelet count with sunitinib (21.9%). Efficacy and safety for patients in the East Asian subset were generally similar to those of the global population, except as noted.

Atezolizumab Plus PEGPH20 Versus Chemotherapy in Advanced Pancreatic Ductal Adenocarcinoma and Gastric Cancer: MORPHEUS Phase Ib/II Umbrella Randomized Study Platform.

BACKGROUND: The MORPHEUS platform comprises multiple open-label, randomized, phase Ib/II trials designed to identify early efficacy and safety signals of treatment combinations across cancers. Atezolizumab (anti-programmed cell death 1 ligand 1 [PD-L1]) was evaluated in combination with PEGylated recombinant human hyaluronidase (PEGPH20). METHODS: In 2 randomized MORPHEUS trials, eligible patients with advanced, previously treated pancreatic ductal adenocarcinoma (PDAC) or gastric cancer (GC) received atezolizumab plus PEGPH20, or control treatment (mFOLFOX6 or gemcitabine plus nab-paclitaxel [MORPHEUS-PDAC]; ramucirumab plus paclitaxel [MORPHEUS-GC]). Primary endpoints were objective response rates (ORR) per RECIST 1.1 and safety. RESULTS: In MORPHEUS-PDAC, ORRs with atezolizumab plus PEGPH20 (n = 66) were 6.1% (95% CI, 1.68%-14.80%) vs. 2.4% (95% CI, 0.06%-12.57%) with chemotherapy (n = 42). In the respective arms, 65.2% and 61.9% had grade 3/4 adverse events (AEs); 4.5% and 2.4% had grade 5 AEs. In MORPHEUS-GC, confirmed ORRs with atezolizumab plus PEGPH20 (n = 13) were 0% (95% CI, 0%-24.7%) vs. 16.7% (95% CI, 2.1%-48.4%) with control (n = 12). Grade 3/4 AEs occurred in 30.8% and 75.0% of patients, respectively; no grade 5 AEs occurred. CONCLUSION: Atezolizumab plus PEGPH20 showed limited clinical activity in patients with PDAC and none in patients with GC. The safety of atezolizumab plus PEGPH20 was consistent with each agent's known safety profile. (ClinicalTrials.gov Identifier: NCT03193190 and NCT03281369).

INTEGRATE II: randomised phase III controlled trials of regorafenib containing regimens versus standard of care in refractory Advanced Gastro-Oesophageal Cancer (AGOC): a study by the Australasian Gastro-Intestinal Trials Group (AGITG).

BACKGROUND: Advanced gastro-oesophageal cancer (AGOC) carries a poor prognosis. No standard of care treatment options are available after first and second-line therapies. Regorafenib is an oral multi-targeted tyrosine kinase inhibitor targeting angiogenic, stromal, and oncogenic receptor tyrosine kinases. Regorafenib 160 mg daily prolonged progression free survival compared to placebo (INTEGRATE, phase 2). Regorafenib 80 mg daily in combination with nivolumab 3 mg/kg showed promising objective response rates (REGONIVO). METHODS/DESIGN: INTEGRATE II (INTEGRATE IIa and IIb) platform comprises two international phase III randomised controlled trials (RCT) with 2:1 randomisation in favor of experimental intervention. INTEGRATE IIa (double-blind) compares regorafenib 160 mg daily on days 1 to 21 of each 28-day cycle to placebo. INTEGRATE IIb (open label) compares REGONIVO, regorafenib 90 mg days 1 to 21 in combination with intravenous nivolumab 240 mg days 1 and 15 each 28-day cycle with investigator's choice of chemotherapy (control). Treatment continues until disease progression or intolerable adverse events as per protocol. Eligible participants include adults with AGOC who have failed two or more lines of treatment. Stratification is by location of tumour (INTEGRATE IIa only), geographic region, prior VEGF inhibitor and prior immunotherapy use (INTEGRATE IIb only). Primary endpoint is overall survival. Secondary endpoints are progression free survival, objective response rate, quality of life, and safety. Tertiary/correlative objectives include biomarker and pharmacokinetic evaluation. DISCUSSION: INTEGRATE II provides a platform to evaluate the clinical utility of regorafenib alone, as well as regorafenib in combination with nivolumab in treatment of participants with refractory AGOC. TRIAL REGISTRATION: INTEGRATE IIa prospectively registered 1 April 2016 Australia New Zealand Clinical Trial Registry: ACTRN12616000420448 (ClinicalTrials.gov NCT02773524). INTEGRATE IIb prospectively registered 10 May 2021 ClinicalTrials.gov: NCT04879368.

Epigenetic promoter alterations in GI tumour immune-editing and resistance to immune checkpoint inhibition.

OBJECTIVES: Epigenomic alterations in cancer interact with the immune microenvironment to dictate tumour evolution and therapeutic response. We aimed to study the regulation of the tumour immune microenvironment through epigenetic alternate promoter use in gastric cancer and to expand our findings to other gastrointestinal tumours. DESIGN: Alternate promoter burden (APB) was quantified using a novel bioinformatic algorithm (proActiv) to infer promoter activity from short-read RNA sequencing and samples categorised into APBhigh, APBint and APBlow. Single-cell RNA sequencing was performed to analyse the intratumour immune microenvironment. A humanised mouse cancer in vivo model was used to explore dynamic temporal interactions between tumour kinetics, alternate promoter usage and the human immune system. Multiple cohorts of gastrointestinal tumours treated with immunotherapy were assessed for correlation between APB and treatment outcomes. RESULTS: APBhigh gastric cancer tumours expressed decreased levels of T-cell cytolytic activity and exhibited signatures of immune depletion. Single-cell RNAsequencing analysis confirmed distinct immunological populations and lower T-cell proportions in APBhigh tumours. Functional in vivo studies using 'humanised mice' harbouring an active human immune system revealed distinct temporal relationships between APB and tumour growth, with APBhigh tumours having almost no human T-cell infiltration. Analysis of immunotherapy-treated patients with GI cancer confirmed resistance of APBhigh tumours to immune checkpoint inhibition. APBhigh gastric cancer exhibited significantly poorer progression-free survival compared with APBlow (median 55 days vs 121 days, HR 0.40, 95% CI 0.18 to 0.93, p=0.032). CONCLUSION: These findings demonstrate an association between alternate promoter use and the tumour microenvironment, leading to immune evasion and immunotherapy resistance.

Health-related quality-of-life outcomes in patients with advanced renal cell carcinoma treated with lenvatinib plus pembrolizumab or everolimus versus sunitinib (CLEAR): a randomised, phase 3 study.

BACKGROUND: Results from the phase 3 CLEAR study showed that lenvatinib plus pembrolizumab improved progression-free survival and overall survival compared with sunitinib in patients with advanced renal cell carcinoma. We aimed to assess the health-related quality-of-life (HRQOL) outcomes from the CLEAR study. METHODS: This open-label, randomised, phase 3 study was done across 200 hospitals and cancer centres in 20 countries. Patients were required to be 18 years or older, with advanced clear-cell renal cell carcinoma, and a Karnofsky performance status of 70% or higher. Patients who had received previous systemic anticancer therapy for renal cell carcinoma were not eligible. Patients were randomly assigned (1:1:1) to lenvatinib (oral 20 mg per day) plus pembrolizumab (intravenous 200 mg every 21 days), lenvatinib (oral 18 mg per day) plus everolimus (oral 5 mg per day) in 21-day cycles, or sunitinib (oral 50 mg per day, 4 weeks on followed by 2 weeks off). Patients were assigned to treatments with a computer-generated randomisation scheme and were stratified by geographical region and Memorial Sloan Kettering Cancer Center prognostic groups. The primary endpoint, previously reported, was progression-free survival, and HRQOL was a secondary endpoint. Most HRQOL analyses were done in patients who underwent randomisation, received at least one dose of study treatment, and had any HRQOL data. Completion and compliance analyses were done in the full analysis set. Functional Assessment of Cancer Therapy Kidney Symptom Index-Disease-Related Symptoms (FKSI-DRS), European Organisation for the Research and Treatment of Cancer Quality of Life Questionnaire-Core 30 (EORTC QLQ-C30), and the EQ-5D-3 Level (EQ-5D-3L) preference questionnaire were administered at baseline and on day 1 of each subsequent 21-day cycle. This study is registered with ClinicalTrials.gov, NCT02811861, and is closed to new participants. FINDINGS: Between Oct 13, 2016, and July 24, 2019, 355 patients were randomly assigned to the lenvatinib plus pembrolizumab group, 357 to the lenvatinib plus everolimus group, and 357 to the sunitinib group. Median follow-up for HRQOL analyses was 12·9 months (IQR 5·6-22·3). Because of the promising efficacy and safety results of lenvatinib plus pembrolizumab in the first-line setting, we focus the HRQOL results in this report on that combination versus sunitinib. Mean change from baseline in the lenvatinib plus pembrolizumab group compared with the sunitinib group was -1·75 (SE 0·59) versus -2·19 (0·66) for FKSI-DRS, -5·93 (0·86) versus -6·73 (0·94) for EORTC QLQ-C30 global health status/quality of life (GHS/QOL), and -4·96 (0·85) versus -6·64 (0·94) for the EQ-5D visual analogue scale (VAS). Median time to first deterioration in the lenvatinib plus pembrolizumab group compared with the sunitinib group was 9·14 weeks (95% CI 6·43-12·14) versus 12·14 weeks (9·14-15·29; HR 1·13 [95% CI 0·94-1·35], log-rank p=0·20) for FKSI-DRS, 12·00 weeks (7·29-15·14) versus 9·14 weeks (6·29-12·14; 0·88 [0·74-1·05], log-rank p=0·17) for EORTC QLQ-C30 GHS/QOL, and 9·43 weeks (6·43-12·29) versus 9·14 weeks (6·29-12·00; 0·83 [0·70-0·99], log-rank p=0·041) for the EQ-5D VAS. Median time to definitive deterioration in the lenvatinib plus pembrolizumab group compared with the sunitinib group was 134·14 weeks (95% CI 120·00-not estimable) versus 117·43 weeks (90·14-131·29; HR 0·70 [95% CI 0·53-0·92], log-rank p=0·0081) for FKSI-DRS, 114·29 weeks (102·14-153·29) versus 75·14 weeks (57·29-105·14; 0·60 [0·47-0·77], log-rank p<0·0001) for EORTC QLQ-C30 GHS/QOL, and 124·86 weeks (94·71-134·57) versus 74·86 weeks (54·14-96·00; 0·67 [0·53-0·85], log-rank p=0·0012) for the EQ-5D VAS. No outcomes on any of the instruments significantly favoured sunitinib over lenvatinib plus pembrolizumab. Most HRQOL comparisons of lenvatinib plus everolimus versus sunitinib were similar or favoured sunitinib. INTERPRETATION: These HRQOL results demonstrate that patients given lenvatinib plus pembrolizumab treatment had similar or favourable scores compared with patients given sunitinib, particularly with respect to time to definitive deterioration. These results support the efficacy and safety profile of lenvatinib plus pembrolizumab as first-line therapy for patients with advanced renal cell carcinoma. FUNDING: Eisai (Nutley, NJ, USA) and Merck Sharp & Dohme, a subsidiary of Merck & Co (Kenilworth, NJ, USA).

Zanidatamab, a novel bispecific antibody, for the treatment of locally advanced or metastatic HER2-expressing or HER2-amplified cancers: a phase 1, dose-escalation and expansion study.

BACKGROUND: HER2-targeted therapies have substantially improved outcomes for patients with HER2-positive breast and gastric or gastro-oesophageal junction cancers. Several other cancers exhibit HER2 expression or amplification, suggesting that HER2-targeted agents can have broader therapeutic impact. Zanidatamab is a humanised, bispecific monoclonal antibody directed against two non-overlapping domains of HER2. The aim of this study was to evaluate the safety and anti-tumour activity of zanidatamab across a range of solid tumours with HER2 expression or amplification. METHODS: This first-in-human, multicentre, phase 1, dose-escalation and expansion trial included patients aged 18 years and older, with a life expectancy of at least 3 months, with an Eastern Cooperative Oncology Group performance status of 0 or 1, and locally advanced or metastatic, HER2-expressing or HER2-amplified solid tumours of any kind who had received all available approved therapies. The primary objectives of part 1 were to identify the maximum tolerated dose, optimal biological dose, or recommended dose of zanidatamab; all patients were included in the primary analyses. Part 1 followed a 3 + 3 dose-escalation design, including different intravenous doses (from 5 mg/kg to 30 mg/kg) and intervals (every 1, 2, or 3 weeks). The primary objective of part 2 was to evaluate the safety and tolerability of zanidatamab monotherapy in solid tumours. This trial is registered with ClinicalTrials.gov (NCT02892123), and parts 1 and 2 of the trial are complete. Part 3 of the study evaluates the use of zanidatamab in combination with chemotherapy and is ongoing. FINDINGS: Recruitment took place between Sept 1, 2016, and March 13, 2021. In Part 1 (n=46), no dose-limiting toxicities were detected and the maximum tolerated dose was not reached. The recommended dose for part 2 (n=22 for biliary tract cancer; n=28 for colorectal cancer; and n=36 for other HER2-expressing or HER2-amplified cancers excluding breast or gastro-oesophageal cancers; total n=86) was 20 mg/kg every 2 weeks. The most frequent treatment-related adverse events in part 1 of the study were diarrhoea (24 [52%] of 46 patients; all grade 1-2) and infusion reactions (20 [43%] of 46 patients; all grade 1-2). The most frequent treatment-related adverse events in part 2 of the study were diarrhoea (37 [43%] of 86 patients; all grade 1-2 except for one patient) and infusion reactions (29 [34%] of 86 patients; all grade 1-2). A total of six grade 3 treatment-related adverse events were reported in four (3%) of 132 patients. In part 2, 31 (37%; 95% CI 27·0-48·7) of 83 evaluable patients had a confirmed objective response. There were no treatment-related deaths. INTERPRETATION: These results support that HER2 is an actionable target in various cancer histologies, including biliary tract cancer and colorectal cancer. Evaluation of zanidatamab continues in ongoing studies. FUNDING: Zymeworks.

The Essentials of Multiomics.

Within the last decade, the science of molecular testing has evolved from single gene and single protein analysis to broad molecular profiling as a standard of care, quickly transitioning from research to practice. Terms such as genomics, transcriptomics, proteomics, circulating omics, and artificial intelligence are now commonplace, and this rapid evolution has left us with a significant knowledge gap within the medical community. In this paper, we attempt to bridge that gap and prepare the physician in oncology for multiomics, a group of technologies that have gone from looming on the horizon to become a clinical reality. The era of multiomics is here, and we must prepare ourselves for this exciting new age of cancer medicine.

Analytical and Clinical Validation of Cell-Free Circulating Tumor DNA Assay for the Estimation of Tumor Mutational Burden.

BACKGROUND: Ultra-deep sequencing to detect low-frequency mutations in circulating tumor-derived DNA (ctDNA) increases the diagnostic value of liquid biopsy. The demand for large ctDNA panels for comprehensive genomic profiling and tumor mutational burden (TMB) estimation is increasing; however, few ctDNA panels for TMB have been validated. Here, we designed a ctDNA panel with 531 genes, named TMB500, along with a technical and clinical validation. METHODS: Synthetic reference cell-free DNA materials with predefined allele frequencies were sequenced in a total of 92 tests in 6 batches to evaluate the precision, linearity, and limit of detection of the assay. We used clinical samples from 50 patients with various cancers, 11 healthy individuals, and paired tissue samples. Molecular barcoding and data analysis were performed using customized pipelines. RESULTS: The assay showed high precision and linearity (coefficient of determination, r2 =0.87) for all single nucleotide variants, with a limit of detection of 0.24%. In clinical samples, the TMB500 ctDNA assay detected most variants present and absent in tissues, showing that ctDNA could assess tumor heterogeneity in different tissues and metastasis sites. The estimated TMBs correlated well between tissue and blood, except in 4 cases with extreme heterogeneity that showed very high blood TMBs compared to tissue TMBs. A pilot evaluation showed that the TMB500 assay could be used for disease monitoring. CONCLUSIONS: The TMB500 assay is an accurate and reliable ctDNA assay for many clinical purposes. It may be useful for guiding the treatment of cancers with diverse genomic profiles, estimating TMB in immune therapy, and disease monitoring.