Alterations In Cell Cycle Genes Research Articles

Concomitant genomic features stratify prognosis to patients with advanced EGFR mutant lung cancer.

This study aimed to explore the clinical significance of genomics features including tumor mutation burden (TMB) and copy number alteration (CNA) for advanced EGFR mutant lung cancer. We retrospectively identified 1378 patients with advanced EGFR mutant lung cancer and next-generation sequencing tests from three cohorts. Multiple co-occurring genomics alternations occurred in a large proportion (97%) of patients with advanced EGFR mutant lung cancers. Both TMB and CNA were predictive biomarkers for these patients. A joint analysis of TMB and CNA found that patients with high TMB and high CNA showed worse responses to EGFR-TKIs and predicted worse outcomes. TMBhighCNAhigh, as a high-risk genomic feature, showed predictive ability in most of the subgroups based on clinical characteristics. These patients had larger numbers of metastatic sites, and higher rates of EGFR copy number amplification, TP53 mutations, and cell-cycle gene alterations, which showed more potential survival gain from combination treatment. Furthermore, a nomogram based on genomic features and clinical features was developed to distinguish prognosis. Genomic features could stratify prognosis and guide clinical treatment for patients with advanced EGFR mutant lung cancer.

Olmutinib Reverses Thioacetamide-Induced Cell Cycle Gene Alterations in Mice Liver and Kidney Tissues, While Wheat Germ Treatment Exhibits Limited Efficacy at Gene Level.

Background and Objectives: TAA is potent hepatic/renal toxicant. Conversely, WGO is a potent dietary supplement with impressive antioxidant properties. Olmutinib is an apoptotic chemotherapy drug that does not harm the liver or kidney. This study investigated the impact of olmutinib and wheat germ oil (WGO) on Thioacetamide (TAA)-induced gene alterations in mice liver and kidney tissues. Materials and Methods: Adult male C57BL/6 mice were exposed to 0.3% TAA in drinking water for 14 days, followed by the oral administration of olmutinib (30 mg/kg) and WGO (1400 mg/kg) for 5 consecutive days. Treatment groups included the following: groups I (control), II (TAA-exposed), III (TAA + olmutinib), IV (TAA + WGO), and V (TAA + olmutinib + WGO). Results: The findings revealed that TAA exposure increased MKi67 and CDKN3 gene expression in liver and kidney tissues. Olmutinib treatment effectively reversed these TAA-induced effects, significantly restoring MKi67 and CDKN3 gene expression. WGO also reversed MKi67 effects in the liver but exhibited limited efficacy in reversing CDKN3 gene alterations induced by TAA exposures in both the liver and kidney. TAA exposure showed the tissue-specific expression of TP53, with decreased expression in the liver and increased expression in the kidney. Olmutinib effectively reversed these tissue-specific alterations in TP53 expression. While WGO treatment alone could not reverse the gene alterations induced by TAA exposure, the co-administration of olmutinib and WGO exhibited a remarkable potentiation of therapeutic effects in both the liver and kidney. The gene interaction analysis revealed 77.4% of physical interactions and co-localization between MKi67, CDKN3, and TP53 expressions. Protein-protein interaction networks also demonstrated physical interactions between MKi67, TP53, and CDKN3, forming complexes or signaling cascades. Conclusions: It was predicted that the increased expression of the MKi67 gene by TAA leads to the increase in TP53, which negatively regulates the cell cycle via increased CDKN3 expression in kidneys and the restoration of TP53 levels in the liver. These findings contribute to our understanding of the effects of olmutinib and WGO on TAA-induced gene expression changes and highlight their contrasting effects based on cell cycle alterations.

Cell cycle gene alterations associate with a redistribution of mutation risk across chromosomal domains in human cancers.

Mutations in human cells exhibit increased burden in heterochromatic, late DNA replication time (RT) chromosomal domains, with variation in mutation rates between tissues mirroring variation in heterochromatin and RT. We observed that regional mutation risk further varies between individual tumors in a manner independent of cell type, identifying three signatures of domain-scale mutagenesis in >4,000 tumor genomes. The major signature reflects remodeling of heterochromatin and of the RT program domains seen across tumors, tissues and cultured cells, and is robustly linked with higher expression of cell proliferation genes. Regional mutagenesis is associated with loss of activity of the tumor-suppressor genes RB1 and TP53, consistent with their roles in cell cycle control, with distinct mutational patterns generated by the two genes. Loss of regional heterogeneity in mutagenesis is associated with deficiencies in various DNA repair pathways. These mutation risk redistribution processes modify the mutation supply towards important genes, diverting the course of somatic evolution.

The Rise and Fall of Leukemia Clones in Longitudinal Samples from Diagnosis to Relapse in KMT2A-rearranged Infant and Childhood Leukemia

The genomic landscape, evolutionary trajectories and the mechanisms driving relapse in KMT2A-rearranged (KMT2A-r) infant and childhood acute lymphoblastic (ALL) and acute myeloid leukemia (AML) are not completely understood. We therefore studied 30 KMT2A-r ALL (n=16) and AML (n=14) patients of which 19 relapsed. Eleven diagnose-relapse-germline trios were analyzed by whole genome (WGS) and whole exome sequencing (WES), and all patients by ultra-deep resequencing (average 3400X) of patient-specific mutations identified by WGS/WES on the diagnostic (n=30) and relapse (n=11) samples, at diagnosis (n=30), during treatment (n=203) and at relapse (n=30, 1-4 bone marrow (BM) relapses in 17 patients). The mutational burden increased from diagnosis to relapse in the diagnose-relapse-germline trios. The combined analysis of WGS/WES and ultra-deep data showed that the leukemias typically evolved through branching evolution, and that a selective sweep occurred at first relapse in 76% of patient. Further, additional subclones were often detected at relapse and in 69% the relapse clone was detected already at diagnosis as a subclone. Eight pathways were recurrently altered including signalling, cell cycle, epigenetic, B-cell development, transcription factor, glucocorticoid receptor signalling, purine metabolism and cohesin. Signalling mutations were the most common type of mutations at diagnosis (50%) and at relapse (41%) and the diagnostic frequency was similar in patients that remained in complete remission (CR) and in those that relapsed (55% versus 47%). Signalling mutations were often subclonal (60%) and 30% had more than one mutation and mutations in signalling genes were usually gained or maintained at relapse in ALL but mainly lost or reduced in size in AML. Alterations in cell cycle genes were enriched at relapse and either gained (60%) or maintained (27%), and included TP53 in ALL and AML, CDKN2A/B in ALL and CCND3 in AML. The TP53 alterations were typically biallelic at relapse. Only 2/30 patients had diagnostic TP53 mutations and both relapsed. Further, relapse-specific mutations in genes involved in glucocorticoid signalling and purine metabolism (CREBBP, NT5C2,PRPS2, NR3C1) were detected in all ALL patients with early relapse (n=4, during maintenance therapy). These relapse samples also had co-occurring IKZF1 deletions with 3 acquired at relapse and 1 maintained, as well as acquired TP53 mutations (3/4 patients). By contrast, infant ALL with a very early relapse (n=4, before maintenance therapy) lacked cell cycle and chemoresistance-associated mutations at relapse. Ultra-deep sequencing did not detect the CREBBP, NT5C2, PRPS2 or TP53 mutations at diagnosis and manual inspection of the WGS reads failed to detect the NR3C1 deletion. In AML, gain of WT1 mutations was seen in patients with late relapse (after >1 year in CR1). Longitudinal sequencing on the 30 patients showed that at day 15, ALL patients that remained in CR (n=5) had higher levels of molecularly detectable leukemia cells as compared to those that relapsed (n=8), however at day 29, the reverse was seen with relapse patients having higher levels (n.s). Further, infants with very early relapse (n=3) had a higher leukemic burden at both time points as compared to those with early relapse (n=5). Patients with very early relapse typically reached CR later and the relapse clone could be detected at diagnosis in all 4 patients compared to 2/4 with early relapse. In 7 patients, 4 that relapsed and 3 that remained in CR, low-frequency mutations and KMT2A-fusion positive leukemic clones (DNA-level) were found at remission. Moreover, 4 patients had molecularly detectable clones across all investigated time points until relapse (6-13 time points), 2 had persistent disease but 2 entered CR. Finally, the relapse clone could be detected 46-126 days before relapse in 6 patients. Combined, early relapse in infant and childhood KMT2A-r ALL was characterized by relapse-specific TP53, IKZF1, CREBBP, NR3C1, NTC52 or PRPS2 alterations. By contrast, very early relapse KMT2A-r infant ALL likely arises from pre-existing chemo resistant cells with a paucity of genetic alterations in relapse and chemoresistance-associated genes at relapse. Further, longitudinal molecular tracking provided biological insight into clonal response to treatment and captured the complexity of the temporal evolution in very early and early relapse patients.

EP08.02-147 Clinical Outcomes in Patients with or without Cell Cycle Gene Alterations in EGFR mutated Non-Small Cell Lung Cancer

Epidermal Growth Factor Receptor (EGFR) exon 19 deletions and L858R mutations are oncogenic drivers in a subset of non-small cell lung cancers (NSCLC). While tyrosine kinase inhibitor (TKI) therapies against mutant EGFR have significantly improved response rates and progression free survival (PFS) compared to cytotoxic chemotherapy for patients with metastatic EGFR-mutated NSCLC, responses to therapy are incomplete and resistance to EGFR-targeted therapies is inevitable. Somatic and copy-number alterations in cell cycle genes concurrent with EGFR mutations have been suggested to play a role in EGFR-TKI resistance, however this has not been completely characterized.

Targeting Acquired and Intrinsic Resistance Mechanisms in Epidermal Growth Factor Receptor Mutant Non-Small-Cell Lung Cancer

Over the past 2 decades, rapid advances in molecular profiling and the development of targeted therapies have dramatically improved the clinical course of advanced non-small-cell lung cancer (NSCLC). Mutations in the epidermal growth factor receptor (EGFR) gene are found in about a third of patients with advanced NSCLC, and the approval of first-generation EGFR targeted kinase inhibitors significantly improved survival when compared with platinum-based doublet chemotherapy (PBC), the previous standard of care. Inevitably, selective pressure from first-generation EGFR inhibitors led to acquired resistance mechanisms, such as the T790M mutation. The advent of third-generation EGFR inhibitors (e.g., osimertinib) successfully overcame the T790M resistance mechanism, and osimertinib subsequently became the first-line therapy for EGFR mutant NSCLC. Currently, research in EGFR mutant NSCLC is primarily focused on targeting resistance mechanisms to osimertinib. Over the past several years, many important acquired and intrinsic mechanisms of resistance to osimertinib have been identified. Acquired resistance mechanisms include C797X, mesenchymal epithelial transition factor (MET) amplification, HER2/HER3 amplification, phosphoinositide 3-kinase (PI3K) pathway mutations, RAS/mitogen-activated protein kinase (MAPK) pathway mutations, cell-cycle gene alterations, oncogenic fusions, and histologic transformations. An important intrinsic resistance mechanism to osimertinib is the EGFR exon 20 insertion mutation, which is sensitive to the newly Food and Drug Administration (FDA)-approved tyrosine kinase inhibitor mobocertinib and the EGFR/MET bispecific antibody amivantamab. This review article aims to (1) summarize the advances in the treatment of EGFR mutant NSCLC, (2) delineate known resistance mechanisms to the current first-line therapy, osimertinib, and (3) describe the development of targeted drugs that aim to overcome these resistance mechanisms.

Genomic profiling of gastrointestinal cancers by comprehensive tumor-normal sequencing.

109 Background: Gastrointestinal cancers (GIC) account for 26% of global cancer incidence and 35% of cancer-related deaths. We investigated the molecular landscape and therapeutic targets across 18 types of GIC using whole exome (WES) and whole transcriptome sequencing (WTS). Methods: GEM ExTra assay was performed on 603 paired samples (ages 18-90 years, median = 61 years). Targeted sequence coverage was 180X for germline DNA and 400X for tumor DNA. Reportable somatic alterations included single base substitutions, indels, Copy Number Alterations, gene fusions, alternate transcripts, as well as tumor mutational burden (TMB) and microsatellite instability (MSI) status. Germline subtraction identified somatic-specific alterations. Results: Analysis of 603 GIC patient samples, including esophageal, gastric cancer (GC), biliary tract (BT), pancreatic cancer (PC), colorectal cancer (CRC), and other cancers, identified 434 actionable targets. The median number of alterations was 3 per GIC patient. The 5 most common actionable alterations were in APC, KRAS, CDKN2A, ARID1A and PIK3CA. Activation of Wnt signaling was found in 264/603 (44%), with the majority being in CRC cases. Alterations in cell cycle genes including CDKN2A, CDK4/6 and others were noted in 129/603 (21%) cases, with the majority in PC, suggesting benefit from CDK4/6 inhibitors. Activation of PI3K/PTEN/Akt/mTOR pathway was noted in 105/603 (17%), with the majority harbored in CRC, suggesting benefit from targeting this pathway. ERBB2 amplification and mutations were noted in 22/603 (4%) across different GIC tumor types. Alterations in homologous recombination genes predicting platinum and PARP inhibitor response was noted in 181/603 (30%) samples distributed across GIC subtypes. KRAS (G12C) mutation was found in 7% of all KRAS mutations across GIC subtypes, thus allowing patients to enroll in clinical trials with G12C-specific inhibitors. The majority of cases with MSI- and TMB-high status were identified in GC and CRC tumors and may be predictive of response to immunotherapy. WTS identified actionable fusions, including FGFR1/2/3 and novel NRG1 fusions in BT cancers. Conclusions: Our study revealed actionable targets used in patient selection for precision therapies, in addition to other mutational profiles of clinical significance. Overall, comprehensive genomic profiling enabled detection of established and novel actionable alterations, including fusions, which may have gone undetected using hotspot panels.

Lummus wins master licensor contract for DRPIC's new PC complex in Oman

The Lung Cancer Master Protocol (Lung-MAP; S1400) is a completed biomarker-driven master protocol designed to address an unmet need for better therapies for squamous non-small-cell lung cancer. Lung-MAP (S1400) was created to establish an infrastructure for biomarker screening and rapid regulatory intent evaluation of targeted therapies and was the first biomarker-driven master protocol initiated with the US National Cancer Institute (NCI).Lung-MAP (S1400) was done within the National Clinical Trials Network of the NCI using a public–private partnership. Eligible patients were aged 18 years or older, had stage IV or recurrent squamous non-small-cell lung cancer, had previously been treated with platinum-based chemotherapy, and had an Eastern Cooperative Oncology Group (ECOG) performance status of 0–2. The study included a screening component using the FoundationOne assay (Foundation Medicine, Cambridge, MA, USA) for next-generation sequencing, and a clinical trial component with biomarker-driven substudies and non-match substudies for patients who were ineligible for biomarker-driven substudies. Patients were pre-screened and received their substudy assignment upon progression, or they were screened at progression and received their substudy assignment upon completion of testing. Patients could enrol onto additional substudies after progression on a substudy. The study is registered with ClinicalTrials.gov, NCT02154490, and all research related to Lung-MAP (S1400) is completed.Between June 16, 2014, and Jan 28, 2019, 1864 patients enrolled and 1841 (98·9%) submitted tissue. 1674 (90·9%) of 1841 patients had biomarker results, and 1404 (83·9%) of 1674 patients received a substudy assignment. Of the assigned patients, 655 (46·7%) registered to a substudy. The biomarker-driven substudies evaluated taselisib (targeting PIK3CA alterations), palbociclib (cell cycle gene alterations), AZD4547 (FGFR alteration), rilotumumab plus erlotinib (MET), talazoparib (homologous recombination repair deficiency), and telisotuzumab vedotin (MET). The non-match substudies evaluated durvalumab, and nivolumab plus ipilimumab for anti-PD-1 or anti-PD-L1-naive disease, and durvalumab plus tremelimumab for anti-PD-1 or anti-PD-L1 relapsed disease. Combining data from the substudies, ten (7·0%) of 143 patients responded to targeted therapy, 53 (16·8%) of 315 patients responded to anti-PD-1 or anti-PD-L1 therapy for immunotherapy-naive disease, and three (5·4%) of 56 responded to docetaxel in the second line of therapy. Median overall survival was 5·9 months (95% CI 4·8–7·8) for the targeted therapy groups, 7·7 months (6·7–9·2) for the docetaxel groups, and 10·8 months (9·4–12·3) for the anti-PD-1 or anti-PD-L1-containing groups. Median progression-free survival was 2·5 months (95% CI 1·7–2·8) for the targeted therapy groups, 2·7 months (1·9–2·9) for the docetaxel groups, and 3·0 months (2·7–3·9) for the anti-PD-1 or anti-PD-L1-containing groups.Lung-MAP (S1400) met its goal to quickly address biomarker-driven therapy questions in squamous non-small-cell lung cancer. In early 2019, a new screening protocol was implemented expanding to all histological types of non-small-cell lung cancer and to add focus on immunotherapy combinations for anti-PD-1 and anti-PD-L1 therapy-relapsed disease. With these changes, Lung-MAP continues to meet its goal to focus on unmet needs in the treatment of advanced lung cancers.US National Institutes of Health, and AbbVie, Amgen, AstraZeneca, Bristol Myers Squibb, Genentech, and Pfizer through the Foundation for the National Institutes of Health.

The interplay of cell cycle and DNA repair gene alterations in upper tract urothelial carcinoma: predictive and prognostic implications.

Upper tract urothelial carcinoma (UTUC) is rare but can occur sporadically outside the context of Lynch syndrome. In these cases, knowing whether non-mismatch repair (MMR), DNA damage response and repair (DDR), and cell cycle gene alterations may predict responses to chemotherapy or immunotherapy and survival is of clinical importance. This study examined the germline and somatic mutational landscape of two UTUC patients with differential responses to programmed death 1 (PD-1)/PD-ligand 1 (PD-L1) immune checkpoint inhibitors and queried three independent UTUC cohort studies for co-occurrence of key cell cycle and DDR genes, as well as for their associations with overall survival (OS). TP53 and RB1 emerged as potential determinants of shorter OS in UTUC cohort patients, regardless of concurrent DDR alterations, and if prospectively assessed in larger studies they might also explain resistance to PD-1/PD-L1 blockade despite PD-L1 expression.

Cell cycLe inhibitiON to target the EVolution of urOthelial cancer (CLONEVO): A single-arm, open-label window-of-opportunity trial of neoadjuvant abemaciclib in platinum-ineligible muscle invasive bladder cancer patients.

TPS5096 Background: The standard of care for clinically localized muscle-invasive bladder cancer (MIBC) is neoadjuvant platinum-based combination chemotherapy followed by radical cystectomy (RC). Up to 40% of patients (pts) are ineligible to receive cisplatin and proceed to RC without any neoadjuvant therapy. We and others have demonstrated enrichment of molecular alterations in cell cycle genes in MIBC, including copy number losses of CDKN2A in 41% of pts. Abemaciclib is a unique CDK4/6 inhibitor with single agent activity and a target kinome distinct from other CDK4/6 inhibitors. We have demonstrated that CRISPR knockout of CDKN2A increases susceptibility to abemaciclib in bladder cancer cell lines. Beyond tumor-intrinsic effects, abemaciclib also modulates the tumor microenvironment (TME) via upregulating human endogenous retroviral elements and increasing T cell infiltration. Methods: Cell cycLe inhibitiON to target the EVolution of urOthelial cancer (CLONEVO) is a single arm, window-of-opportunity trial of neoadjuvant abemaciclib which will evaluate tumor cell and TME changes in response to abemaciclib. Enrolled pts must be ineligible for platinum-based neoadjuvant therapy for resectable MIBC. Pts receive abemaciclib (200 mg BID PO) for 4 weeks prior to RC. Tumor tissue collected via transurethral resection of bladder tumor (TURBT) and residual tumor at RC undergo single cell RNA sequencing and whole-exome sequencing. Patient-derived organoids and xenografts are generated for a co-clinical trial of abemaciclib alone or in combination. The primary endpoint is the measurement of changes in cell cycle dynamics. Secondary objectives are assessment of toxicity via NCI CTCAE v 5.0 and pathologic downstaging of MIBC. We will perform targeted sequencing of a panel of cell cycle genes in serial plasma and urine cell free DNA to evaluate changes in the variant allele fractions of somatic alterations. The novel design of this trial allows dynamic in vivo assessment of tumor changes and creates a new paradigm for studying tumor evolution in real time. Clinical trials information: NCT03837821. Clinical trial information: NCT03837821 .

Molecular characterization and prognostic analysis of non-small cell lung cancer: A single centric real-world study.

e21646 Background: Identifying targetable genetic variants may help maximize clinical benefit of non-small cell lung cancer (NSCLC). Here, we retrospectively analyzed the benefit of next-generation sequencing (NGS) for NSCLC in routine clinical practice. Methods: A total of 59 NSCLC patients were enrolled in the study from Jun. 2018 to Dec. 2019. Formalin-fixed paraffin-embedded tissues samples were collected and sequenced by NGS, which detects single-nucleotide variants, small insertions/deletions, rearrangements, and copy-number alterations of either 59 or 1021 tumor related genes. The tumor response was evaluated using RECIST v1.1. Results: The detection rate of targetable variants was 59.32% (35/59), including EGFR mutation 49.15% (29/59), ALK rearrangement 5.08% (3/59), ROS1 rearrangement 1.69% (1/59), BRAF V600E mutation 1.69% (1/59), and ERBB2 mutation 1.69% (1/59). Of 29 patients with EGFR mutations, 5 patients (17.24%) had multiple EGFR mutations, including 2 patients (6.89%) with uncommon mutations. 25patients (86.21%) harbored concurrent mutations that may limit the efficacy of EGFR-TKIs, including EGFR amplification, activation of bypass signaling pathways, TP53 exon8 mutation, BCL2L11 deletion polymorphism, and PI3K-AKT-mTOR and cell cycle gene alterations. Among 25 patients received EGFR-TKIs, TKI effectiveness was evaluated in 18 patients. The disease control rate was 100% and overall response rate was 27.78%. The proportion of patients with TMB-H and/or PD-L1≥1, and targetable variants negative was 33.89% (20/59). Only one patient of this group received immunotherapy with pembrolizumab and achieved SD at the 2nd month. The proportion of patients with targetable variants negative, TMB-L, and PD-L1 < 1 or unknow patients was 6.78% (4/59), two patients of them left hospital without additional treatment, two patients received chemotherapy and the KRAS mutation present in one of them suggested that he might be resistant to EGFR-TKIs. Conclusions: More than half of NSCLC patients have targetable genetic variants and more than 30% patients have a molecular signature that can be treated with immunotherapy. Compared with immunotherapy, targeted therapy has higher acceptability in real-world. NGS-based genetic testing may have clinical value to predict the effectiveness of targeted therapy and immunotherapy. It can be widely implemented and standardized into clinical use.

Alterations of cell cycle genes in cancer: unmasking the role of cancer stem cells.

The cell cycle is a complex and strictly controlled process, consisting of different phases. Cell cycle regulation depends on phase-specific transcriptions of cell cycle genes. The alterations of cell cycle genes can predispose normal cells to have a cancerous phenotype. Indeed, several mechanisms underlying the deregulation of the cell cycle have been identified in different types of cancer. Cancer stem cells (CSCs), a fraction of tumor cells, are selectively capable of initiating tumor development. However, the deregulation of the cell cycle progression in CSCs still remains incompletely understood. This review describes epigenetic alterations and aberrant transcriptional regulation of cell cycle genes in CSCs as well as cell cycle patterns of CSCs.

Cell cycLe inhibitiON to target the EVolution of urOthelial cancer (CLONEVO): A single-arm, open-label window-of-opportunity trial of neoadjuvant abemaciclib in platinum-ineligible muscle invasive bladder cancer patients.

TPS606 Background: The standard of care for clinically localized muscle-invasive bladder cancer (MIBC) is neoadjuvant platinum-based combination chemotherapy followed by radical cystectomy (RC). Up to 40% of patients (pts) are ineligible to receive cisplatin and proceed to RC without any neoadjuvant therapy. We and others have demonstrated enrichment of molecular alterations in cell cycle genes in MIBC, including copy number losses of CDKN2A in 41% of pts. Abemaciclib is a unique CDK4/6 inhibitor with single agent activity and a target kinome distinct from other CDK4/6 inhibitors. We have demonstrated that CRISPR knockout of CDKN2A increases susceptibility to abemaciclib in bladder cancer cell lines. Beyond tumor-intrinsic effects, abemaciclib also modulates the tumor microenvironment (TME) via upregulating human endogenous retroviral elements and increasing T cell infiltration. Methods: Cell cycLe inhibitiON to target the EVolution of urOthelial cancer (CLONEVO) is a single arm, window-of-opportunity trial of neoadjuvant abemaciclib which will evaluate tumor cell and TME changes in response to abemaciclib. Enrolled pts must be ineligible for platinum-based neoadjuvant therapy for resectable MIBC. Pts receive abemaciclib (200 mg BID PO) for 4 weeks prior to RC. Tumor tissue collected via transurethral resection of bladder tumor (TURBT) and residual tumor at RC undergo single cell RNA sequencing and whole-exome sequencing. Patient-derived organoids and xenografts are generated for a co-clinical trial of abemaciclib alone or in combination. The primary endpoint is the measurement of changes in cell cycle dynamics. Secondary objectives are assessment of toxicity via NCI CTCAE v 5.0 and pathologic downstaging of MIBC. We will perform targeted sequencing of a panel of cell cycle genes in serial plasma and urine cell free DNA to evaluate changes in the variant allele fractions of somatic alterations. The novel design of this trial allows dynamic in vivo assessment of tumor changes and creates a new paradigm for studying tumor evolution in real time. Clinical trial information: NCT03837821.

Bevacizumab Plus Erlotinib in Chinese Patients with Untreated, <i>EGFR</i>-Mutated, Advanced NSCLC (ARTEMIS-CTONG1509): A Multicenter Phase 3 Study

Most patients with advanced non-small-cell lung cancer (NSCLC) develop resistance to first-line treatment. However, dual epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) pathway inhibition may delay therapeutic resistance. We assessed erlotinib plus bevacizumab versus erlotinib alone in an open-label, phase 3 study of Chinese patients with untreated EGFR-mutated metastatic NSCLC. The primary endpoint was progression-free survival (PFS) assessed by an independent review committee (IRC). Safety was assessed in all treated patients. In total, 311 patients received bevacizumab plus erlotinib (n=157) or erlotinib only (n=154). Median IRC-assessed PFS was 17.9 months (95% confidence interval [CI] 15.2–19.9) for bevacizumab plus erlotinib and 11.2 months (95% CI 9.7–13.8) for erlotinib only (Hazard Ratio 0.55 [95% CI 0.41–0.73], p < 0.001). Grade ≥3 treatment-related adverse events occurred in 86 (54.8%) and 40 (26.1%) patients, respectively. Bevacizumab plus erlotinib significantly improved PFS in patients with untreated EGFR-mutated NSCLC. The baseline mutation landscape was comparable between the two groups with 41% of patients in bevacizumab plus erlotinib group and 45% of patients in erlotinib group harboring concurrent mutations including amplification, oncogenic frusions, MAPK/PI3K alterations and cell cycle gene alterations. The addition of bevacizumab[WT1] did not alter the molecular dynamics of mutations during the course of treatment. At disease progression, EGFR T790M was the major resistance mechanism, acquired by 40% of patients in bevacizumab plus erlotinib group and 61% of patients in erlotinib only group, with a comprable detection rate in both groups (p=0.188).

Abstract B004: Revealing the mechanisms of acquired resistance to osimertinib from re-biopsy specimens in advanced non-small cell lung cancer with EGFR T790M mutation (LOGIK1607)

Abstract Background: Although treatment with osimertinib confers survival benefits in the second-line treatment of epidermal growth factor receptor (EGFR)-mutated lung cancer with the EGFR T790M mutation, the mechanism of acquired resistance to osimertinib has not been well understood. Here, we planned a multicenter, prospective observational study in order to clarify the possible acquired resistance mechanisms to second-line treatment using osimertinib in Japanese non-small cell lung cancer patients. Methods: EGFR T790M positive advanced non-small cell lung cancer patients were included in this study. We excluded EGFR tyrosine kinase inhibitor naïve patients, patients whose EGFR T790M proved only by liquid biopsy, and patients whose tumor specimens were not adequate for targeted resequencing analysis. Re-biopsy was planned after the patient developed acquired resistance to osimertinib under written informed consent. Nucleic acids were extracted from formalin-fixed paraffin embedded tumor sections and targeted resequencing was performed using a panel consisting of 143 genes (Oncomine Comprehensive Assay, OCA, ThermoFisher Scientific, USA) in a CLIA-certified laboratory (SRL Inc., Japan), and then analyzed using IonReporter software (ThermoFisher Scientific, USA). Results: Between November 2016 and February 2019, 86 patients from 25 institutions were screened. Among 71 patients who were eligible, 31 patients were still on osimertinib and 33 patients developed acquired resistance. Among the 33 patients who developed acquired resistance, 16 patients proceeded to re-biopsy (4 males, 8 Del19 and 7 L858R EGFR mutations). 15/16 samples from re-biopsies and 9/16 samples from pre-osimertinib biopsies proceeded to OCA analyses. Others were excluded because of low quality of extracted nucleic acids. The acquired resistance mechanisms were as follows: T790M maintained in 10 patients with EGFR G796S being found in 1 patient. T790M loss observed in 5 patients with 1 patient having copy number gain in growth-arrest specific gene 6 (GAS6). Another patient possibly had copy number alterations in cell cycle genes. All patients had equal or reduced mutation counts during the therapeutic course, suggesting clonal selections by the therapies. Conclusions: EGFR C797S mutation was not observed in this series. Copy number alteration in GAS6-AXL pathway and cell cycle pathway could be targets of therapy. Citation Format: Atsushi Osoegawa, Masafumi Yamaguchi, Tomomi Nakamura, Ryotaro Morinaga, Kentaro Tanaka, Kosuke Kashiwabara, Kenichi Taguchi, Kazuki Nabeshima, Junji Kishimoto, Kenji Sugio. Revealing the mechanisms of acquired resistance to osimertinib from re-biopsy specimens in advanced non-small cell lung cancer with EGFR T790M mutation (LOGIK1607) [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B004. doi:10.1158/1535-7163.TARG-19-B004

SWOG S1400C (NCT02154490)—A Phase II Study of Palbociclib for Previously Treated Cell Cycle Gene Alteration–Positive Patients with Stage IV Squamous Cell Lung Cancer (Lung-MAP Substudy)

ObjectiveLung-MAP (SWOG S1400) is a master platform trial assessing targeted therapies in squamous NSCLC. The objective of study C (S1400C) was to evaluate the response rate to palbociclib, a cyclin-dependent kinase 4 and cyclin-dependent kinase 6 inhibitor, in patients with cell cycle gene abnormalities. MethodsPatients with squamous NSCLC, a performance status of 0 to 2, and normal organ function who had progressed after at least one prior platinum-based chemotherapy with cyclin-dependent kinase 4 gene (CDK4) or cyclin D1 gene (CCND1), cyclin D2 gene (CCND2), or cyclin D3 gene (CCND3) amplifications on tumor specimens were eligible. The study was originally designed as a phase II/III trial comparing palbociclib with docetaxel, but it was modified to a single-arm phase II trial with the primary end point of response when immunotherapy was approved. If two or fewer responses were seen in the first 20 patients, then the study would cease enrollment. ResultsA total of 88 patients (9% of patients screened) were assigned to S1400C, and 53 patients enrolled (including 17 to receive docetaxel). One patient who had been registered to receive docetaxel was re-registered to receive palbociclib after progression while taking docetaxel. The frequencies of cell cycle gene alterations in the eligible patients taking palbociclib (n = 32) were as follows: CCND1, 81% (n = 26); CCND2, 9% (n = 3); CCND3, 6% (n = 2); and CDK4, 3% (n = 1). In all, 32 eligible patients received palbociclib. There were two partial responses (response rate 6% [95% confidence interval (CI): 0%–15%]), both with CCND1 amplification. Twelve patients had stable disease (38% [95% CI: 21%–54%]). The median progression-free survival was 1.7 months (95% CI: 1.6–2.9 months) and the median overall survival was 7.1 months (95% CI: 4.2–12.5). ConclusionPalbociclib as monotherapy failed to demonstrate the prespecified criteria for advancement to phase III testing.

Abstract 4415: CDK4/6 inhibition with lerociclib (G1T38) delays acquired resistance to targeted therapies in preclinical models of non-small cell lung cancer

Abstract Tyrosine kinase inhibitors (TKIs) targeting oncogenes such as EGFR, ALK, or RET have dramatically improved anti-tumor efficacy in patients with non-small cell lung cancer (NSCLC). Despite the significant clinical benefit offered by these agents, tumor responses to single-agent TKIs nevertheless remain limited in their magnitude and duration. Here we investigate combination therapy approaches with lerociclib (G1T38), a selective, oral, and potential best-in-class inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6). Consistent with previous studies reporting frequent cell-cycle gene alterations in NSCLC, we show that lerociclib synergizes with TKIs targeting EGFR, ALK, and RET in lung cancer cell lines and enhances the efficacy of osimertinib and crizotinib in PDX models harboring EGFR or ALK mutations, respectively. Moreover, in a well-characterized murine xenograft model of EGFR-mutant NSCLC, seven days of treatment with a clinically-relevant dose of osimertinib (25 mg/kg p.o. QDx7) resulted in 60% complete tumor responses, versus 100% complete tumor responses in the osimertinib plus lerociclib (25 mg/kg osi + 100 mg/kg lero, p.o. QDx7) group. Strikingly, complete tumor responses in the combination group persisted through an additional 35 days (or more) of observation, by which time all tumors in the osimertinib monotherapy group had re-emerged - suggesting a role for lerociclib in extending the duration of antitumor response. Based on this result, we assessed the ability of lerociclib to delay the emergence of resistance by chronically treating NSCLC cell lines with TKI +/- lerociclib. These experiments revealed that combination treatment with lerociclib significantly delayed acquired TKI resistance in multiple NSCLC cell lines. The same result was achieved in vivo in an EGFR-mutant NSCLC xenograft model known to develop resistance to EGFR TKI therapy, wherein combination treatment with lerociclib delayed the outgrowth of osimertinib-resistant tumors. Studies to characterize the mechanism by which lerociclib delays TKI resistance in NSCLC are ongoing. Finally, we addressed whether combination therapy with lerociclib can treat TKI resistance after it develops. In EGFR-mutant NSCLC xenografts harboring MET amplification - one of the more common mechanisms of resistance observed to EGFR TKIs in the clinic - the combination of lerociclib plus osimertinib caused significant tumor growth inhibition (90%) compared to osimertinib monotherapy (34%) after 46 days of continuous daily dosing. Collectively, our results suggest a compelling rationale for utilizing lerociclib as a backbone therapy for multiple combination regimens in NSCLC. A phase 1b/2 clinical trial evaluating lerociclib plus osimertinib in EGFR-mutant NSCLC is ongoing (NCT03455829). Citation Format: Daniel M. Freed, Claire R. Hall, Jay C. Strum, Patrick J. Roberts. CDK4/6 inhibition with lerociclib (G1T38) delays acquired resistance to targeted therapies in preclinical models of non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4415.

First survival outcomes and additional secondary analyses from PURE-01: Pembrolizumab (pembro) before radical cystectomy (RC) in muscle-invasive urothelial bladder carcinoma (MIBC).

391 Background: PURE-01 is a phase 2 study of Pembro before RC in MIBC: the primary endpoint (pathologic complete response [pT0]) was met in advance ( Necchi et al. JCO 2018). While the study is still recruiting patients (pts), we analyzed the interim long-term outcomes in terms of relapse-free survival (RFS) and the role of multiparametric bladder magnetic resonance imaging (mpMRI) in predicting major pathologic response (pT&lt;2) in RC. Methods: Pts with clinical stage T2-3bN0M0 received 3 courses of 200mg Pembro, before RC. Pts were staged and re-assessed with mpMRI before RC. 1-y RFS (overall and by pathologic stage) after Pembro and RC was compared with that from clinical stage-matched cohort of San Raffaele Urological Institute (URI, n=1021) and of the RISC multicenter database (n=719, Bandini et al, Eur Urol Oncol 2018). mpMRI were performed in all pts through bladder catheterization. The apparent diffusion coefficient (ADC) changes post-Pembro were matched with pathologic response, qPCR and genomic data from RC samples (FoundationONE). Results: 70 pts have completed treatment thus far: with 27 pT0 (38.6%) and 44 pT&lt;2 (62.9%), activity was confirmed. 21 pts had ≥1y follow-up and were analyzed. Overall, 1-y RFS (95% CI) in PURE-01, URI and RISC were: 95.2 (86.6-100), 79.0 (76-82), and 69.7 (66.2-73.4). 1-y RFS after neoadjuvant chemotherapy and RC in URI and RISC were: 76.7 (61-97), and 76.5 (71-83). 1-y RFS for pT3-4 and/or pN+ pts were: 88.9 (71-100), 71.2 (67-76), and 57.1 (52-62). 39/70 pts had measurable disease for pre-post mpMRI changes. Pts with pT&lt;2 (n=16) on RC showed a median “mean ADC” of 0.98 mm2/s vs 0.84 mm2/s of non-responders (NR, n=23, p=0.098), despite an increased T-cell infiltrate. Pts with cell-cycle gene alterations in RC (n=8) were all NR and showed lower median “mean ADC” values (p=0.074). Conclusions: The early efficacy data further support the role of single-agent IO as neoadjuvant therapy in MIBC. Pembro may be beneficial at long-term also in pathologically high-risk pts, thus questioning the value of PD-L1 for pt selection. mpMRI and genomic data might help identifying those pts who may need re-TURB only instead of RC. Clinical trial information: NCT02736266.

Abstract PD2-02: Longitudinal ctDNA sequencing using an expanded genomic panel in the PALOMA3 trial of palbociclib plus fulvestrant

Abstract Background: CDK4/6 inhibition combined with endocrine therapy (ET) is now the standard of care for advanced hormone receptor (HR) positive breast cancer patients (pts). Previous ctDNA analysis of paired pre-treatment (pre-T) and end of treatment (EoT) plasma samples from the PALOMA-3 trial of the CDK4/6 inhibitor P with/without F demonstrated that acquired PIK3CA, ESR1, RB1 and rare growth factor receptor mutations (mut) likely contribute to resistance (Turner et al, ASCO 2018, Abstract 1001). We now report results from an extended ctDNA analysis of paired PALOMA-3 plasma samples using hybridization capture-based, next-generation sequencing (NGS) assay covering 87 breast cancer, HR signaling, and cell cycle-related genes. Methods: Pre-/post-menopausal pts whose disease progressed after prior ET (N=521) were randomized 2:1 to receive F 500 mg + P (125 mg/d, 3 wk on/1 wk off) or placebo. Cell-free DNA extracted from paired plasma samples (n=194) was analyzed with a custom 87-gene NGS assay, to identify single nucleotide variants and copy number amplification (CNA), with molecular barcoding, high NGS coverage (10,000-20,000 reads per nucleotide position), and background error correction. Differences in ctDNA mut frequencies detected in P+F compared to F alone were assessed and their association to clinical outcome estimated. Results: Blinded assay qualification and droplet digital PCR validation suggested a mut detection frequency cutoff at 0.3%. A high coefficient of correlation with previously presented data was observed for ESR1 and PIK3CA variants (r=0.94 and 0.97, respectively), with acquisition of PIK3CA and ESR1 mutations at EoT in P+F and F groups. Gene level mut analysis of EoT plasma revealed no significant difference between P+F vs F alone (Table), although there was an increase in RB1 mutations in P+F consistent with previous data. Other acquired muts at EoT (SMAD4, NOTCH2, and CDKN1B) were observed at low frequencies. Muts in CDK4 and CDK6 were rarely observed (&amp;lt; 1% of pts), regardless of treatment arm. Detected Variants, Frequency, n (%) P+FF GenePre-TEoTPre-TEoTPre-T pvalEoT pvalPIK3CA47(37)51(40)19(28)22(33)0.2660.352ESR136(28)45(35)19(28)24(36)11TP5330(24)33(26)23(34)25(37)0.1280.137RB12(2)9(7)2(3)2(3)NA0.336PTEN5(4)7(6)3(4)3(4)11AKT17(6)7(6)2(3)2(3)0.7210.721 Conclusions: Our results corroborate the previously reported results demonstrating that genomic profiles of treatment resistant cancer are similar between P + F and F therapy. Expanded analysis of cell-cycle genes identified no new recurrently mutated genes, and confirmed that RB1 mutations are selected at low frequency after P+F treatment. Alterations in cell cycle genes may not be a common mechanism of resistance to CDK4/6 inhibitors in HR+ HER2- advanced breast cancer. Sponsor: Pfizer Citation Format: O'Leary B, Lira ME, Huang S, Deng S, Xie T, Kinong JK, Hardwick J, Rejto P, Liu Y, Huang X, Huang Bartlett C, André F, Loibl S, DeMichele A, Cristofanilli M, Turner NC. Longitudinal ctDNA sequencing using an expanded genomic panel in the PALOMA3 trial of palbociclib plus fulvestrant [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD2-02.

Landscape of EGFR-Dependent and -Independent Resistance Mechanisms to Osimertinib and Continuation Therapy Beyond Progression in EGFR-Mutant NSCLC

Osimertinib was initially approved for T790M-positive non-small cell lung cancer (NSCLC) and, more recently, for first-line treatment of EGFR-mutant NSCLC. However, resistance mechanisms to osimertinib have been incompletely described. Using cohorts from The University of Texas MD Anderson Lung Cancer Moonshot GEMINI and Moffitt Cancer Center lung cancer databases, we collected clinical data for patients treated with osimertinib. Molecular profiling analysis was performed at the time of progression in a subset of the patients. In the 118 patients treated with osimertinib, 42 had molecular profiling at progression. T790M was preserved in 21 (50%) patients and lost in 21 (50%). EGFR C797 and L792 (26%) mutations were the most common resistance mechanism and were observed exclusively in T790M-preserved cases. MET amplification was the second most common alteration (14%). Recurrent alterations were observed in 22 genes/pathways, including PIK3CA, FGFR, and RET. Preclinical studies confirmed MET, PIK3CA, and epithelial-to-mesenchymal transition as potential resistance drivers. Alterations of cell-cycle genes were associated with shorter median progression-free survival (PFS, 4.4 vs. 8.8 months, P = 0.01). In 76 patients with progression, osimertinib was continued in 47 cases with a median second PFS (PFS2) of 12.6 months; 21 patients received local consolidation radiation with a median PFS of 15.5 months. Continuation of osimertinib beyond progression was associated with a longer overall survival compared with discontinuation (11.2 vs. 6.1 months, P = 0.02). Osimertinib resistance is associated with diverse, predominantly EGFR-independent genomic alterations. Continuation of osimertinib after progression, alone or in conjunction with radiotherapy, may provide prolonged clinical benefit in selected patients.See related commentary by Devarakonda and Govindan, p. 6112.