Cyclin D1 Amplification Research Articles

OR05-03 Treatment Of Parathyroid Neoplasia By Pharmacologic cdk4/6 Inhibition

Abstract Disclosure: J. Costa-Guda: None. J. Bellizzi: None. S. Gorka: None. C. Titarenko: None. A. Arnold: None. Parathyroid carcinoma (PC) is a rare, life-threatening malignancy. Despite relatively slow growth, recurrent or metastatic disease is typically incurable and often fatal due to cumulative complications of sustained elevation of parathyroid hormone (PTH) and accompanying hypercalcemia. Medical therapy aids in managing sequelae of hyperparathyroidism (HPT) but non-surgical interventions to reduce tumor burden have been largely ineffective in PC. Cyclin D1 amplification is seen in nearly 30% of PC, making it a key potentially actionable therapeutic target. Importantly, in contrast to other tissue contexts, cyclin D1-driven parathyroid neoplasia has been experimentally demonstrated to depend on cyclin D1’s ability to bind and activate its partner cyclin dependent kinases (cdk). This suggests that parathyroid tumor cells may be more sensitive to alterations in cdk-mediated proliferation control, and thus more responsive to pharmacologic cdk inhibition, compared with other tumor types. We sought to test the efficacy of cdk4/6 inhibitor therapy in cyclin D1-overexpressing parathyroid tumors in a pre-clinical system. We utilized a mouse model, harboring a PTH-CCND1 (PCD) transgene to drive parathyroid-specific cyclin D1 overexpression, which develops hormonally-active parathyroid tumors with known kinetics. Transgenic mice with established hyperparathyroidism and wild type littermates were treated with the cdk4/6 inhibitors palbociclib or abemaciclib for three weeks. While vehicle-treated PCD mice showed a 7% increase in serum calcium (Ca) levels over the course of treatment, Ca decreased by 1.8% and 0.5% in palbociclib- and abemaciclib-treated PCD mice, respectively, from pre-treatment levels. This treatment effect persisted for at least 60 days after discontinuation of treatment. Decreased parathyroid proliferation, as evidenced by 4.7-fold and 17.3-fold fewer Ki-67-positive cells in palbociclib- and abemaciclib-treated PCD mice, respectively, compared with vehicle-treated PCD mice, was also seen. Cdk inhibitor treatment had no effect on Ca or parathyroid cell proliferation in wild type mice. These results demonstrate that palbociclib and abemaciclib can significantly slow the progression of hypercalcemia and parathyroid proliferation in a cyclin D1-driven parathyroid tumor model, suggesting that cyclin D1 overexpressing parathyroid cells may be particularly sensitive to cdk4/6 inhibition. Our findings provide the first evidence of a direct pharmacologic effect in a relevant preclinical system to support the hypothesis that selected HPT patients, eg those suffering from advanced PC with activated cyclin D1, could substantially benefit from cdk4/6 inhibitor therapy. This evidence may aid clinicians in selecting therapeutic interventions, including when considering such drugs for off-label use or inclusion of PC patients in “basket” clinical trials. Presentation: Thursday, June 15, 2023

CCNB2 as a potential biomarker of bladder cancer via the high throughput technology.

Bladder cancer and oral squamous cell carcinoma (OSCC) seriously affect people's health. However, the relationship between bladder cancer and OSCC remains unclear. Got GSE138206, GSE146483, GSE184616, and bladder cancer datasets GSE65635, GSE100926 from Gene Expression Omnibus database. Weighted gene co-expression network analysis was used to identify the significant module. Functional enrichment analysis was performed via the Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes. Furthermore, the Gene Set Enrichment Analysis was also used to complete the enrichment analysis. Comparative Toxicogenomics Database found most relevant diseases to core genes. TargetScan is used to forecast analysis of microRNA and target genes. In Gene Ontology analysis, differentially expressed genes were mostly concentrated in cell differentiation, extrallular region, structural molecule activity, and actin binding. In Kyoto Encyclopedia of Genes and Genomes analysis, the differentially expressed genes were mainly enriched in PI3K-Akt signaling pathway, pathway in cancer, and extracellular matrix-receptor interaction. Seven hub genes (cyclin B2 [CCNB2], TK1, CDC20, PCNA, CKS1B, CDCA5, MCM4) were obtained. Hub genes (CCNB2, CDC20) are highly expressed in OSCC and bladder cancer samples. CCNB2 was one common oncogene of bladder cancer and OSCC.

CCND1 Amplification in Pancreatic Ductal and Ampullary Adenocarcinoma and Its Impact on Patients' Survival: a Single-Center Observational Study.

Studies conducted worldwide in the last few decades have demonstrated that various high- and low-frequency inherited and somatic mutations play important roles in the pathogenesis of periampullary carcinoma. Owing to relatively inaccessible location and lack of specific early diagnostic signs, majority of periampullary tumors are difficult to manage. Limited chemotherapeutic options that are available are highly toxic and not very efficacious. CyclinD1, if found to be amplified in these malignancies, might become an important gene to be targeted for monoclonal antibody therapy. An analytical retrospective-prospective study was done on 35 patients of operable periampullary carcinoma, in Medical College, Kolkata from January 2019 to July 2020. After isolating DNA from tumor and corresponding normal tissue by Qiagen DNEasy kit, CyclinD1 amplification was assessed by RT-PCR using Taqman DNA copy number in the laboratory of Indian Statistical Institute. Survival analysis was done by Kaplan-Meier estimator and all statistical calculations performed through SPSS software. Six (17%) out of 35 patients were found to have > twofold amplifications of CyclinD1 gene. However, no positive correlation was found between CyclinD1 amplification and overall survival of the patients (p value 0.21). Positive correlation was not found in our study between CCND1 amplification and periampullary malignancy. However, a single large study conducted in Japan by Yamazaki showed reduced survival and higher metastasis in CyclinD1 positive periampullary carcinoma. So there is reasonable scope in future for large-scale population-based studies to establish similar association in our subcontinent as well.

Abstract PS5-01: Biomarkers of resistance to palbociclib in ER+ primary breast cancer in the PALLET trial

Abstract Background: CDK4/6 inhibitors are being used in combination with aromatase inhibitors as therapy for advanced ER+ breast cancer (BC) and are being explored for use in primary BC. Few mechanisms driving resistance to added CDK4/6 inhibitors have been defined. The PALLET phase II randomized neoadjuvant trial of letrozole (LET) ± palbociclib (PALBO) in postmenopausal ER+HER2- primary BC reported that clinical response rate over 14wks was not significantly increased by adding PALBO to LET but suppression of Ki67 was significantly increased (Johnston et al, JCO 2018, 37, 178): after 14wks complete cell cycle arrest (CCCA, Ki67<2.7%) was present in 59% on LET and 90% on LET+PALBO. Here we sought to identify biomarkers of de novo resistance to allow selection of patients most likely to benefit from added PALBO. Methods: 307 patients were randomized to LET (n=103) or LET+PALBO (n=204) for 14 wks. The first 2wks of LET+PALBO patients were randomised to LET, PALBO, or LET+PALBO. Biopsies were taken at baseline, 2wks and 14wks. Biomarker data are presented here for baseline only, other than Ki67 at both baseline and 14wks. IHC analyses were conducted on FFPE biopsies for ER, PgR, RB1, cyclin-E1, and cyclin-D1 (also FISH). RNA-seq was performed on fresh frozen biopsies. Association of each biomarker with CCCA was determined by logistic regression. Differentially expressed genes (DEGs) were identified between patients sensitive (CCCA) (n=94) and resistant (non-CCCA) (n=10) to treatments with or without PALBO at 14wks by DESeq2. Fifty hallmark gene sets were tested for significant enrichment with DEGs and differential gene sets were identified by using Gene Set Enrichment Analysis (GSEA). Results: The association of IHC biomarkers with CCCA is shown in the table. Lower levels of ER, higher levels of cyclin-E1, and amplification of cyclin-D1 were each significantly associated with a greater chance of non-CCCA with LET+PALBO. High cyclin-E1 levels were also associated with reduced chance of CCCA with LET only. Patients with high baseline Ki67 also exhibited higher non-CCCA with LET+PALBO at 14wks (p=0.0002). In the RNAseq data we identified 1973 DEGs between the 14wk CCCA and non-CCCA patients for LET+PALBO. E2F and MYC targets, PI3K/AKT/MTOR signalling and interferon response gene sets were among the hallmark gene sets enriched for genes with higher expression in non-CCCA patients at 14wks for LET+PALBO (FDR<0.05). For LET-only, 311 DEGs were identified and the “Estrogen Response Early” gene set was significantly enriched in genes with higher expression in CCCA samples. At the individual gene level, genes significantly associated with non-CCCA after 14wks LET+PALBO included CCNE1, CDK2 and several E2F-related genes (p<0.05). Their expression was not significantly different between non-CCCA and CCCA patients with LET alone. Conclusion: Biomarkers associated with response/resistance to added PALBO were different from LET only. PALBO resistance was associated with higher baseline expression of cyclin-E1 (both IHC and RNA), CDK2, and genes related to E2F, MYC, interferon and MTOR signalling. These results suggest that multiple identifiable mechanisms of de novo resistance to PALBO are likely to exist in primary ER+ BC. On-going WES analyses will allow the significance of alterations at the DNA level to be presented. Table 1.Continuous measurement in a logistic regression for CCCA at 14 weeks; Oddsratio calculated separately for group A and groups B,C,D and were adjusted forregion (UK vs NA); * amplified vsnon-amplifiedContinuous measurement in a logistic regression for CCCA at 14 weeksBiomarkerLET LET+PALBOOdds ratio95% CIpOdds ratio95% CIpER1.120.36, 3.480.844.471.62, 12.380.004PgR4.381.03, 18.580.053.050.50, 18.560.23RB13.010.24, 38.560.400.420.05, 38.490.83Cyclin-E10.100.01, 0.840.030.020.00, 0.200.001Cyclin-D1 IHC3.090.51, 18.490.222.560.28, 23.330.40CyclinD1 FISH*1.470.43, 4.990.530.280.06, 0.860.03 Citation Format: Eugene F Schuster, Hui Xiao, Elena Lopez-Knowles, Lucy Kilburn, Mothaffar Rimawi, David A Wheeler, Katherine Pogue-Geile, Yuan Lui, Samuel A Jacobs, Chet Cornman, Shannon Puhalla, Maggie Cheang, Judith Bliss, Stephen Johnston, Mitch Dowsett, On behalf of the PALLET Trialists. Biomarkers of resistance to palbociclib in ER+ primary breast cancer in the PALLET trial [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-01.

Dinaciclib, a cyclin-dependent kinase inhibitor, suppresses cholangiocarcinoma growth by targeting CDK2/5/9

Cholangiocarcinoma (CCA) is a highly invasive cancer, diagnosed at an advanced stage, and refractory to surgical intervention and chemotherapy. Cyclin-dependent kinases (CDKs) regulate cell cycle progression and transcriptional processes, and are considered potential therapeutic targets for cancer. Dinaciclib is a small molecule multi-CDK inhibitor targeting CDK 2/5/9. In this study, the therapeutic efficacy of dinaciclib was assessed using patient-derived xenograft cells (PDXC) and CCA cell lines. Treatment with dinaciclib significantly suppressed cell proliferation, induced caspase 3/7 levels and apoptotic activity in PDXC and CCA cell lines. Dinaciclib suppressed expression of its molecular targets CDK2/5/9, and anti-apoptotic BCL-XL and BCL2 proteins. Despite the presence of cyclin D1 amplification in the PDXC line, palbociclib treatment had no effect on cell proliferation, cell cycle or apoptosis in the PDXC as well as other CCA cell lines. Importantly, dinaciclib, in combination with gemcitabine, produced a robust and sustained inhibition of tumor progression in vivo in a PDX mouse model, greater than either of the treatments alone. Expression levels of two proliferative markers, phospho-histone H3 and Ki-67, were substantially suppressed in samples treated with the combination regimen. Our results identify dinaciclib as a novel and potent therapeutic agent alone or in combination with gemcitabine for the treatment of CCA.

MEK blockade overcomes the limited activity of palbociclib in head and neck cancer.

Head and neck cancer (HNC) is characterized with multiple aberrations in cell cycle pathways, including amplification of cyclin D1. Palbociclib (PAL), a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor, has been reported to regulate cell cycle progression in HNC. However, recent studies have revealed the acquired resistance of certain cells to PAL through activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. Therefore, we investigated whether the inhibition of MEK/ERK pathway by trametinib (TRA) may overcome the limited efficacy of PAL in HNC. We evaluated the effect of PAL alone and in combination with TRA on the viability of HNC cells, and found that the combination treatment synergistically inhibited the proliferation of HNC cells. The combination treatment induced G0/G1 cell cycle arrest and apoptotic cell death. In particular, apoptosis mediated by the combination treatment was accompanied with an increase in caspase-3 activity and the number of TUNEL-positive apoptotic cells. These results were consistent with the decrease in cell cycle progression and mitogen-activated protein kinase (MAPK) pathway activation. In a xenograft mouse model of HNC, PAL and TRA synergistically inhibited tumor growth and enhanced tumor cell apoptosis, consistent with the increase in the number of TUNEL-positive cells. The anti-proliferative effects were evident in tumor tissues subjected to the combination treatment as compared with those treated with single drug. Taken together, our study demonstrates that the combination of PAL and TRA exerts synergistic anticancer effects and inhibits cell cycle check points and MEK/ERK pathway in HNC, suggestive of their potential application for HNC treatment.

WITHDRAWN: Circ_0001946 facilitates the proliferation and represses apoptosis of ox-LDL-stimulated Vascular Smooth Muscle Cells via regulating miR-641/CCND1 pathway

Abstract Recently, circRNAs are reported to exhibit significant regulation on atherosclerosis development. Circ_0001946 has been identified in several diseases and our present study was aimed to investigate its biological roles on Vascular Smooth Muscle Cells (VSMCs) incubated with ox-LDL. In our research, we validated circ_0001946 was obviously increased in human atherosclerosis patient serums. ox-LDL induced circ_0001946 expression in VSMCs. Meanwhile, we reported miR-641 was obviously decreased in atherosclerosis serum samples compared to the health controls. It was indicated silence of circ_0001946 repressed VSMCs proliferation and contributed to the cell apoptosis. In addition, we observed that down-regulation of circ_0001946 induced miR-641 expression greatly and their direct correlation was proved using dual-luciferase reporter assay. Inhibitors of miR-641 rescued the effects of depletion of circ_0001946 on proliferation and apoptosis in VSMCs treated by ox-LDL. In addition, amplification and overexpression of cyclin D1 (CCND1), which can alter cell cycle progression, is dys-regulated in a variety of diseases. It was showed that CCND1 could be a target of miR-641 and circ_0001946 acted as a sponge of miR-641 to increase CCND1 level. In conclusion, our current data suggested circ_0001946 silence inhibited VSMCs proliferation and increased cell apoptosis through modulating miR-641 and CCND1.

Abstract 2553: Integration of genomic studies and the patient-derived xenograft model in nasopharyngeal carcinoma precision medicine research

Abstract Patient-derived xenograft (PDX) tumor model has become a new approach to identify druggable tumor mutations, and to screen and evaluate personalized anti-cancer drugs. Here, we established five nasopharyngeal carcinoma (NPC) PDXs in mouse model and performed whole-exome sequencing (WES) and copy number variations (CNVs) analyses. Relatively high incident rate of CNV aberration in cell cycle pathway was detected. Among these five NPC-PDXs, three had cyclin D1 (CCND1) amplification while four had cyclin-dependent kinase inhibitor CDKN2A deletion. Selected inhibitors were tested for their anti-tumor effects in two NPC-PDX mouse models. Palbociclib, a cyclin-dependent kinase inhibitor, revealed efficient anti-tumor effects by inducing G1 arrest in the PDX tumor according to RNA sequencing. In this study, cyclin D1 overexpression was also observed in >90% clinical metastatic NPC tumors (87/91) and was associated with poor outcomes. One NPC patient with liver metastatic who was prescribed with Palbociclib had stable disease and a drop in Epstein Barr virus (EBV) DNA titer. CNV analysis disclosed that plasma CCND1/CDKN2A ratio is correlated with EBV DNA load in NPC patients’ plasma and could serve as a screening test to select potential CDK4/6 inhibitors treatment candidates. Taken together, our findings indicate that integrated information from sequencing-based genomic studies and tumor transcriptomes before and after drug treatment in NPC-PDX models provide novel guidelines for personalized precision treatments. Citation Format: Cheng-Lung Hsu, Hsin-Pai Li. Integration of genomic studies and the patient-derived xenograft model in nasopharyngeal carcinoma precision medicine research [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 2553.

Effects of palbociclib on oral squamous cell carcinoma and the role of PIK3CA in conferring resistance.

ObjectiveLack of effective therapies remains a problem in the treatment of oral squamous cell carcinoma (OSCC), especially in patients with advanced tumors. OSCC development is driven by multiple aberrancies within the cell cycle pathway, including amplification of cyclin D1 and loss of p16. Hence, cell cycle inhibitors of the CDK4/6-cyclin D axis are appealing targets for OSCC treatment. Here, we determined the potency of palbociclib and identified genetic features that are associated with the response of palbociclib in OSCC.MethodsThe effect of palbociclib was evaluated in a panel of well-characterized OSCC cell lines by cell proliferation assays and further confirmed by in vivo evaluation in xenograft models. PIK3CA-mutant isogenic cell lines were used to investigate the effect of PIK3CA mutation towards palbociclib response. ResultsWe demonstrated that 80% of OSCC cell lines are sensitive to palbociclib at sub-micromolar concentrations. Consistently, palbociclib was effective in controlling tumor growth in mice. We identified that palbociclib-resistant cells harbored mutations in PIK3CA. Using isogenic cell lines, we showed that PIK3CA mutant cells are less responsive to palbociclib as compared to wild-type cells with concurrent upregulation of CDK2 and cyclin E1 protein levels. We further demonstrated that the combination of a PI3K/mTOR inhibitor (PF-04691502) and palbociclib completely controlled tumor growth in mice. ConclusionsThis study demonstrated the potency of palbociclib in OSCC models and provides a rationale for the inclusion of PIK3CA testing in the clinical evaluation of CDK4/6 inhibitors and suggests combination approaches for further clinical studies.

Integrated genomic analyses in PDX model reveal a cyclin-dependent kinase inhibitor Palbociclib as a novel candidate drug for nasopharyngeal carcinoma

BackgroundPatient-derived xenograft (PDX) tumor model has become a new approach in identifying druggable tumor mutations, screening and evaluating personalized cancer drugs based on the mutated targets.MethodsWe established five nasopharyngeal carcinoma (NPC) PDXs in mouse model. Subsequently, whole-exome sequencing (WES) and genomic mutation analyses were performed to search for genetic alterations for new drug targets. Potential drugs were applied in two NPC PDX mice model to assess their anti-cancer activities. RNA sequencing and transcriptomic analysis were performed in one NPC PDX mice to correlate with the efficacy of the anti-cancer drugs.ResultsA relative high incident rate of copy number variations (CNVs) of cell cycle-associated genes. Among the five NPC-PDXs, three had cyclin D1 (CCND1) amplification while four had cyclin-dependent kinase inhibitor CDKN2A deletion. Furthermore, CCND1 overexpression was observed in > 90% FFPE clinical metastatic NPC tumors (87/91) and was associated with poor outcomes. CNV analysis disclosed that plasma CCND1/CDKN2A ratio is correlated with EBV DNA load in NPC patients’ plasma and could serve as a screening test to select potential CDK4/6 inhibitor treatment candidates. Based on our NPC PDX model and RNA sequencing, Palbociclib, a cyclin-dependent kinase inhibitor, proved to have anti-tumor effects by inducing G1 arrest. One NPC patient with liver metastatic was treated with Palbociclib, had stable disease response and a drop in Epstein Barr virus (EBV) EBV titer.ConclusionsOur integrated information of sequencing-based genomic studies and tumor transcriptomes with drug treatment in NPC-PDX models provided guidelines for personalized precision treatments and revealed a cyclin-dependent kinase inhibitor Palbociclib as a novel candidate drug for NPC.

Abstract OT3-05-09: A randomized phase II trial of fulvestrant with or without ribociclib after progression on aromatase inhibition plus cyclin-dependent kinase 4/6 inhibition in patients with unresectable or metastatic hormone receptor positive, HER2 negative breast cancer (Maintain trial)

Abstract Background Cyclin dependent kinase 4/6 inhibitors (CDK4/6i), including palbociclib and ribociclib (R), have demonstrated remarkable benefit in progression free survival (PFS) in patients (pts) with hormone receptor positive (HR+), HER2- metastatic breast cancer (MBC) when combined with anti-estrogen therapy. Switching between anti-estrogen therapies at disease progression is standard of care in the treatment of HR+ MBC. We evaluate the strategy of switching anti-estrogen therapy to fulvestrant (F) and maintaining CDK4/6 inhibition with R in pts with HR+, HER2- MBC who have progressed on an aromatase inhibitor (AI) + CDK4/6i. Trial Design Phase II, multi-center, randomized, double-blind, placebo-controlled trial to evaluate F +/- R in pts with HR+, HER2- MBC who have previously progressed on any AI + CDK4/6i. Pts can be screened and registered at two different time points: Scenario 1: Before receiving any CDK4/6i Scenario 2: At the time of progression of disease (POD) while being treated with an AI + CDK4/6i In scenario 1, the study will provide pts with letrozole + R, but pts will not be randomized until they demonstrate POD. At randomization, pts will be assigned 1:1 to either a) F + R or b) F + placebo, with treatment given in 4-week cycles. F will be given as a 500 mg dose intramuscularly every 2 weeks for 3 times and then every 4 weeks, as per standard of care. R or placebo will be given orally at 600 mg daily, 3 weeks on/1 week off. CT scans and bone scan are to be performed prior to every third cycle. Serum and whole blood samples and optional tissue biopsies for biomarker assessment will be performed prior to study treatment (scenario 1), prior to randomization to R +/- F, and when the patient goes off study. Main Eligibility Criteria 1. Age ≥ 18 years with unresectable or metastatic BC 2. Estrogen and/or progesterone receptor positive, HER2 negative, as per ASCO-CAP 3. Postmenopausal status or receiving ovarian suppression 4. Measurable or unmeasurable disease; stable CNS disease allowed Specific Aims Primary: Progression free survival (PFS), defined as the time from randomization to POD or death. Secondary: Objective response rate (ORR), clinical benefit rate (CBR = ORR + stable disease rate), overall survival (OS), and duration of response. Pts in scenario 1 will also be assessed for PFS, OS, CBR, and safety while receiving AI + R (pre-randomization). Biomarker assessment will include amplification of cyclin D1 and cyclin E, phosphoRb and TK1 expression, Rb1 and p16 loss, and ctDNA for ESR1 and PIK3CA mutations. Target Accrual 132 pts accrued from 11 academic medical centers in the U.S, with a goal of completing accrual in two years (˜60 to 72 pts in each scenario). Statistical Methods Assuming a median PFS of 3.8 months with F alone, we predict that F + R will lead to a median PFS of at least 6.5 months. A one-sided log-rank test with a sample size of N=120 and alpha=0.025, achieves 80% power to detect a difference in PFS of 2.7 months. N=132 pts allows for a 10% drop-out rate. Contact Kevin Kalinsky, MD, MS 212-305-1945 kk2693@cumc.columbia.edu. Citation Format: Kalinsky K, Mundi PS, Chiuzan C, Accordino MK, Trivedi MS, Sparano JA, Oh SY, Tiersten A, O'Regan R, Esteva FJ, Jain S, Mayer IA, Forero A, Vaklavas C, Baer L, Crew K, Hershman DL. A randomized phase II trial of fulvestrant with or without ribociclib after progression on aromatase inhibition plus cyclin-dependent kinase 4/6 inhibition in patients with unresectable or metastatic hormone receptor positive, HER2 negative breast cancer (Maintain trial) [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr OT3-05-09.

Amplification of EGFR and cyclin D1 genes associated with human papillomavirus infection in oral squamous cell carcinoma.

Human papillomavirus (HPV) infection is associated with several genetic alterations including oncogene amplification, leading to increased aggression of tumors. Recently, a relationship between HPV infection and oncogene amplification has been reported, but this finding remains controversial. This study therefore investigated relationships between HPV infection and amplification of genes in the epidermal growth factor receptor (EGFR) signaling cascade in oral squamous cell carcinoma (OSCC). Extracted DNA from 142 formalin-fixed paraffin-embedded (FFPE) OSCC tissues was performed to investigate the copy number of EGFR, KRAS, c-myc and cyclin D1 genes using real-time polymerase chain reaction (RT-PCR) and compared with calibrators. A tissue microarray of OSCC tissues was used for detection of c-Myc expression and HPV infection by immunohistochemistry and HPV E6/E7 RNA in situ hybridization, respectively. HPV infection was also investigated using PCR and RT-PCR. Of the 142 OSCC samples, 81 (57%) were HPV-infected cases. The most frequently amplified gene was c-myc (55.6%), followed by cyclin D1 (26.1%), EGFR (23.9%) and KRAS (19.7%). Amplification of c-myc was significantly associated with levels of its protein product. EGFR amplification was also significantly associated with amplification of genes in the signaling cascade: KRAS (50.0%), c-myc (34.2%) and cyclin D1 (46.0%). Interestingly, HPV infection was significantly associated with amplification of both EGFR (76.5%) and cyclin D1 (73.0%). Only cyclin D1 amplification was significantly associated with severity of OSCC histopathology. HPV infection may play an important synergistic role in amplification of genes in the EGFR signaling cascade, leading to increased aggression in oral malignancies.

A randomized phase II trial of fulvestrant with or without ribociclib after progression on aromatase inhibition plus cyclin-dependent kinase 4/6 inhibition in patients with unresectable or metastatic hormone receptor positive, HER2 negative breast cancer.

TPS1112 Background: CDK4/6i, including palbociclib and ribociclib (R), have demonstrated remarkable benefit in progression free survival (PFS) in patients (pts) with HR+, HER2- MBC with anti-estrogen therapy. Switching between anti-estrogen therapies at disease progression is standard of care in the treatment of HR+ MBC. We evaluate the strategy of switching anti-estrogen therapy to fulvestrant (F) and maintaining CDK4/6 inhibition with R in pts with HR+, HER2- MBC who have progressed on an AI + CDK4/6i. Methods: Trial Design Phase II, multi-center, randomized, double-blind, placebo-controlled trial to evaluate F +/- R in pts with HR+, HER2- MBC who have previously progressed on any AI + CDK4/6i: Screened at 2 different scenarios: Scenario 1: Before receiving any CDK4/6i Scenario 2: Time of progression of disease (POD) while being treated with an AI + CDK4/6i Intervention At randomization, pts assigned 1:1 to either a) F + R or b) F + placebo, with treatment given in 4-week cycles. Major Eligibility Criteria 1, Metastatic BC, 2. HR+ HER2-, 3. Measurable or unmeasurable disease Specific Aims Primary: PFS. Secondary: Objective response rate, clinical benefit rate, overall survival, and duration of response. Biomarker assessment: amplification of cyclin D1 and cyclin E, phosphoRb and TK1 expression, Rb1 and p16 loss, and ctDNA for ESR1 and PIK3CA mutations. Statistical Methods Assuming a median PFS of 3.8 months with F alone, we predict that F + R will lead to a median PFS of at least 6.5 months. A one-sided log-rank test with a sample size of N = 120 and alpha = 0.025, achieves 80% power to detect a difference in PFS of 2.7. With a 10% dropout, n = 132. Clinical trial registry number NCT02632045.

Abstract OT2-01-19: A randomized phase II trial of fulvestrant with or without ribociclib after progression on aromatase inhibition plus cyclin-dependent kinase 4/6 inhibition in patients with unresectable or metastatic hormone receptor positive, HER2 negative breast cancer

Abstract Background Cyclin dependent kinase 4 and 6 inhibitors (CDK4/6i), including palbociclib and ribociclib (R), have demonstrated remarkable benefit in progression free survival (PFS) in patients (pts) with hormone receptor positive (HR+), HER2- metastatic breast cancer (MBC) when combined with anti-estrogen therapy. Switching between anti-estrogen therapies at disease progression is standard of care in the treatment of HR+ MBC. We evaluate the strategy of switching anti-estrogen therapy to fulvestrant (F) and maintaining CDK4/6 inhibition with R in pts with HR+, HER2- MBC who have progressed on an aromatase inhibitor (AI) + CDK4/6i. Trial Design Phase II, multi-center, randomized, double-blind, placebo-controlled trial to evaluate F +/- R in pts with HR+, HER2- MBC who have previously progressed on any AI + CDK4/6i. Pts can be screened and registered at two different time points: Scenario 1: Before receiving any CDK4/6i Scenario 2: At the time of progression of disease (POD) while being treated with an AI + CDK4/6i In scenario 1, the study will provide pts with letrozole + R, but pts will not be randomized until they demonstrate POD. At randomization, pts will be assigned 1:1 to either a) F + R or b) F + placebo, with treatment given in 4-week cycles. F will be given as a 500 mg dose intramuscularly every 2 weeks for 3 times and then every 4 weeks, as per standard of care. R or placebo will be given orally at 600 mg daily, 3 weeks on/1 week off. CT scans and bone scan are to be performed prior to every third cycle. Serum and whole blood samples and optional tissue biopsies for biomarker assessment will be performed prior to study treatment (scenario 1), prior to randomization to R +/- F, and when the patient goes off study. Main Eligibility Criteria: 1. Age ≥ 18 years with unresectable or metastatic BC 2. Estrogen and/or progesterone receptor positive, HER2 negative, as per ASCO-CAP 3. Postmenopausal status or receiving ovarian suppression 4. Measurable or unmeasurable disease; stable CNS disease allowed 5. No clinically significant cardiac disease 6. No concomitant CYP3A4/5 inducer or inhibitor Specific Aims Primary: Progression free survival (PFS), defined as the time from randomization to POD or death. Secondary: Objective response rate (ORR), clinical benefit rate (CBR = ORR + stable disease rate), overall survival (OS), and duration of response. Pts in scenario 1 will also be assessed for PFS, OS, CBR, and safety while receiving AI + R (pre-randomization). Biomarker assessment will include amplification of cyclin D1 and cyclin E, phosphoRb and TK1 expression, Rb1 and p16 loss, and ctDNA for ESR1 and PIK3CA mutations. Target Accrual 132 pts accrued from 11 academic medical centers in the U.S, with a goal of completing accrual in two years (∼60 to 72 pts in each scenario). Statistical Methods Assuming a median PFS of 3.8 months with F alone, we predict that F + R will lead to a median PFS of at least 6.5 months. A one-sided log-rank test with a sample size of N=120 and alpha=0.025, achieves 80% power to detect a difference in PFS of 2.7 months. N=132 pts allows for a 10% drop-out rate. Citation Format: Mundi PS, Codruta C, Accordino MK, Sparano J, Andreopoulou E, Vadhat LT, Tiersten A, Esteva F, O'Regan R, Jain S, Mayer I, Forero A, Crew KD, Hershman DL, Kalinsky KM. A randomized phase II trial of fulvestrant with or without ribociclib after progression on aromatase inhibition plus cyclin-dependent kinase 4/6 inhibition in patients with unresectable or metastatic hormone receptor positive, HER2 negative breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr OT2-01-19.

Evaluation of Cyclin D1 expression in Head and Neck Squamous Cell Carcinoma.

Squamous Cell Carcinoma (SCC) is an aggressive epithelial malignancy of the upper aero digestive tract and comprises 90% of all Head and Neck Squamous Cell Carcinoma (HNSCC). It is the sixth leading cancer worldwide with approximately 600,000 cases reported annually. It is one of the most common cancers in India. Tumour Lymph Node and Metastases (TNM) staging has been the most useful indicator to predict prognosis in HNSCC but recently various biomolecular markers have potentially offered new methods for early diagnosis and treatment alternatives for HNSCC patients; one amongst them being cyclin D1. This study has been undertaken to evaluate expression of cyclin D1 in HNSCC cases and to find out its association with various pathological prognostic factors. A 48 formalin fixed paraffin embedded tumour sections, stained with Haematoxylin & Eosin were graded and staged. Immunohistochemistry for cyclin D1 was evaluated as Extent Score (ES), Intensity Score (IS) and Total Score (TS) was calculated. All the relevant data collected was transferred on to the excel sheet. Chi square test with and without Yate's correction was used to compare various parameters. The p-value ≤ 0.05 was taken as critical level of significance. A significant association was seen between TS of Cyclin D1 expression with tumour stage and with lymph node metastasis but not with grade. Higher Cyclin D1 expression is associated with higher tumour stage and lymph node metastasis. Therefore, there is value of analysing cyclin D1 amplification and expression, for prognostic evaluation. This may also be further used for targeted therapy in head and neck cancers.

Amplification and protein overexpression of cyclin D1: Predictor of occult nodal metastasis in early oral cancer.

Accurate nodal staging is pivotal for treatment planning in early (stage I-II) oral cancer. Unfortunately, current imaging modalities lack sensitivity to detect occult nodal metastases. Chromosomal region 11q13, including genes CCND1, Fas-associated death domain (FADD), and CTTN, is often amplified in oral cancer with nodal metastases. However, evidence in predicting occult nodal metastases is limited. In 158 patients with early tongue and floor of mouth (FOM) squamous cell carcinomas, both CCND1 amplification and cyclin D1, FADD, and cortactin protein expression were correlated with occult nodal metastases. CCND1 amplification and cyclin D1 expression correlated with occult nodal metastases. Cyclin D1 expression was validated in an independent multicenter cohort, confirming the correlation with occult nodal metastases in early FOM cancers. Cyclin D1 is a predictive biomarker for occult nodal metastases in early FOM cancers. Prospective research on biopsy material should confirm these results before implementing its use in routine clinical practice. © 2016 Wiley Periodicals, Inc. Head Neck 39: 326-333, 2017.

Alterations in the cell cycle checkpoint pathway in breast cancer.

126 Background: Breast cancer is a molecularly diverse disease with molecular subtypes defined by ER, PR and HER2 status. Dysregulation of the cell cycle pathway through activation of cyclin D1 and/or inactivation of CDKN2A is a mechanism of tumor progression in breast cancer and drugs targeting CDK4/6 is undergoing investigation in this disease with promising preliminary results. We investigated several key genetic alterations in cell cycle regulatory proteins in advanced breast cancer patient samples to identify potential targets for cell cycle related treatment options. Methods: A comprehensive genomic profiling was performed on 37 breast cancer samples with the majority being metastatic. Testing included targeted exome sequencing of 562 genes in tumor and paired normal DNA. Results: Patients’ ages ranged from 27-66 years. Analysis of cell cycle regulatory genes revealed focal amplification of cyclin D1/D2 or CDKN2A loss in 9/37 (24%) of cases, of which, 45% were hormone receptor (HR) positive, HER2 negative, 33% were HR positive, HER2 positive and 22% were triple negative (TNBC) subtype. Loss of RB1 was analyzed since it negatively regulates response to CDK4/6 inhibitors. Loss of RB1 was noted in 4/37 (10.8%) of cases, all of which were TNBCs. Only a single TNBC with RB1 loss had concurrent CDK4 amplification and loss of CDKN2A. No evidence of CCNE1 amplification was found in any samples. Incidence of CDK4 and CDK6 amplification was found in samples with cyclin D1 amplification or CDKN2A loss. Our cohort included a single male TNBC patient who harbored cyclin D1 amplification as well as CDKN2A loss. Overall, amplification of cyclin D1, cyclin D2, CDK4 and CDK6 was found in 16%, 5.4%, 2.7% and 2.7% respectively. Loss of CDKN2A was noted in 5.4% of tumors profiled. Conclusions: Exome sequencing identified subsets of patients with mutated cell cycle associated genes that may benefit from CDK 4/6 inhibitors. Our data suggests loss of RB1 is more frequent in TNBC subtype which may confer resistance to CDK4/6 inhibitors. Our analysis suggests that in addition to HR positive, HER2 negative patients, a subset of HR positive, HER2 positive patients could be sensitive to CDK4/6 inhibitors due to activation of the cell cycle pathway as evidenced by mutations in key genes.

Abstract 502: Copy number alterations identify targeted therapies in preclinical models of osteosarcoma

Abstract Osteosarcoma (OS) is an aggressive pediatric bone malignancy that affects 400 children per year in the United States. It has one of the lowest 5 yr. survival rates of any pediatric cancer. OS is characterized by genomic instability and numerous copy number alterations (CNA). We hypothesized that CNA may be drivers of OS and that targeting cancer genes with CNAs may be a novel approach for treatment of OS. We have established a robust approach for generation of patient-derived xenografts (PDX) from patients with OS. To date, 12 PDX have been established from OS patients. Using whole-genome sequencing (WGS), we have identified possible target CNAs for individual patients and are currently testing them in their corresponding PDX. In one patient, an amplification in c-myc suggested possible susceptibility to the bromodomain inhibitor JQ1. In a second patient sample, amplification of Cyclin D1 in the setting of p16 loss and wild-type Rb suggested responsiveness to a CDK4/6 specific inhibitor. This patient sample also has an Aurora kinase B amplification, suggesting susceptibility to inhibition of this kinase as a therapeutic approach. We treated the corresponding PDX with the drug predicted to target the patient sample based on CNA. In one case, JQ1 treatment inhibited tumor growth in the PDX generated from the patient sample with c-myc amplification. In the second case, Aurora kinase B inhibition arrested tumor growth in the PDX harboring the Aurora kinase B amplification. We are currently testing whether CDK4/6 inhibition is sufficient to inhibit tumor growth as predicted by WGS. As a follow up we are currently submitting several more patient samples and paired PDX for WGS as well as RNAseq to determine other potential targeted therapeutics based on CNA. In summary, our work suggests that WGS can be used to identify potential novel therapies for patients with OS. Further work will be needed to determine whether response in PDX models correlates with clinical response in patients. Citation Format: Leanne C. Sayles, Marcus Breese, Alejandro Sweet-Cordero. Copy number alterations identify targeted therapies in preclinical models of osteosarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 502. doi:10.1158/1538-7445.AM2015-502

Abstract 703: A novel ATP-competitive MEK/Aurora kinase inhibitor BI-847325 reverses acquired BRAF inhibitor resistance through suppression of Mcl-1 and inhibition of MEK expression

Abstract BACKGROUND: BRAF/MEK inhibitors have shown promising levels of response in melanomas harboring the BRAF V600E mutation, however responses tend to be short-lived and resistance is a major clinical problem. In the current study, we evaluated the pharmacological activity of BI847325, a dual inhibitor of MEK and Aurora kinases against multiple melanoma cell line models. METHODS: The cytotoxic effect of BI847325 was evaluated in vitro in a panel of selected BRAF-mutant vemurafenib resistant cell lines by Alamar blue and Annexin V binding assay. 3D spheroid model systems and colony formation assays demonstrated the long-term growth inhibitory effect of BI847325. Western blot analysis and qRT-PCR studies were carried out to evaluate the mechanism underlying BI5-mediated cytotoxicity. In vivo studies in Balb SCID mice were performed to assess the suppression of BRAF-mutant xenografts on treatment with BI847325. RESULTS: BI847325 potently reduced the growth and survival of BRAF-mutant melanoma cell lines with acquired and intrinsic BRAF inhibitor resistance (NRAS mutations, BRAF splice forms, Cyclin D1 amplification, RTK upregulation, PTEN loss, COT amplification). We confirmed that BI847325 induced apoptosis through decrease in Mcl-1 mRNA and protein expression and increase in BIM expression. The effects of BI847325 upon BIM and Mcl-1 expression could not be mimicked by the combination of other MEK and aurora kinase inhibitors. For the first time we demonstrated that BI847325 reverses the acquired vemurafenib resistance by inhibiting MEK expression at mRNA and protein level. A strong suppression of MEK expression was observed without recovery following 72 h of washout. In vivo studies revealed complete tumor suppression with no recurrence over a period of 65 days of treatment with 70mg/kg/week dose of BI847325. In contrast, treatment with vemurafenib analog PLX4720 in the same mouse model was associated with tumor relapse after 30 days of treatment. BI847325 also successfully suppressed the long-term growth of xenografts with acquired vemurafenib resistance. Analysis of tumor samples complied with in vitro results demonstrating inhibition of phospho-ERK, phospho-Histone3, Mcl-1 and total MEK. CONCLUSION: In conclusion we report for the first time that BI847325, a novel ATP-competitive MEK/ Aurora kinase inhibitor effectively inhibits BRAF-mutant melanoma and overcomes vemurafenib-resistance by decreasing expression of MEK and Mcl-1; in vitro and in vivo. Further preclinical and clinical investigations towards this would open new avenues in treatment of melanoma. Citation Format: Manali S. Phadke, Patrizia Sini, Keiran Smalley. A novel ATP-competitive MEK/Aurora kinase inhibitor BI-847325 reverses acquired BRAF inhibitor resistance through suppression of Mcl-1 and inhibition of MEK expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 703. doi:10.1158/1538-7445.AM2015-703

Comprehensive analysis of driver mutations in Chinese squamous cell lung carcinomas by targeted next-generation sequencing.

7544 Background: The aim of the study was to analyze the driver mutation profiles in a large cohort of Chinese SqCLCs to identify potential therapeutic targets. Methods: We detected approximately 2,800 COSMIC mutations from 50 oncogenes and tumor suppressor genes on 159 samples by using Ion Torrent semiconductor-based next-generation sequencing.We conducted FISH for fibroblast growth factor receptor 1 (FGFR1) and cyclin D1 (CCND1) amplification on 172 and 246 samples, respectively. In addtion, we analyzed PTEN expression by immunohistochemistry on 172 samples. Results: Somatic mutations were detected in 73.6% (117/159) of patients. The most commonly mutated gene detected in this study was TP53 (89 cases, 56.0%), followed by CDKN2A (14 cases, 8.8%), PI3KCA (14 cases, 8.8%), KRAS (7 cases, 4.4%), and EGFR (5 cases, 3.1%). The incidences of FGFR1 amplification, CCND1 amplification and loss of PTEN expression in this cohort of patients were 16.9% (29/172), 11% (27/246) and 43.6% (75/172), respectively. The fr...