CDK4 Amplification Research Articles

Cell-Free DNA from Ascites and Pleural Effusions: Molecular Insights into Genomic Aberrations and Disease Biology

Collection of cell-free DNA (cfDNA) from the blood of individuals with cancer has permitted noninvasive tumor genome analysis. Detection and characterization of cfDNA in ascites and pleural effusions have not yet been reported. Herein, we analyzed cfDNA in the ascites and pleural effusions from six individuals with metastatic cancer. In all cases, cfDNA copy number variations (CNV) were discovered within the effusate. One individual had a relevant alteration with a high copy amplification in EGFR in a never smoker with lung cancer, who showed only MDM2 and CDK4 amplification in a prior tissue biopsy. Another subject with metastatic breast cancer had cytology-positive ascites and an activating PIK3CA mutation identified in the tissue, blood, and ascites collectively. This individual had tumor regression after the administration of the mTOR inhibitor everolimus and had evidence of chromotripsis from chromosomal rearrangements noted in the cell-free ascitic fluid. These results indicate that cfDNA from ascites and pleural effusions may provide additional information not detected with tumor and plasma cell-free DNA molecular characterization, and a context for important insights into tumor biology and clonal dynamic change within primary tumor and metastatic deposits. Mol Cancer Ther; 16(5); 948-55. ©2017 AACR.

Polμ deficiency induces moderate shortening of P53−/− mouse lifespan and modifies tumor spectrum

Non-homologous end joining (NHEJ) is the main mechanism for double strand break (DSB) DNA repair. The error-prone DNA polymerase mu (Polμ) is involved in immunoglobulin variable region rearrangement and in general, NHEJ in non-lymphoid cells. Deletion of NHEJ factors in P53−/− mice, which are highly prone to development of T cell lymphoma, generally increases cancer incidence and shifts the tumor spectrum towards aggressive pro-B lymphoma. In contrast, Polμ deletion increased sarcoma incidence, proportionally reducing pro-B lymphoma development on the P53-deficient background. Array comparative genomic hybridization (aCGH) analyses showed DNA copy number alterations in both P53−/− and Polμ−/−P53−/− lymphomas. Our results also indicate that the increase in sarcoma incidence in Polμ−/−P53−/− mice could be associated with Cdk4 and Kub3 amplification and overexpression. These results identify a role for Polμ in the prevention of sarcomagenesis on a murine P53-deficient background, in contrast to most other NHEJ factors.

The genomic dynamics during progression of lung adenocarcinomas.

Intra-tumor heterogeneity is a big barrier to precision medicine. To explore the underlying clonal diversity in lung adenocarcinomas, we selected nine individuals with whole-genome sequencing data from primary and matched metastatic tumors as a cohort for study. Similar global pattern of arm-level copy number changes and large variations of somatic single-nucleotide variant between the primary and metastasis are observed in the majority of cases. Importantly, we found breakage-fusion-bridge (BFB) cycles acting as an important mechanism for underlying cancer gene amplification, such as amplification of CDK4, CDKN3 and FGFR1 in early stage. We also identified recurrent focal amplification of gene CCNY derived from BFB in two metastatic tumors, but not in primary tumor. Clonal analysis of case 236T demonstrated that mutational processes are varying with tumor progression. Collectively, our data provide new insights into genetic diversity and potential therapeutic target in lung adenocarcinoma.

Recent translational research into targeted therapy for liposarcoma

Liposarcomas (LPS) are among the most common soft tissue sarcomas, originating from adipocytes. Treatment for LPS typically involves surgical resection and radiation therapy, while the use of conventional cytotoxic chemotherapy for unresectable or metastatic LPS remains controversial. This review summarizes the results of recent translational research and trials of novel therapies targeting various genetic and molecular aberrations in different subtypes of LPS. Genetic aberrations such as the 12q13-15 amplicon, genetic amplification of MDM2, CDK4, TOP2A, PTK7, and CHEK1, point mutations in CTNNB1, CDH1, FBXW7, and EPHA1, as the fusion of FUS-DDIT3/EWSR1-DDIT3 are involved in the pathogenesis LPS and represent potential therapeutic candidates. Tyrosine kinase inhibitors targeting MET, AXL, IGF1R, EGFR, VEGFR2, PDGFR-β and Aurora kinase are effective in certain types of LPS. Abnormalities in the PI3K/Akt signaling pathway deregulation of C/EBP-α and its partner PPAR-γ, and the interaction between calreticulin (CRT) and CD47 are also promising therapeutic targets. These promising new approaches may help to supplement existing treatments for LPS.

Abstract PD1-07: Comprehensive characterization of matched pre-treatment biopsies and residual disease of doxorubicin treated breast cancer

Abstract Background Neoadjuvant chemotherapy is standard of care for locally advanced breast cancer. Unfortunately not all patients benefit from this treatment. Even after decades of research, we still cannot predict which tumor will or will not respond. This may in part be due to tumor heterogeneity, as the sample taken before treatment not necessarily represents the tumor cell population that causes therapy resistance. Methods To test this hypothesis, we collected pre-treatment biopsies, resection specimens, and matched blood from 21 breast cancer patients treated with doxorubicin and cyclophosphamide in a neoadjuvant setting. Specifically, tumors were selected with a tumor percentage >50% after chemotherapy to enrich for resistant samples and ensure high quality data. RNA and whole exome sequencing were performed to characterize somatic mutations, copy number alterations and gene expression profiles. Histopathological characteristics were determined to obtain a comprehensive profile of all tumor samples. Results The comparisons of somatic variants and copy number alterations revealed a very diverse image: in several cases, high-level amplifications, large genomic gains or losses, and mutations in known oncogenes or tumor suppressors such as MAP3K1 and RUNX1 were either lost or gained during treatment, while in other cases no such changes were detected. We observed a remarkable number of genetic alterations involved in cell cycle progression and DNA damage checkpoints, including amplification of MDM2, CCND1 and CDK4, and copy number loss or mutations in CDKN1B and ATM. Strikingly, both cases of CDKN1B loss were identified in pre-treatment biopsies and no longer detectable in the surgery specimen. In contrast, CCND1, CDK4 and MDM2 amplifications were retained, although CCND1 expression decreased significantly in CCND1 amplified tumors. In addition, eighty percent of tumors showed a decreased cell proliferation after chemotherapy, where the high-proliferative ER+ (Luminal B) tumors were most strongly affected. This trend was also visible in a validation cohort of 94 ER+ samples, but the prognosis of Luminal B tumors that showed a decrease in proliferation was still significantly worse than that of Luminal A tumors that did not show an altered proliferation rate. Conclusion Our results confirm that biologically relevant genomic alterations can differ between pre- and post-treatment samples, which greatly impacts biomarker discovery. In addition, our findings emphasize the chemotherapy insensitivity of CCND1 amplified ER+ breast cancers, and stress the need for better treatment regimens for these patients. In contrast, genomic loss of CDKN1B may be a marker for sensitivity to doxorubicin. Citation Format: Lips EH, Hoogstraat M, Mulder L, Nederlof PM, Sonke GS, Rodenhuis S, Wesseling J, Wessels LFA. Comprehensive characterization of matched pre-treatment biopsies and residual disease of doxorubicin treated 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 PD1-07.

CDK4/6 Inhibition in Breast Cancer: Mechanisms of Response and Treatment Failure.

To describe the role of D-type cyclins and CDKs 4 and 6 in breast cancer, and to discuss potential biomarkers for sensitivity or resistance to CDK4/6 inhibitors. A small number of preclinical and clinical studies have explored potential mechanisms of CDK4/6 inhibitor response and resistance in breast cancer. Putative markers of response include ER-positivity, luminal patterns of gene expression, high cyclin D1 levels, and low p16 levels. Possible resistance mechanisms include loss of Rb function, overexpression/amplification of cyclin E, and CDK6 amplification. Most these remain speculative and have not been validated in clinical specimens. If early successes with CDK4/6 inhibitors are to be capitalized upon, it is critical that our understanding of CDK4/6 biology in breast cancer extends beyond its current rudimentary state. Only then we will be able to develop rational therapeutic combinations that further enhance the efficacy of these agents.

Frequent amplification of receptor tyrosine kinase genes in welldifferentiated/ dedifferentiated liposarcoma.

Well-differentiated liposarcoma (WDLPS) and dedifferentiated liposarcoma (DDLPS) are closely related tumors commonly characterized by MDM2/CDK4 gene amplification, and lack clinically effective treatment options when inoperable. To identify novel therapeutic targets, we performed targeted genomic sequencing analysis of 19 WDLPS and 37 DDLPS tumor samples using a panel of 104 cancer-related genes (NCC oncopanel v3) developed specifically for genomic testing to select suitable molecular targeted therapies. The results of this analysis indicated that these sarcomas had very few gene mutations and a high frequency of amplifications of not only MDM2 and CDK4 but also other genes. Potential driver mutations were found in only six (11%) samples; however, gene amplification events (other than MDM2 and CDK4 amplification) were identified in 30 (54%) samples. Receptor tyrosine kinase (RTK) genes in particular were amplified in 18 (32%) samples. In addition, growth of a WDLPS cell line with IGF1R amplification was suppressed by simultaneous inhibition of CDK4 and IGF1R, using palbociclib and NVP-AEW541, respectively. Combination therapy with CDK4 and RTK inhibitors may be an effective therapeutic option for WDLPS/DDLPS patients with RTK gene amplification.

PathTiMEx: Joint Inference of Mutually Exclusive Cancer Pathways and Their Progression Dynamics.

High-throughput sequencing technologies have facilitated the generation of an unprecedented amount of genomic cancer data, opening the way to a more profound understanding of tumorigenesis. In this endeavor, two fundamental questions have emerged, namely (1) which alterations drive tumor progression and (2) in which order do they occur? Answering these questions is crucial for therapeutic decisions involving targeted agents. Because of interpatient heterogeneity, progression at the level of pathways is more reproducible than progression at the level of single genes. In this study, we introduce pathTiMEx, a generative probabilistic graphical model that describes tumor progression as a partially ordered set of mutually exclusive driver pathways. pathTiMEx employs a stochastic optimization procedure to jointly optimize the assignment of genes to pathways and the evolutionary order constraints among pathways. On real cancer data, pathTiMEx recapitulates previous knowledge on tumorigenesis, such as the temporal order among pathways which include APC, KRAS, and TP53 in colorectal cancer, while also proposing new biological hypotheses, such as the existence of a single early causal event consisting of the amplification of CDK4 and the deletion of CDKN2A in glioblastoma. pathTiMEx is available as an R package.

Gene amplification of CCNE1, CCND1, and CDK6 in gastric cancers detected by multiplex ligation-dependent probe amplification and fluorescence in situ hybridization

New and effective treatments for advanced gastric cancer are urgently needed. Cyclins E and D1 form a complex with cyclin-dependent kinase 2, 4, or 6 to regulate G1-S transition. The G1-S regulatory genes encoding cyclin E (CCNE1), cyclin D1 (CCND1), and CDK6 (CDK6) are frequently amplified in gastric cancer and may therefore influence molecularly targeted therapies against ERBB2 or EGFR when coamplified. A total of 179 formalin-fixed and paraffin-embedded gastric cancer specimens were examined for these gene amplifications by multiplex ligation-dependent probe amplification and fluorescence in situ hybridization. Amplification of at least 1 G1-S regulatory gene was found in 35 tumors (CCNE1 amplification, 15% of samples; CCND1, 6%; CDK6, 1%). In 13 of the 35 tumors, dual-color fluorescence in situ hybridization identified coamplification of the G1-S regulatory genes with ERBB2, EGFR, and/or KRAS in single cancer nuclei. The observation that cells with G1-S regulatory gene amplification contained clonal subpopulations with coamplification of ERBB2, EGFR, or KRAS in 5 early and 3 advanced cancers suggests that amplification of the G1-S regulatory genes represents an early event, which precedes ERBB2, EGFR, or KRAS amplification. Amplified CCNE1, CCND1, and CDK6 in advanced gastric cancer may be potentially useful as direct targets for molecular therapy or for combination therapy with ERBB2 or EGFR inhibitors. Multiplex ligation-dependent probe amplification could be a useful tool for identification of patients who would benefit from such therapies.

Acquired CDK6 amplification promotes breast cancer resistance to CDK4/6 inhibitors and loss of ER signaling and dependence

Dysregulated activation of the CDK4/6 kinases is a hallmark of most mammary-derived carcinomas. ATP-competitive inhibitors against this complex have been recently advanced in the clinic and have shown significant activity, particularly against tumors driven by the estrogen receptor (ER). However, resistance to these compounds has begun to emerge often months to years after their initiation. We investigated potential mechanisms of resistance using cell line models that are highly sensitive to this class of drugs. After prolonged exposure to the selective and potent CDK4/6 inhibitor LY2835219, clones emerged and several were found to harbor amplification of the CDK6 kinase. Amplification of CDK6 resulted in a marked increase in CDK6 expression and reduced response of the CDK4/6 target, phospho-Rb (pRb), to CDK4/6 inhibitors. Knockdown of CDK6 restored drug sensitivity, while enforced overexpression of CDK6 was sufficient to mediate drug resistance. Not only did CDK6 overexpression mediate resistance to CDK4/6 inhibitors but it also led to reduced expression of the ER and progesterone receptor (PR), and diminished responsiveness to ER antagonism. The reduced ER/PR expression after CDK4/6 inhibitor resistance was additionally observed in tumor biopsy specimens from patients treated with these drugs. Alternative mechanisms of resistance to CDK4/6 inhibitors such as loss of pRb and cyclin E1 overexpression also exhibited decreased hormone responsiveness, suggesting that the clinical paradigm of sequential endocrine-based therapy may be ineffective in some settings of acquired CDK4/6 resistance.

MDM2 and CDK4 amplifications are rare events in salivary duct carcinomas.

Salivary duct carcinoma (SDC) is an aggressive adenocarcinoma of the salivary glands associated with poor clinical outcome. SDCs are known to carry TP53 mutations in about 50%, however, only little is known about alternative pathogenic mechanisms within the p53 regulatory network. Particularly, data on alterations of the oncogenes MDM2 and CDK4 located in the chromosomal region 12q13-15 are limited in SDC, while genomic rearrangements of the adjacent HMGA2 gene locus are well documented in subsets of SDCs. We here analyzed the mutational status of the TP53 gene, genomic amplification of MDM2, CDK4 and HMGA2 rearrangement/amplification as well as protein expression of TP53 (p53), MDM2 and CDK4 in 51 de novo and ex pleomorphic adenoma SDCs.25 of 51 cases were found to carry TP53 mutations, associated with extreme positive immunohistochemical p53 staining levels in 13 cases. Three out of 51 tumors had an MDM2 amplification, one of them coinciding with a CDK4 amplification and two with a HMGA2 rearrangement/amplification. Two of the MDM2 amplifications occurred in the setting of a TP53 mutation. Two out of 51 cases showed a CDK4 amplification, one synchronously being MDM2 amplified and the other one displaying concurrent low copy number increases of both, MDM2 and HMGA2.In summary, we here show that subgroups of SDCs display genomic amplifications of MDM2 and/or CDK4, partly in association with TP53 mutations and rearrangement/amplification of HMGA2. Further research is necessary to clarify the role of chromosomal region 12q13-15 alterations in SDC tumorigenesis and their potential prognostic and therapeutic relevance.

Differential diagnosis of atypical lipomatous tumor/well-differentiated liposarcoma and dedifferentiated liposarcoma: utility of p16 in combination with MDM2 and CDK4 immunohistochemistry

The differential diagnosis between atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDLPS) and dedifferentiated liposarcoma (DDLPS) from their morphologic counterparts is challenging. Currently, the diagnosis is guided by MDM2 and CDK4 immunohistochemistry (IHC) and is confirmed by the amplification of the corresponding genes. Recently, p16 IHC has been proposed as a useful diagnostic biomarker. The objective was to assess the utility of p16 IHC in the differential diagnosis of ALT/WDLPS and DDLPS. Our series included 101 tumors that were previously analyzed using fluorescence in situ hybridization for MDM2 and CDK4 amplification. We compared sensitivity and specificity of p16 IHC to MDM2 and CDK4 IHC in the differential diagnosis of ALT-WDLPS (n=19) versus benign adipocytic tumors (n=44) and DDLPS (n=18) versus mimicking sarcomas (n=20). In the differential diagnosis of ALT-WDLPS, p16 had a sensitivity of 89.5% but a specificity of 68.2%, which was impaired by false-positive lipomas with secondary changes, especially in biopsies. Likewise, in the differential diagnosis of DDLPS, p16 had a sensitivity of 94.4% and a specificity of 70%, which hampered its use as a single marker. However, adding p16 to MDM2 and/or CDK4 increased diagnostic specificity. Indeed, MDM2+/p16+ tumors were all ALT-WDLPS, and MDM2-/p16- tumors were all benign adipocytic tumors. Moreover, all MDM2+/CDK4+/p16+ tumors were DDLPS, and the MDM2-/CDK4-/p16- tumor was an undifferentiated sarcoma. Although the use of p16 as a single immunohistochemical marker is limited by its specificity, its combination with MDM2 and CDK4 IHC may help discriminate ALT-WDLPS/DDLPS.

Genomewide copy number analysis of Müllerian adenosarcoma identified chromosomal instability in the aggressive subgroup

AbstractMüllerian adenosarcomas are malignant gynecologic neoplasms. Advanced staging and sarcomatous overgrowth predict poor prognosis. Because the genomic landscape remains poorly understood, we conducted this study to characterize the genomewide copy number variations in adenosarcomas. Sixteen tumors, including eight with and eight without sarcomatous overgrowth, were subjected to a molecular inversion probe array analysis. Copy number variations, particularly losses, were significantly higher in cases with sarcomatous overgrowth. Frequent gains of chromosomal 12q were noted, often involving cancer-associated genes CDK4 (six cases), MDM2, CPM, YEATS4, DDIT3, GLI1 (five each), HMGA2 and STAT6 (four), without association with sarcomatous overgrowth status. The most frequent losses involved chromosomes 13q (five cases), 9p, 16q and 17q (four cases each) and were almost limited to cases with sarcomatous overgrowth. MDM2 and CDK4 amplification, as well as losses of RB1 (observed in two cases) and CDKN2A/B (one case), was verified by FISH. By immunohistochemistry, all MDM2/CDK4-coamplified cases were confirmed to overexpress both encoded proteins, whereas all four cases with (plus an additional four without) gain of HMGA2 overexpressed the HMGA2 protein. Both cases with RB1 loss were negative for the immunostaining of the encoded protein. Chromothripsis-like copy number profiles involving chromosome 12 or 14 were observed in three fatal cases, all of which harbored sarcomatous overgrowth. With whole chromosome painting and deconvolution fluorescent microscopy, dividing tumor cells in all three cases were shown to have scattered extrachromosomal materials derived from chromosomes involved by chromothripsis, suggesting that this phenomenon may serve as visual evidence for chromothripsis in paraffin tissue. In conclusion, we identified frequent chromosome 12q amplifications, including loci containing potential pharmacological targets. Global chromosomal instability and chromothripsis were more frequent in cases with sarcomatous overgrowth. To our knowledge, this is the first time that evidence of chromothripsis has been demonstrated in paraffin-embedded clinical tissues and in adenosarcomas.

Abstract 2435: Amplification of CDK4 and MDM2 is associated with atypical clinical features in high risk neuroblastoma patients

Abstract MYCN-amplification and 11q-deletion are important, although incomplete, markers of high-risk neuroblastoma. Thus, characterization of additional genomic alterations that can be used as prognostic and/or predictive markers is of clinical importance in order to provide best treatment possible. By using SNP-microarrays we identified a small group of neuroblastomas with high grade amplification of one or multiple loci on 12q, commonly involving the potential oncogenic target genes CKD4 (12q13-14) and/or MDM2 (12q15). The CDK4 and MDM2 regions were co-amplified in 13/16 samples, two tumors had CDK4-amplification in absence of MDM2-amplification while one tumor had MDM2-amplification without CDK4-amplification. Exome sequencing was performed on seven tumors and whole genome sequencing (WGS) was performed on tumor and constitutional DNA from one patient with 12q-amplification. No novel protein altering SNVs were detected within the amplified regions except a rare INHBE mutation in one patient. The tumor examined with WGS show high degree of structural rearrangements including chromothriptic features on chromosome 12 leading to high grade amplification of CDK4 and MDM2. This sample displayed 21 somatic protein changing alterations although not in any gene with known function in chromatin stability or DNA repair. Interestingly, the majority of the 12q-amplified neuroblastomas were of abdominal origin, some with renal location with initial suspicion of Wilms’ tumor. Atypical metastatic pattern were also seen in this patient group showing low degree of bone marrow involvement favoring other metastatic sites such as lung. The consistent co-amplification of two separate chromosome 12 regions in this subset of neuroblastoma suggests that there are one or more genes with importance in tumor development/progression. Our study indicates that CDK4 appears as main target in this 12q-amplified neuroblastoma subgroup although other genes such as MDM2 and FRS2 also could provide proliferative advantages. The 12q-amplified neuroblastomas exhibit distinct clinical features and may benefit from targeted therapy using a small molecule CDK4/CDK6 inhibitor such as LEE011 (Novartis). Citation Format: Susanne M. Fransson, Hanna Kryh, Niloufar Javanmardi, Inge M. Ambros, Ana P. Berbegall, Ingrid Ora, Rosa Noguera, Jurate Asmundsson, Bengt Sandstedt, Peter F. Ambros, Per Kogner, Tommy Martinsson. Amplification of CDK4 and MDM2 is associated with atypical clinical features in high risk neuroblastoma patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2435.

Abstract 2430: Neuroblastoma: Telomere elongation is responsible for aggressive behavior

Abstract Neuroblastoma (NB) is a highly malignant pediatric tumor of the sympathetic nervous system that commonly displays low overall mutation frequency. We searched for new structural rearrangements in a series of 275 NBs of all stages using high density SNP microarrays. Exome sequencing was also performed on 15 tumor/constitutional DNA pairs and 25 additional tumors, mainly high-risk NBs, using paired-end sequencing on Illumina instrument. Normalized coverage data were used to generate array comparative genomic hybridization (CGH)-like profiles. Structural rearrangements leading to gain of TERT gene were observed in 15 of 275 neuroblastomas as judged by SNP microarray. 12 out of these 15 tumors with TERT rearrangement belong to 11q-deleted subgroup of NB patients that marks tumors with a poor prognosis. These rearrangements occurred only in high-risk NBs in an almost mutually exclusive fashion with MYCN amplifications, which is a known genetic event in this tumor type. Furthermore, in the small series of NBs analyzed by exome sequencing, we detect extensive structural rearrangements of chromosome 2, 5 and 7 leading to amplification of MYCN, TERT and CDK6 respectively in one tumor. The exome sequencing approach detected at average 14 (range 2-77) somatic protein-changing alterations per tumor. Combining structural and mutational data from the exome sequencing show recurrent alterations in ATRX gene. In our NB cohort we see alterations of ATRX in four non MYCN-amplified tumors: two focal deletions, one nonsense mutation and one deleterious missense mutation. ATRX inactivation results in alternative lengthening of telomeres (ALT). This means that high-risk neuroblastomas cancer cells can preserve their telomeres by ALT or by activation of telomerase reverse transcriptase (encoded by TERT). Alterations in TERT and ATRX were mutually exclusive, which is in agreement with the independent activation by these genes of telomere lengthening. Moreover, three cases with chromothripsis of chromosome 5 with rearrangements affecting the TERT were identified through SNP microarrays. These results identify TERT as at least one of the target genes activated by chromothripsis of chromosome 5. This remodeling of the genomic context likely abrogates transcriptional silencing of TERT in high-risk neuroblastoma and places telomerase activation in the centre of transformation in a large fraction of these tumors. This study identifies recurrent TERT and ATRX rearrangements and telomere lengthening as an important mechanism characterizing high-risk tumors and it supports the pharmacological inhibition of these targets to improve the outcome for this patient group. Citation Format: Niloufar Javanmardi, Susanne Fransson, Rose-Marie Sjöberg, Anna Djos, Per Kogner, Tommy Martinsson. Neuroblastoma: Telomere elongation is responsible for aggressive behavior. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2430.

Abstract 4751: Genetic subtype-specific prognostic significance of genetic alterations in lower-grade gliomas

Abstract BACKGROUND: Lower-grade gliomas (WHO grade II and III gliomas) are usually slowly progressive but rarely curable brain caners, for which optimal therapy is still controversial. Recently, we have delineated a comprehensive picture of genetic alterations in lower-grade gliomas and revealed that they could be clearly classified into three genetic subtypes characterized by distinct genetic and clinical features: IDH1/2 mutated tumors with 1p/19q co-deletion (Type−I), IDH1/2 mutated tumors without 1p/19q co-deletion (Type−II) and IDH1/2 wild-type tumors (Type−III). Median survival of each genetic subtype is 16.4, 7.77, and 2.12 years in Type−I, −II, and −III tumors, respectively. Given a higher layer of inter-tumor heterogeneity inferred from the presence of additional genetic lesions on each subtype, there still remains a possibility that within each subtype, we could find one or more subgroups showing distinct biological behaviors and clinical outcomes. In the current study, we analyzed a large data set of patients with lower-grade gliomas fully genotyped for major genetic lesions to determine the prognostic significance of additional genetic alterations.. METHODS: We analyzed genetic alterations detected by exome and/or targeted sequencing as well as SNP-array karyotyping obtained from clinically well-annotated 724 patients aged ≥18 years with supratentorial lower-grade gliomas from the Japan and The Cancer Genome Atlas (TCGA) cohorts. We performed univariate and multivariate Cox hazard proportional analyses for overall survival; multivariate models incorporated age at the time of surgery (≥60 vs. <60 years old), WHO grade (grade II vs. III), and extent of resection (gross total resection vs. partial resection or biopsy) with stratification of cohorts (Japan or TCGA) and were performed with stepwise selection of variables based on the Akaike information criterion. RESULTS: In the multivariate Cox proportional-hazards regression analysis, we revealed that several genetic alterations have subtype-specific prognostic significance: NOTCH1 mutation in Type-I [hazard ratio (HR) = 2.57, 95% confidence interval (CI) = 1.40-4.70], high-level focal amplification of CDK4 [HR = 9.40, 95% CI = 3.08-28.7], PIK3R1 mutation [HR = 8.13, 95% CI = 2.70-24.52], and loss of chromosome 9p [HR = 1.98, 95% CI = 1.16-3.38] in Type-II, and loss of chromosome 10 [HR = 2.26, 95% CI = 1.28-3.97] in Type-III tumors. In Type-III tumors, WHO grade [HR = 5.79, 95% CI = 2.79-12.0] showed a much higher prognostic value than other prognostic factors. Most of these genetic alterations are components of three major signaling pathways or copy number variations frequently affected in glioblastomas, suggesting a close link of disease progression to glioblastoma pathophysiology. CONCLUSIONS: Subtype-specific genetic lesions can be used to further stratify patients in each genetic subtype for overall survival to improve management of lower-grade gliomas. Citation Format: Kosuke Aoki, Hiromichi Suzuki, Hideo Nakamura, Keitaro Matsuo, Kazuya Motomura, Fumiharu Ohka, Masahiro Mizoguchi, Yasutomo Momii, Yasuhiro Muragaki, Satoru Miyano, Toshihiko Wakabayashi, Atsushi Natsume, Seishi Ogawa. Genetic subtype-specific prognostic significance of genetic alterations in lower-grade gliomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4751.

Abstract 1397: Oncomine focus assay: Simultaneous detection of clinically relevant hotspot mutations, CNVs, and gene fusions in 52 oncogenes relevant to solid tumors

Abstract Introduction: The Oncomine™ Focus Assay (OFA) is aimed to simultaneously detect and report hotspot mutations, Copy Number Variants (CNVs) and gene fusions in 52 oncogenes clinically relevant to solid tumors. With minimal DNA/RNA input, OFA employs AmpliSeq™ library preparation chemistry to enrich target regions for Ion Torrent™ Next Generation Sequencing. Variants are annotated for actionability by Oncomine® Knowledgebase. Here we report an analytical validation of OFA. Methods: DNA and RNA, extracted from the FFPE processed GM12878, was used as the negative control to evaluate the specificity of OFA. A RNA sample containing multiple oncogenic gene fusions and a DNA sample containing multiple hotspot SNV and indels were used as the positive controls. Fresh DNA from cancer cell lines (HCC1143 or NCI-H2122) along with DNA from matched normal cell lines, HorizonDx NGS standards TruQ-1, TruQ-2, several engineered FFPE samples with gene fusions or copy number changes, and 43 clinical FFPE samples of a variety of solid tumor types (non-small cell lung cancer, colorectal cancer, gastrointestinal stromal tumor, ovarian cancer, pancreatic cancer, and melanoma) were used to evaluate the OFA performance. The analysis of the sequencing data was primarily performed with the OFA pipeline integrated with the Oncomine® Knowledgebase. Only the genetic alterations with clinical utility were selected as the final output from the data pipeline. The SBS and indels detected by OFA were confirmed by Sanger sequencing. Any CNVs detected were confirmed by FISH, if possible, and detected fusions were confirmed by RT-PCR or FISH. Results: No clinically relevant genetic alterations were detected from the negative control FFPE-GM12878, indicating the high specificity of the OFA. The LOD for SBS and short indel detection was 5%, as assessed by TruQ-1 and TruQ-2, each containing 9-10 variants. The assay can detect gene fusion RNA present as 0.1%-1% of the total RNA, and gene amplifications are detected with a minimum of 50% tumor cell content. A known 12X CCND1 amplifications in HCC1143 and a 13X MYC amplification in NCI-H2122 were detected. In addition, a 13X EGFR amplification and 11X CDK4 amplification were detected in a lung cancer FFPE sample and confirmed by FISH. The analytical specificity for detection of SBS and indels was 100% (25/25). The analytical specificity for detection of CNV gain and gene fusions, and the assay sensitivity are currently under assessment. Conclusions: These results demonstrate the high sensitivity and specificity of OFA. With as little as 10 ng RNA and 10 ng DNA, OFA provides biomarker analysis of patient FFPE tumor specimens focusing on the detection of genetic alterations that have been targeted by oncology drugs or supported by published clinical evidence. Citation Format: Peng Fang, Zhenyu Yan, Paul Labrousse, Weihua Liu, Jennifer Biroschak, Jennifer Wright, Selby Weeks, Cindy Spittle, Chad Galderisi, Jin Li. Oncomine focus assay: Simultaneous detection of clinically relevant hotspot mutations, CNVs, and gene fusions in 52 oncogenes relevant to solid tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1397.

Preclinical evaluation of potential therapeutic targets in dedifferentiated liposarcoma

Sarcomas are rare cancers with limited treatment options. Patients are generally treated by chemotherapy and/or radiotherapy in combination with surgery, and would benefit from new personalized approaches. In this study we demonstrate the potential of combining personal genomic characterization of patient tumors to identify targetable mutations with in vitro testing of specific drugs in patient-derived cell lines. We have analyzed three metastases from a patient with high-grade metastatic dedifferentiated liposarcoma (DDLPS) by exome and transcriptome sequencing as well as DNA copy number analysis. Genomic aberrations of several potentially targetable genes, including amplification of KITLG and FRS2, in addition to amplification of CDK4 and MDM2, characteristic of this disease, were identified. We evaluated the efficacy of drugs targeting these aberrations or the corresponding signaling pathways in a cell line derived from the patient. Interestingly, the pan-FGFR inhibitor NVP-BGJ398, which targets FGFR upstream of FRS2, strongly inhibited cell proliferation in vitro and induced an accumulation of cells into the G0 phase of the cell cycle. This study indicates that FGFR inhibitors have therapeutic potential in the treatment of DDLPS with amplified FRS2.

BEZ235 (PIK3/mTOR inhibitor) Overcomes Pazopanib Resistance in Patient-Derived Refractory Soft Tissue Sarcoma Cells

BACKGROUND: Although pazopanib treatment has become the standard chemotherapy in salvage setting for metastatic sarcoma patients, most patients progress after pazopanib treatment in 4 to 6 months. After failure to pazopanib, patients have limited options for treatment. Therefore, subsequent therapy in patients who failed to pazopanib is urgently needed and the use of patient derived cells or patient derived tumors for accompanying testing with various pharmacological inhibitors could offer additional treatment options for these patients. METHODS: Patient derived tumor cells were collected from ascites at the time of progression to pazopanib and a 13-drug panel was tested for drug sensitivity. We confirmed the results using in vitro cell viability assay and immunoblot assay. We also performed the genomic profiling of PDX model. RESULTS: The growth of patient derived tumor cells was significantly reduced by exposure to 1.0 μM AZD2014 compared with control (control versus AZD2014, mean growth = 100.0% vs 16.04%, difference = 83.96%, 95% CI = 70.01% to 97.92%, P = .0435). Similarly, 1.0 μM BEZ235 profoundly inhibited tumor cell growth in vitro when compared to control (control versus BEZ235, mean growth = 100.0% vs 7.308%, difference = 92.69%, 95% CI = 78.87% to 106.5%, P < .0001). Despite the presence of CDK4 amplification in the patient-derived tumor cells, LEE011 did not considerably inhibit cell proliferation when compared with control (control vs LEE011, mean growth = 100.0% vs 80.23%, difference = 19.77%, 95% CI = 1.828% to 37.72%, P = .0377). The immunoblot analysis showed that BEZ235 treatment decreased pAKT, pmTOR and pERK whereas AZD2014 decreased only pmTOR. CONCLUSION: Taken together, upregulation of mTOR/AKT pathway in sarcoma patient derived cells was considerably inhibited by the treatment of AZD2014 and BEZ235 with downregulation of AKT pathway (greater extent for BEZ235). These molecules may be considered as treatment option in STS patient who have failed to pazopanib in the context of clinical trials.

Giant cell rich osteosarcoma revisited-diagnostic criteria and histopathologic patterns, Ki67, CDK4, and MDM2 expression, changes in response to bisphosphonate and denosumab treatment.

Defining giant cell-rich osteosarcoma (GCRO) as "an osteosarcoma in which more than 50% of the tumor consists of numerous uniformly distributed osteoclastic giant cells amidst oval or spindle mononuclear cells embedded in a fibrovascular stroma," eight such cases identified among 265 cases of osteosarcoma were analysed. Their age ranges from 11 to 33years, with peak incidence in the second decade and equal sex distribution. Seventy-five percent presented with pain, commonest in the knee, affecting the metaphysis. Most appeared radiologically as well-circumscribed expansile multiloculated osteolytic lesions, and many are displayed periosteal reaction. They showed several distinct histologic patterns: the stromal and giant cell, fibrohistiocytic, aneurysmal-cystic, osteoblastoma-like, and parosteal and fibrous dysplasia-like patterns. Focal subtle lacelike osteoid deposition, permeative infiltration into adjacent native bony trabeculae and over 30% Ki67 proliferative index were characteristic. There was no CDK4 and MDM2 amplification. In those having bisphosphonate and denosumab treatment, there was limited focal necrosis with reduction in the number of giant cells and broad trabecular woven bone formation but no giant osteoclast was seen. Two patients with initial diagnosis of giant cell tumor treated by curettage and local resection pursued aggressive clinical courses, died after 14 and 21months. The others survived 12 to 110months. GCRO accounts for about 3% of all osteosarcomas and apart from its more frequent diaphyseal location and associated normal bone-specific alkaline phosphate levels; it shares with conventional high-grade osteosarcoma the same patient demographics, sites of occurrence, absence of CDK4 and MDM2 amplification, and probably clinical course.