CDK4 Amplification Research Articles

Liposarcoma in the axilla developed from a longstanding lipoma.

Liposarcoma in the axilla developed from a longstanding lipoma.

CDK4 amplification predicts recurrence of well-differentiated liposarcoma of the abdomen.

BackgroundThe absence of CDK4 amplification in liposarcomas is associated with favorable prognosis. We aimed to identify the factors associated with tumor recurrence in patients with well-differentiated (WD) and dedifferentiated (DD) liposarcomas.MethodsFrom 2000 to 2010, surgical resections for 101 WD and DD liposarcomas were performed. Cases in which complete surgical resections with curative intent were carried out were selected. MDM2 and CDK4 gene amplification were analyzed by quantitative real-time polymerase chain reaction (Q-PCR).ResultsThere were 31 WD and 17 DD liposarcomas. Locoregional recurrence was observed in 11 WD and 3 DD liposarcomas. WD liposarcomas showed better patient survival compared to DD liposarcomas (P<0.05). Q-PCR analysis of the liposarcomas revealed the presence of CDK4 amplification in 44 cases (91.7%) and MDM2 amplification in 46 cases (95.8%). WD liposarcomas with recurrence after surgical resection had significantly higher levels of CDK4 amplification compared to those without recurrence (P = 0.041). High level of CDK4 amplification (cases with CDK4 amplification higher than the median 7.54) was associated with poor recurrence-free survival compared to low CDK4 amplification in both univariate (P = 0.012) and multivariate analyses (P = 0.020).ConclusionsLevel of CDK4 amplification determined by Q-PCR was associated with the recurrence of WD liposarcomas after surgical resection.

Fluorescent biosensors for probing CDK4/cyclin D activity and developing non‐ATP pocket Inhibitors for melanoma, lung cancer and lymphoma (972.2)

CDK4/cyclin D kinase constitutes an attractive pharmacological target in lung cancer, melanoma and lymphoma, associated with mutation or amplification of CDK4, cyclin D or p16INK4a, but efforts to develop tools for detection of this kinase in its native environment, as well as selective inhibitors for therapeutic purposes have remained limited.To this aim we have engineered a fluorescent polypeptide biosensor that reports on CDK4/cyclin D activity in a sensitive and continuous fashion in vitro, in living cells and in biopsies and which allows to monitor response to therapeutics in animal tumour models. We have further designed two biosensors which have been applied to identify competitors of essential protein/protein interfaces between CDK4 and Cyclin D, and allosteric inhibitors that perturb the conformational dynamics of CDK4, respectively, by high throughput screening. These studies highlight the importance of fluorescent biosensors for fundamental research, biomedical developments and drug discovery programmes, providing novel and sensitive approaches to monitor cancer‐associated alterations in protein kinase activities and develop non‐ATP pocket inhibitors.Grant Funding Source: Grants to MCM “Chercheuse d’Avenir” Région Languedoc‐Roussillon, ARC and INCA

Comprehensive Genomic Profiling of Relapsed and Metastatic Adenoid Cystic Carcinomas by Next-generation Sequencing Reveals Potential New Routes to Targeted Therapies

We hypothesized that next-generation sequencing could reveal actionable genomic alterations (GAs) and potentially expand treatment options for patients with advanced adenoid cystic carcinoma (ACC). Genomic profiling using next-generation sequencing was performed on hybridization-captured, adapter ligation libraries derived from 28 relapsed and metastatic formalin-fixed paraffin-embedded ACC. The 3230 exons of 182 cancer-related genes and 37 introns of 14 genes frequently rearranged in cancer were fully sequenced using the Illumina HiSeq 2000. All classes of GAs were evaluated. Actionable GAs were defined as those impacting targeted anticancer therapies on the market or in registered clinical trials. A total of 44 GAs were identified in the 28 ACC tumors, with 12 of 28 (42.9%) of tumors harboring at least 1 potentially actionable GA. The most common nonactionable GAs were identified in KD6MA (5 cases; 18%), ARID1A (4 cases; 14%), RUNX1 (2 cases; 7%), and MYC (2 cases; 7%). Actionable GAs included NOTCH1 (3 cases; 11%), MDM2 (2 cases; 7%), PDGFRA (2 cases; 7%), and CDKN2A/B (p16) (2 cases; 7%). Other potentially actionable GAs identified in a single case included: mutations in AKT1, BAP1, EGFR, and PIK3CA, homozygous deletion of FBXW7, and amplifications of CDK4, FGFR1, IGF1R, KDR, KIT, and MCL1. The frequency of GA in ACC is lower than that seen in the more common solid tumors. Comprehensive genomic profiling of ACC can identify actionable GAs in a subset of patients that could influence therapy for these difficult-to-treat progressive neoplasms.

Aberrant CDK4 Amplification in Refractory Rhabdomyosarcoma as Identified by Genomic Profiling

Rhabdomyosarcoma (RMS) is the most commonly occurring type of soft tissue tumor in children. However, it is rare in adults, and therefore, very little is known about the most appropriate treatment strategy for adult RMS patients. We performed genomic analysis of RMS cells derived from a 27-year-old male patient whose disease was refractory to treatment. A peritoneal seeding nodule from the primary tumor, pleural metastases, malignant pleural effusion, and ascites obtained during disease progression, were analyzed. Whole exome sequencing revealed 23 candidate variants, and 10 of 23 mutations were validated by Sanger sequencing. Three of 10 mutations were present in both primary and metastatic tumors, and 3 mutations were detected only in metastatic specimens. Comparative genomic hybridization array analysis revealed prominent amplification in the 12q13–14 region, and more specifically, the CDK4 proto-oncogene was highly amplified. ALK overexpression was observed at both protein and RNA levels. However, an ALK fusion assay using NanoString technology failed to show any ALK rearrangements. Little genetic heterogeneity was observed between primary and metastatic RMS cells. We propose that CDK4, located at 12q14, is a potential target for drug development for RMS treatment.

Dedifferentiated liposarcoma and pleomorphic liposarcoma

Dedifferentiated liposarcoma (DDLPS) and pleomorphic liposarcoma (PLPS) are distinct high-grade liposarcomas. DDLPS is a nonlipogenic sarcoma characterized by amplification of MDM2 and CDK4. PLPS is a high-grade sarcoma containing lipoblasts, characterized by a complex karyotype and a more aggressive clinical course. Rarely, DDLPS shows lipogenic differentiation, mimicking PLPS. The cytomorphologic features of DDLPS and PLPS and the utility of ancillary studies have not been systemically analyzed. Cytologic preparations of 25 DDLPS and 13 PLPS, all histologically confirmed, were retrospectively reviewed along with clinical and cytogenetic data. Sample cellularity, vascular architecture, background material, predominant cell morphology, quality of the cytoplasm, and nuclear pleomorphism were compared for both tumor types. Immunohistochemistry for MDM2 and CDK4 was performed on cell blocks and/or core needle biopsies. Fine-needle aspirate smears from both DDLPS and PLPS were variably cellular, composed of cellular clusters and noncohesive cells. Abundant myxoid stroma was present in ∼25% of DDLPS and PLPS cases, whereas branching curvilinear vessels were more common in DDLPS than in PLPS (7 of 25 versus 2 of 13). Tumors were composed of predominantly spindled (18 of 25 DDLPS versus 3 of 13 PLPS) or epithelioid cells (7 of 25 DDLPS versus 6 of 13 PLPS). Pleomorphic cells were predominant in 3 PLPS, and were frequent in both (13 of 25 DDLPS versus 10 of 13 PLPS). The cytoplasm was mostly fibrillary and often vacuolated in both entities. Other features included necrosis, mitoses, and a prominent inflammatory infiltrate. The main cytologic differences were the presence of marked pleomorphism, abundant lipoblasts, and cells with microvacuolated cytoplasm in most PLPS. A total of 24 (96%) and 20 (80%) cases of DDLPS expressed MDM2 and CDK4, respectively, whereas none of the PLPS expressed both markers. Six DDLPS tested showed ring or giant marker chromosomes and/or MDM2 amplification by fluorescence in situ hybridization; 2 PLPS had complex karyotypes. DDLPS and PLPS exhibit variable and occasionally overlapping cytologic features. The presence of lipoblasts, cells with microvacuolated cytoplasm, and marked pleomorphism are more suggestive of PLPS, but these characteristics can be present in DDLPS. Coexpression of MDM2 and CDK4 distinguishes DDLPS from PLPS.

Abstract C81: BBB efflux pump activity limits brain penetration of palbociclib (PD0332991) in glioblastoma.

Abstract Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults and is associated with a poor prognosis. Progress in developing effective therapies for this disease is significantly limited by the blood-brain barrier (BBB), which limits the delivery of many anti-cancer agents to infiltrative tumor cells. In addition to physical barriers, such as tight junctions, the efflux proteins bcrp and P-gp in the BBB limit the brain distribution of numerous anti-cancer agents. Palbociclib (PD0332991) is a potent Cdk4/6 inhibitor which has shown remarkable efficacy in treating peripheral (non-brain) tumors. The Cdk4 pathway is dysregulated in approximately 75% of GBM; most commonly, the pathway is hyperactivated through the homozygous deletion of p16 (52%), amplification of Cdk4 (18%), or amplification of Cdk6 (1%). The purpose of this study is to define the role of the efflux transporters P-gp and bcrp in the brain distribution of palbociclib and to examine if an intact BBB limits efficacy. Palbociclib brain distribution studies were performed in FVB wild-type, P-gp knockout (PKO; Mdr1a/b(-/-)), bcrp knockout (BKO; Bcrp1(-/-)), and triple knockout (TKO; Mdr1a/b(-/-)Bcrp1(-/-)) mice after an oral dose (10mg/kg). The concentrations of palbociclib from all distribution studies were determined by a sensitive and specific LC-MS/MS assay. Survival studies were conducted in patient-derived primary GBM xenograft models in athymic nu/nu mice. The brain exposure of palbociclib (AUCbrain-to-AUCplasma ratio) was ∼ 33.5, 3.2, and 150-fold higher as compared to WT mice (WT: .044; PKO: 1.34; BKO: 0.13; TKO: 6.24). Further, the steady-state brain-to-plasma ratio (B/P) of palbociclib after a constant intra-peritoneal infusion of 10 µg/hr for 48hrs was ∼120-fold higher in the TKO mice than the WT mice [WT: (0.21 ± 0.07); PKO: (2.48 ± .13); BKO: (0.43 ± 0.12); TKO: (26.5 ± 5.4) p &amp;lt; 0.0001]. Inhibition of P-gp and bcrp with elacridar (10 mg/kg IP) resulted in a marked increase in palbociclib brain distribution [control B/P (0.06 ± 0.02); elacridar treatment (2.0 ± 1.4)]. For survival studies, palbociclib was dosed at 150 mg/kg/day continuously. Consistent with limited brain penetration, palbociclib did not improve the median survival of an orthotopic GBM xenograft model. In contrast, treatment of GBM22 xenografts grown as flank tumors resulted in profound efficacy with a 70 day prolongation in the time for tumor volume to reach 1000mm3. These data suggest the clinical paradigm of a potent anti-cancer agent (for instance, palbociclib) used in the treatment of peripheral disease is less effective in the treatment of brain tumors due to the BBB and active efflux by P-gp and bcrp. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C81. Citation Format: Karen E. Parrish, Jenny L. Pokorny, Rajendar K. Mittapalli, Katrina Bakken, Jann N. Sarkaria, William F. Elmquist. BBB efflux pump activity limits brain penetration of palbociclib (PD0332991) in glioblastoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C81.

High level of CDK4 amplification is a poor prognostic factor in well-differentiated and dedifferentiated liposarcoma.

The amplification of MDM2 and CDK4 is the main molecular feature of well-differentiated liposarcomas (WDLS) and dedifferentiated liposarcomas (DDLS). Although the diagnostic usefulness of this molecular characteristic in liposarcomas has been investigated, its prognostic utility of quantitative gene level has not been explored. The aim of this study was to assess the prognostic significance of level of CDK4 amplification in MDM2-amplified WDLS/DDLS. MDM2 amplification in liposarcomas was confirmed by fluorescence in situ hybridization. The copy number of MDM2 and CDK4 was further determined by quantitative PCR (qPCR) and multiplex ligation-dependent probe amplification. Among 56 MDM2-amplified liposarcomas, 30 cases were assigned as WDLS, and 26 as DDLS. When liposarcomas were classified by qPCR-determined CDK4 amplification levels, the high-CDK4 group showed significantly poorer progression free survival (P=0.001) and disease specific survival (P=0.033) than the low-CDK4 group. However, MDM2 amplification level did not show prognostic significance. In WDLS/DDLS, the level of CDK4 amplification was useful for prognosis prediction and precise stratification of patients for targeted therapy.

Phase II trial of the CDK4 inhibitor PD0332991 in patients with advanced CDK4-amplified liposarcoma.

10512 Background: Approximately 90% of well-differentiated / de-differentiated liposarcomas (WD/DDLS) have CDK4 amplification. The selective CDK4/CDK6 inhibitor PD0332991 inhibits growth and induces senescence in liposarcoma cell lines and xenografts. Our prior phase II study demonstrated that treatment with PD0332991 (200mg daily x 14d every 21d) results in clinical benefit in WD/DDLS but moderate hematologic toxicity (48% Grade 3/4 neutropenia; dose reduction in 24%). Aiming to reduce toxicity, we conducted a phase II study to assess progression-free survival (PFS) and toxicity with PD0332991 at a new dose and schedule, 125mg daily x 21d every 28d. Methods: Participants were patients with advanced WD/DDLS. Eligibility criteria were age ≥ 18 years, measurable WD/DDLS (RECIST 1.1), documented progression on at least one systemic therapy directly before enrollment, CDK4 amplification by fluorescence in situ hybridization and retinoblastoma protein expression by immunohistochemistry (≥1+). Pts received oral PD0332991 at 125mg daily for 21 days in 28-day cycles. The primary endpoint was PFS at 12 weeks. Based on historical data, a promising result was defined as a 12-week PFS of ≥40% and not promising as ≤20%. The sample size was up to 28 evaluable patients. If 9 patients were progression free at 12 weeks, then PD0332991 would be considered to have activity in WD/DDLS. Results: 29 pts were enrolled and 25 were evaluable for the primary endpoint. Median age was 62 (range 42-85); 55% were male; median ECOG score was 0 (range 0-1). PFS at 12 weeks was 56% (14/25 patients; 90% CI 41-100%), and thus the study significantly exceeded its primary endpoint. Median PFS was 23.6 weeks (95% CI: 11.6 to Not Reached). There was 1 confirmed partial response lasting &gt; 1 year. Grade 3 and 4 adverse events included anemia (grade 3, 21%), thrombocytopenia (grade 3, 7%; grade 4, 3%), and neutropenia (grade 3, 34%). Dose reduction was required in only 1 patient. Conclusions: In patients with WD/DDLS with CDK4 amplification, PD0332991 treatment was associated with a favorable PFS and objective tumor response. This dose and schedule appears active and may have less toxicity than 200mg x 14d. The 125mg x 21d schedule warrants evaluation in a phase 3 study. Clinical trial information: NCT01209598.

Abstract 3186: Characterizing the antitumor effects of inhibiting translation initiation in glioblastoma multiforme.

Abstract Oncogene addiction is the process by which a tumor cell becomes increasingly dependent on the expression of a particular gene for the maintenance of its tumorigenicity. Alleviating this dependence via genetic or pharmacological means may profoundly inhibit tumor growth. However many cancers are not dependent on a single gene for survival. Glioblastoma Multiforme (GBM) presents itself with many genetic aberrations contributing to its aggressive phenotype. These include loss of PTEN, amplification of CDK4 and EGFR among others, making targeting a single genetic node ineffective where other oncogenic networks may buffer any therapeutic benefit. Over the last 10 years it has been shown that targeting protein translation initiation results in the simultaneous targeting of many oncogenic pathways. Translation initiation is highly implicated in tumorigenesis with over 10 initiation factors acting as either proto-oncogenes or tumor suppressors, the most well described being the eIF4E. The overexpression of eIF4E in many cancers results in messenger RNA discrimination giving rise to an increased translation of a subset of oncogenic mRNAs with highly structured 5’UTRs. However the precise role translation initiation plays in maintaining tumorigenicity in GBM and the subsequent anti-neoplastic properties of its inhibition are not known. Here, we characterize the anti-tumor effects of targeting protein translation in three glioblastoma cell lines U87MG, U251N and the highly aggressive U87ΔEGFR, using a small molecule inhibitor of eIF4E, 4EGI-1. We show that 4EGI-1 severely impairs cell survival over a 72 hour time interval, in a dose-dependent manner. This is marked by an arrest of cell proliferation starting at 24 hours, induction of apoptosis at 48 hours with increased annexin V staining and a decrease in cell motility. We found these effects coincide with a decrease in protein expression of several oncogenes with highly structured 5’UTRs after treatment with 4EGI-1 including cell proliferation proteins c-Myc and Cyclin D1 which are completely lost by 48 hours and regulators of apoptosis such as Bcl-xL, Mcl-1 and Survivin which decrease and are lost by 72 hours, while not affecting overall transcription of these genes. Our results demonstrate the sensitivity of gliomas towards inhibition of translation initiation. This further highlights translation regulation control as a strong force in cancer therapy, particularly in glioblastoma where options are limiting. Citation Format: Arjuna K. Rajakumar, Josephine Nalbantoglu. Characterizing the antitumor effects of inhibiting translation initiation in glioblastoma multiforme. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3186. doi:10.1158/1538-7445.AM2013-3186

Abstract 2744: CDK4/CDK6 inhibition is potently active in a definable subset of human neuroblastomas.

Abstract Background: Neuroblastoma is a pediatric embryonal cancer for which the survival of patients with high-risk disease is less than 50% and has not dramatically changed over the last several years. Recently, a number of cell cycle genes_particularly those within the Cyclin D/CDK4/CDK6/RB network_have been identified as oncogenic vulnerabilities in neuroblastoma, suggesting that their therapeutic exploitation might improve survivability. Indeed, genomic amplifications of CDK4, CDK6, and CCND1 have been reported in primary neuroblastomas, and we have previously shown via an unbiased loss of function screen that CDK4 depletion is associated with potent anti-tumor activity (Cole, PNAS 2011). Here, we sought to translate these findings into novel therapies for children with neuroblastoma by evaluating the effect of pharmacologic Cdk4/Cdk6 inhibition on neuroblastoma viability. Methods: We analyzed the effect of combined Cdk4/6 inhibition in a comprehensive panel of human-derived neuroblastoma cell lines using LEE011, a highly specific Cdk4/6 small molecule inhibitor. Anti-tumor activity was also determined in vivo in three neuroblastoma xenograft models, and integrative genomics was used to identify biomarkers of drug sensitivity. Results: Treatment with LEE011 significantly inhibited proliferation in 10 of 15 human neuroblastoma-derived cell lines by inducing cytostasis at nanomolar concentrations (mean IC50 = 361 ± 97 nM, considering sensitive lines only), as evidenced by significant cell cycle arrest and senescence that were likely attributed to dose-dependent decreases in phosphorylated RB and FOXM1. In addition, responsiveness of neuroblastoma xenografts to LEE011 was reflective of in vitro data in that there was a direct correlation of IC50 values with degree of subcutaneous xenograft growth delay, with the most sensitive lines in vitro showing profound growth inhibition in vivo. While our data indicate that neuroblastomas sensitive to LEE011 were more likely to contain genomic amplification of MYCN (p= 0.04, student's t test), a supervised hierarchical clustering of gene expression data identified several potential gene signatures that could explain the observed differential sensitivity to Cdk4/6 inhibition. Conclusions: Our data show that LEE011 is highly active in a large subset of neuroblastoma cell lines and xenograft models, and therefore support the clinical development of LEE011 as a therapy for neuroblastoma as well as efforts to validate biomarkers of drug activity. Citation Format: JulieAnn Rader, Lori Hart, Mike Russell, Michael Nakazawa, Lili Belcastro, Daniel Martinez, Erica Carpenter, Sunkyu Kim, Sudha Parasuraman, Giordano Caponigro, Robert Schnepp, Andrew Wood, Bruce Pawel, Deborah Watson, Patrick Warren, Kristina Cole, John Maris. CDK4/CDK6 inhibition is potently active in a definable subset of human neuroblastomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2744. doi:10.1158/1538-7445.AM2013-2744

Abstract S3-6: Profiling of triple-negative breast cancers after neoadjuvant chemotherapy identifies targetable molecular alterations in the treatment-refractory residual disease

Abstract Background: Neoadjuvant chemotherapy (NAC) is increasingly used in patients with triple-negative breast cancer (TNBC). NAC can induce a pathologic complete response (pCR) in ∼30% of patients which portends a favorable prognosis. In contrast, patients with residual disease (RD) in the breast at surgical resection exhibit worse outcomes. Objective: We hypothesized that profiling residual TNBCs after NAC would identify molecularly targetable lesions in the chemotherapy-resistant component of the tumor and that the persistent tumor cells would mirror micro-metastases which ultimately recur in such patients. Methods: We utilized targeted next generation sequencing (NGS) for 182 oncogenes and tumor suppressors in a CLIA certified lab (Foundation Medicine, Cambridge, MA) and gene expression profiling (NanoString) of the RD after NAC in 102 patients with TNBC. The RD was stained for Ki67, which has been reported to predict outcome after NAC in unselected breast cancers. Results: Thirteen tumors were not evaluable due to low tumor cellularity. Of 89 evaluable post-NAC tumors, 57 (64%) were basal-like; 19% HER2-enriched; 6% luminal A; 6% luminal B and 5% normal-like. Mean depth of coverage was 635 (range: 135–1207). Of 81 tumors evaluated by NGS, 72/81 (89%) demonstrated mutations in TP53, 22 were MCL1-amplified (27%), and 17 were MYC-amplified (21%). Alterations in the PI3K/mTOR pathway (AKT1-3, PIK3CA, PIK3R1, RAPTOR, PTEN, and TSC1) were identified in 27 tumors (33%). Cell cycle genes were altered in 25 tumors (31%), including amplifications of CDK2, CDK4, and CDK6, CCND1-3, and CCNE1 as well as RB loss. Alterations in the DNA repair pathway (BRCA1/2, ATM; 16 tumors; 20%) and the Ras/MAPK pathway (KRAS, RAF1, NF1; 10 tumors; 12%) were also common. Sporadic growth factor receptor amplifications occurred in EGFR, KIT, PDGFRA, PDGFRB, MET, FGFR1, FGFR2, and IGF1R. NGS identified 7 patients with ERBB2 gene amplification in the RD which was confirmed by FISH in both the pre- and post-treatment tissue, suggesting NGS could assist in the identification of ERBB2-overexpressing tumors misclassified at the time of diagnosis. In general, the gene amplifications identified by NGS corresponded to enhanced gene expression levels. Amplifications of MYC were independently associated with poor recurrence-free survival (RFS) and overall survival (OS). An interaction effect on survival was observed between MEK activation (assayed by a gene expression signature) and MYC amplification, suggesting cooperation between these pathways. Alterations in DNA repair also identified a subgroup with poor RFS and OS. In contrast, high post-NAC Ki67 score did not predict poor RFS or OS in this predominantly TNBC cohort. Conclusions: The diversity of lesions in residual TNBCs after NAC underscores the need for powerful and broad molecular approaches to identify actionable molecular alterations and, in turn, better inform personalized therapy of this aggressive disease. Incorporation of this platform into clinical studies and eventually standards of care should aid in the prioritization of patients with RD after NAC into rational adjuvant studies. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr S3-6.

Sensitivity of MDM2 amplification and unexpected multiple faint alphoid 12 (alpha 12 satellite sequences) signals in atypical lipomatous tumor

This study assessed whether analysis of MDM2 copy number by fluorescence in situ hybridization (FISH) would help distinguish lipomas from atypical lipomatous tumors, otherwise referred to as well-differentiated liposarcomas, using a commercially available MDM2 FISH kit. 227 lipomatous and 201 non-lipomatous tumors were analyzed to assess its sensitivity and specificity. Of 178 mature lipomatous tumors, 86 were classified histologically as lipoma and 92 as atypical lipomatous tumor. Two of the lipomas harboring MDM2 amplification were reclassified as atypical lipomatous tumors. Overall, 13 atypical lipomatous tumors did not reveal MDM2 or CDK4 amplification, although this was reduced to 12 following analysis of multiple slides. Three of these cases revealed very occasional tumor cells harboring high-level MDM2 amplification, two had a dedifferentiated component, and MDM2 amplification was detected when one tumor recurred. The remaining six cases exhibited reactive/inflammatory features and were reclassified as lipomas. The findings indicate that MDM2 amplification is 93.5% sensitive for diagnosing atypical lipomatous tumor. A total of 2 of the 20 dedifferentiated liposarcomas failed to reveal MDM2 amplification. All atypical lipomatous tumors measured >10 cm, two dedifferentiated liposarcoma presented de novo at <10 cm, and ∼50% of lipomas measured >10 cm. Spindle cell lipomas, lipoblastomas, hibernomas and pleomorphic liposarcomas did not reveal MDM2 amplification. Of 201 non-lipomatous tumors, eight revealed MDM2 amplification or multiple faint alphoid 12 signals and were reclassified as dedifferentiated liposarcoma. Multiple faint alphoid 12 signals were observed in nine tumors from seven patients, an observation not previously reported on paraffin sections: these included four atypical lipomatous tumors, and three dedifferentiated liposarcomas, one previously diagnosed as a myxofibrosarcoma, all of which also revealed amplification of CDK4, although two lacked MDM2 amplification. MDM2 FISH test is a useful adjunct to histology for distinguishing lipoma from atypical lipomatous tumor. The limitations of molecular genetic tests must be known before introducing them into a clinical service.

Attenuation of the Retinoblastoma Pathway in Pancreatic Neuroendocrine Tumors Due to Increased Cdk4/Cdk6

In mice, genetic changes that inactivate the retinoblastoma tumor suppressor pathway often result in pancreatic neuroendocrine tumors (Pan-NETs). Conversely, in humans with this disease, mutations in genes of the retinoblastoma pathway have rarely been detected, even in genome-wide sequencing studies. In this study, we took a closer look at the role of the retinoblastoma pathway in human Pan-NETs. Pan-NET tumors from 92 patients were subjected to immunohistochemical staining for markers of the retinoblastoma pathway. To search for amplifications of retinoblastoma pathway genes, genomic DNAs from 26 tumors were subjected to copy number analysis. Finally, a small-molecule activator of the retinoblastoma pathway was tested for effects on the growth of two Pan-NET cell lines. A majority of tumors expressed high amounts of Cdk4 or its partner protein cyclin D1. High amounts of phosphorylated Rb1 were present in tumors that expressed high levels of Cdk4 or cyclin D1. The copy numbers of Cdk4 or the analogous kinase gene Cdk6 were increased in 19% of the tumors. Growth of the human Pan-NET cell line QGP1 was inhibited in a xenograft mouse model by the Cdk4/6 inhibitor, PD 0332991, which reactivates the retinoblastoma pathway. Inactivation of the retinoblastoma pathway was indicated for most Pan-NETs. Gene amplification and overexpression of Cdk4 and Cdk6 suggests that patients with Pan-NETs may respond strongly to Cdk4/6 inhibitors that are entering clinical trials.

Primary dediffentiated liposarcoma of the dura mater: case report

Dear Editor, We report the case of a 62-year-old man who was referred to our institution in June 2008 for a depression of the temporoparietal cranial vault on the right side. This deformation had been known for 10 years with no occurrence of neurological symptoms. Lateral X-ray of the cranium showed an extensive zone of osteolysis of the temporoparietal area on the right side. A brain computed tomography (CT) showed an osteolytic lesion of the parietal bone, involving the inner table and extending to the diploe and the outer table with presence of fat. Brain magnetic resonance imaging (MRI) demonstrated a T2 hypersignal tumor mass. The lateral part of the lesion displayed a T1 hypersignal, and a low signal on the fatsaturated, T1-weighted sequence, signaling the existence of fat within the tumor. No brain edema or brain displacement, dural thickening or abnormal enhancement was observable. The surgical resection was justified by the recent progression of the lytic process as shown on sequential plain radiographs. Peroperatively the dura mater was involved by a yellowish compact tumor mass. After the resection, no evidence of leptomeningeal and subpial infiltration was observed. While complete removal was achieved, closure and reconstruction were obtained by a duraplasty (Goretex) and a methylmetacrylate cranioplasty. The size of the resection specimen was 5 cm. The patient had microscopic complete resection. Microscopically, with HES (hematoxylineosin-saffron) coloration ×100, the tumor was composed of a proliferation of spindled cells arranged in a myxoid or fibrous background. Tumorous cells were mildly atypical and showed few mitoses corresponding to the low-grade dedifferentiated component. Very focally, one could observe well adipose tissue with eccentric hyperchromatic nuclei and vacuolated cytoplasm corresponding to the welldifferentiated component of liposarcoma. Few areas of reactive osteogenesis were present as the tumor infiltrated bone. According to immunohistochemistry, the spindle cells were negative for α-smooth actin, CD34, PS100, CD68 and EMA. Focal nuclear positivity was seen with anti-CDK4 antibodies in well-differentiated adipocytes and in some spindle cells. MDM2 showed more widespread nuclear staining in spindle cells. Amplifications of the oncogenes CDK4 and MDM2 were detected by FISH (fluorescent in situ hybridization) performed on representative paraffin sections (Fig. 1). The final diagnosis was consistent with a primary low-grade dedifferentiated liposarcoma of the dura. Full staging imaging with CT did not show any additional tumor mass. Thanks to Nomeharisoa Rodrigue Emile Hasiniatsy, MD, Frederic Cohen, MD, Frederic Chibon, PhD, Nathalie Stock, MD, Jean-Michel Coindre, MD, PhD, Jean-Laurent Deville, MD, Florence Duffaud, MD, PhD S. Garciaz : S. Salas Department of Medicine, Division of Adult Oncology, APHM, Timone Hospital, 13005 Marseille, France

P16-Cdk4-Rb axis controls sensitivity to a cyclin-dependent kinase inhibitor PD0332991 in glioblastoma xenograft cells

Deregulation of the p16(INK4a)-Cdk4/6-Rb pathway is commonly detected in patients with glioblastoma multiforme (GBM) and is a rational therapeutic target. Here, we characterized the p16(INK4a)-Cdk4/6-Rb pathway in the Mayo panel of GBM xenografts, established from primary tissue samples from patients with GBM, and evaluated their response to PD0332991, a specific inhibitor of Cdk4/6. All GBM xenograft lines evaluated in this study had disruptions in the p16(INK4a)-Cdk4/6-Rb pathway. In vitro evaluation using short-term explant cultures from selected GBM xenograft lines showed that PD0332991 effectively arrested cell cycle in G1-phase and inhibited cell proliferation dose-dependently in lines deleted for CDKN2A/B-p16(INK4a) and either single-copy deletion of CDK4 (GBM22), high-level CDK6 amplification (GBM34), or deletion of CDKN2C/p18(INK4c) (GBM43). In contrast, 2 GBM lines with p16(INK4a) expression and either CDK4 amplification (GBM5) or RB mutation (GBM28) were completely resistant to PD0332991. Additional xenograft lines were screened, and GBM63 was identified to have p16(INK4a) expression and CDK4 amplification. Similar to the results with GBM5, GBM63 was resistant to PD0332991 treatment. In an orthotopic survival model, treatment of GBM6 xenografts (CDKN2A/B-deleted and CDK4 wild-type) with PD0332991 significantly suppressed tumor cell proliferation and prolonged survival. Collectively, these data support the concept that GBM tumors lacking p16(INK4a) expression and with nonamplified CDK4 and wild-type RB status may be more susceptible to Cdk4/6 inhibition using PD0332991.

Phase II trial of the CDK4 inhibitor PD0332991 in CDK4-amplified liposarcoma.

10002 Background: CDK4 is amplified in approximately 90% of well-differentiated/de-differentiated liposarcomas (WD/DDLS). The selective CDK4/CDK6 inhibitor PD0332991 (PD) inhibits growth and induces senescence in liposarcoma cell lines and xenografts. In a phase I trial of PD, several patients with progressive WD/DDLS had prolonged stable disease for several years. To determine the safety and efficacy of PD, a phase II study was performed. Methods: Participants were patients with advanced WD/DDLS. Eligibility criteria were age≥18 years, measurable WD/DDLS (RECIST 1.1), documented progression on at least one systemic therapy directly before enrollment, CDK4 amplification by fluorescence in situ hybridization and retinoblastoma protein (RB) expression by immunohistochemistry (≥1+). Pts received oral PD 200mg daily for 14 consecutive days in 21-day cycles. The primary endpoint was progression-free survival (PFS) at 12 weeks. Based on historical data, a promising result was defined as a 12-week PFS of ≥40% and not promising as ≤20%. The sample size was up to 28 evaluable patients. If 9 patients were progression free at 12 weeks, then PD would be considered to have activity in WD/DDLS. Results: Of 44 patients screened (42/44 CDK4 amplified; 41/44 RB+), 29 were enrolled and 27 were evaluable for the primary endpoint. Median age was 65 (range 37-83); 52% were male; ECOG scores were 0 (69%) or 1 (31%), and the median number of prior regimens was 1 (range 1-5). PFS at 12 weeks was 70% (19/27 patients; 90% CI 56-100%), and thus the study significantly exceeded its primary endpoint. At the data cutoff, the median PFS was 18 weeks. Seven patients remain on study with stable disease at 18-48 weeks of followup. Grade 3 and 4 events included anemia (grade 3, 14%), thrombocytopenia (grade 3, 17%; grade 4, 14%), neutropenia (grade 3, 41%; grade 4, 7%) and febrile neutropenia (3%). Dose reductions were required in 24% of patients. Conclusions: Among patients with WD/DDLS with CDK4 amplification and RB expression who had actively progressing disease despite prior systemic therapy, treatment with the CDK4 inhibitor PD0332991 was associated with improved PFS. A randomized phase 3 trial is planned.

Multiple CDK/CYCLIND genes are amplified in medulloblastoma and supratentorial primitive neuroectodermal brain tumor

Embryonal brain tumors, which include medulloblastoma and the more aggressive supratentorial primitive neuroectodermal tumor (sPNET), comprise one of the largest group of malignant pediatric brain tumors. We observed in high resolution array comparative genomic hybridization and polymerase chain reaction analyses that several different components of the CDK/CYCLIND/pRB regulatory complex, including the CDK4/6 and CCND1/2 loci, are targets of gene amplification in medulloblastoma and sPNET. CDK6 and CCND1 gene amplification were respectively most common and robust, and overall CDK/CYCLIND gene amplification was more commonly observed in sPNET (25%) than medulloblastoma (1-5%). CDK6 overexpression enhanced in vitro and in vivo oncogenicity and endogenous CDK6 or CCND1 knockdown decreased pRB phosphorylation and impaired cell cycle progression in both medulloblastoma and sPNET cell lines. Although animal models implicate the pRB tumor suppressor pathway in medulloblastoma and sPNET, mutations of RB1 or the related INK4 tumor suppressor loci are rare in primary human tumors. Our data suggest that CDK/CYCLIND gene amplification may represent important mechanisms for functional inactivation of pRB in medulloblastoma and sPNET.

Amplification of the STOML3, FREM2, and LHFP Genes Is Associated with Mesenchymal Differentiation in Gliosarcoma

Gliosarcoma is a rare glioblastoma variant characterized by a biphasic tissue pattern with alternating areas that display either glial (glial fibrillary acidic protein-positive) or mesenchymal (reticulin-positive) differentiation. Previous analyses have shown identical genetic alterations in glial and mesenchymal tumor areas, suggesting that gliosarcomas are genetically monoclonal, and mesenchymal differentiation was considered to reflect the elevated genomic instability of glioblastomas. In the present study, we compared genome-wide chromosomal imbalances using array comparative genomic hybridization in glial and mesenchymal tumor areas of 13 gliosarcomas. The patterns of gain and loss were similar, except that the gain at 13q13.3-q14.1 (log(2) ratio >3.0), containing the STOML3, FREM2, and LHFP genes, which was restricted to the mesenchymal tumor area of a gliosarcoma. Further analyses of 64 cases of gliosarcoma using quantitative PCR showed amplification of the STOML3, FREM2, and LHFP genes in 14 (22%), 10 (16%), and 7 (11%) mesenchymal tumor areas, respectively, but not in glial tumor areas. Results of IHC analysis confirmed that overexpression of STOML3 and FREM2 was more extensive in mesenchymal than in glial tumor areas. These results suggest that the mesenchymal components in a small fraction of gliosarcomas may be derived from glial cells with additional genetic alterations.

Abstract LB-270: Genomic amplification of c-Jun activates genes that promote proliferation and cell migration in liposarcoma

Abstract Introduction: Activating protein 1 (AP-1) is a dimeric transcription factor comprising proteins whose common denominator is the possession of basic leucine zipper (bZIP) domains that are essential for dimerization and DNA binding. Among this family, the c-Jun protein is a transcription factor that is activated among the earliest responses to mitogenic signaling. It can promote cell division and differentiation but also can act as a potent inducer of apoptosis in other contexts. Recently, c-Jun was directly implicated as a proto-oncogene in a human cancer when it was found to be genomically amplified in ∼30% of dedifferentiated liposarcomas (DDLPS). It was hypothesized that c-Jun overexpression might directly impair adipogenesis, thereby mediating the transition from well differentiated liposarcomas (WDLPS) to DDLPS. However, recent work from our lab has suggested that c-Jun may regulate other cellular processes in DDLPS, including proliferation. We are currently elucidating the precise mechanisms by which c-Jun amplification drives tumorigenesis in DDLPS. Methods We used lentiviral-mediated RNA interference to inhibit c-Jun in two DDLPS cell lines, one with c-Jun amplification and overexpression (LP6) and one with normal c-Jun copy number (LPS141). We then analyzed changes in mRNA levels by Affymetrix Exon array and changes in miRNA levels via Nanostring. Ingenuity Pathways (IPA) was used to screen the results for biological trends. We validated changes in gene expression via qRT-PCR and immunoblotting. Results: Analysis of 754 microRNAs revealed that 85 (11%) miRNAs exhibited a &amp;gt;2 fold decrease, and 23 (3%) exhibited &amp;gt;2 fold increase after c-Jun knockdown in LP6 cells. mRNA analysis showed that 116 genes were downregulated and 12 genes were upregulated &amp;gt;2 fold after c-Jun knockdown in LP6 cells. In LPS141 cells, 341 genes were downregulated and 57 upregulated. We identified only 10 common differentially expressed genes between these 2 cell lines, suggesting that c-Jun may acquire additional functions when genomically amplified. Pathway analysis showed that c-Jun promotes cell proliferation in both cell lines. However, genes involved in “cellular movement and connective tissue development” were significantly downregulated by c-Jun knockdown only in LP6 cells. We have validated these gene expression changes and are testing the hypothesis that c-Jun amplification promotes cell migration in DDLPS. Discussion: Liposarcomas exhibit nearly universal genomic amplification of MDM2 and CDK4, two oncogenes which are readily targeted by small molecules. Transcription factors such as c-Jun are difficult to target directly by small molecule inhibitors. Our studies suggest that amplified c-Jun may regulate genes that stimulate both proliferation and cell migration in DDLPS. We expect that this work will provide a starting point for developing targeted therapies for c-Jun-amplified DDLPS. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-270. doi:1538-7445.AM2012-LB-270