Targeted pharmacologic inhibition of S-phase kinase-associated protein 2 (SKP2) mediated cell cycle regulation in lung and other RB-Related cancers: A brief review of current status and future prospects.

Small cell lung cancer (SCLC) often exhibits Rb deficiency, TRß and p130 deletion, and SKP2 amplification, suggesting TRß inactivation and SKP2 activation. It is reported that SKP2 targeted therapy is effective in some cancers in vitro and in vivo, but it is not reported for the treatment of SCLC and retinoblastoma. SKP2 is the synthetic lethal gene in SCLC and retinoblastoma, so SKP2 can be used for targeted therapy in SCLC and retinoblastoma. RB1 knockout mice develop several kinds of tumors, but Rb1 and SKP2 double knockout mice are healthy, suggesting that SKP2 targeted therapy may have significant effects on Rb deficient cancers with less side effects, and if successful in SCLC and retinoblastoma in vitro and in animal model, such compounds may be promising for the clinical treatment of SCLC, retinoblastoma, and variety of Rb deficient cancers. Previously our studies showed that retinoblastomas exhibit retinal cone precursor properties and depend on cone-specific thyroid hormone receptor ß2 (TRß2) and SKP2 signaling. In this study, we sought to suppress SCLC and retinoblastoma cell growth by SKP2 inhibitors as a prelude to targeted therapy in vitro and in vivo. We knocked down TRß2 and SKP2 or over-expressed p27 in SCLC and retinoblastoma cell lines to investigate SKP2 and p27 signaling alterations. The SCLC cell lines H209 as well as retinoblastoma cell lines Y79, WERI, and RB177 were treated with SKP2 inhibitor C1 at different concentrations, following which Western blotting, Immunostaining, and cell cycle kinetics studies were performed to study SKP2 and p27 expression ubiquitination, to determine impact on cell cycle regulation and growth inhibition. TRß2 knockdown in Y79, RB177 and H209 caused SKP2 downregulation and degradation, p27 up-regulation, and S phase arrest, whereas, SKP2 knockdown or p27 over-expression caused p27 accumulation and G1-S phase arrest. In the cell lines Y79, WERI, RB177, and H209 treatment with C1 caused SKP2 ubiquitination and degradation, p27 de-ubiquitination and accumulation, and cell growth arrest. SKP2 inhibitor C1 significantly suppressed retinoblastoma as well as SCLC cell growth by SKP2 degradation and p27 accumulation. In vivo study also showed inhibition of tumor growth with C1 treatment. Potential limitations of the success of such a therapeutic approach and its translational application in human primary tumors, and alternative approaches to overcome such limitations are briefly discussed for the treatment of retinoblastoma, SCLC and other RB-related cancers.

Somatic Epigenetic Silencing of RIPK3 Inactivates Necroptosis and Contributes to Chemoresistance in Malignant Mesothelioma.

PURPOSE: Receptor-interacting protein kinase 3 (RIPK3) phosphorylates effector molecule MLKL to trigger necroptosis. Although RIPK3 loss is seen in several human cancers, its role in malignant mesothelioma is unknown. This study aimed to determine whether RIPK3 functions as a potential tumor suppressor to limit development of malignant mesothelioma. EXPERIMENTAL DESIGN: RIPK3 expression was examined in 66 malignant mesothelioma tumors and cell lines. Promoter methylation and DNMT1 siRNA studies were performed to assess the mode of RIPK3 silencing in RIPK3-deficient malignant mesothelioma cells. Restoration of RIPK3 expression in RIPK3-negative malignant mesothelioma cells, either by treatment with 5-aza-2'-deoxycytidine or lentiviral expression of cDNA, was performed to assess effects on cell viability, necrosis, and chemosensitization. RESULTS: Loss of RIPK3 expression was observed in 42/66 (63%) primary malignant mesotheliomas and malignant mesothelioma cell lines, and RT-PCR analysis demonstrated that downregulation occurs at the transcriptional level, consistent with epigenetic silencing. RIPK3-negative malignant mesothelioma cells treated with 5-aza-2'-deoxycytidine resulted in reexpression of RIPK3 and chemosensitization. Ectopic expression of RIPK3 also resulted in chemosensitization and led to necroptosis, the latter demonstrated by phosphorylation of downstream target MLKL and confirmed by rescue experiments. Mining of RIPK3 expression and survival outcomes among patients with malignant mesothelioma available from The Cancer Genome Atlas repository revealed that promoter methylation of RIPK3 is associated with reduced RIPK3 expression and poor prognosis. CONCLUSIONS: These data suggest that RIPK3 acts as a tumor suppressor in malignant mesothelioma by triggering necroptosis and that epigenetic silencing of RIPK3 by DNA methylation impairs necroptosis and contributes to chemoresistance and poor survival in this incurable disease.

Cancer genomics of lung cancer including malignant mesothelioma: A brief overview of current status and future prospects.

Cancer as a genetic disease is by now well recognized. Genomic analysis of cancer cells, therefore, has greatly enhanced our ability to identify genetic alterations associated with various cancer types, including both lympho-hematopoietic as well as solid tumors. Chronic myeloid leukemia (CML), based on the specific diagnostic genetic abnormality has served as a prototype disease to clearly demonstrate the significance of the genomic analysis of cancer in identifying targeted therapy. Such a success has provided extra ordinary opportunities to investigate the role of genetic abnormalities and the pathways amenable to targeted therapy, not only in blood cancers but solid tumors such as Lung, Brain, Colon, Renal, Breast cancers as well as other epithelial and mesenchymal tumors. The main focus of this presentation is to illustrate the role of genomic analysis in targeting lung cancer, based on abnormalities or the pathways deregulated in tumor cells from individual patients. Lung cancer is one of the most common epithelial cancers associated with chronic inflammation due to cigarette smoking and other environmental carcinogens, and includes four distinct histologic type; non-small cell lung cancer (NSCLC); small cell lung cancer (SCLC) and squamous cell lung cancer. According to current estimates, 1.3 million cases of lung cancer are expected to be diagnosed worldwide annually, resulting in one million deaths. Since the discovery that patient's tumors with specific mutations in the EGFR may be sensitive to targeted therapeutic approach and the subsequent realization that the such mutations in the gene are not as prevalent, several cancer centers including ours initiated intense efforts to find other mutations or genomic alterations, which may serve as targets of specific therapy. Such efforts have successfully resulted in a battery of genes such as KRAS, ALK, C-MET, HER-2/neu, ROS1, etc., which have helped oncologists to triage the patients for personalized therapies. A significant proportion of patients with lung cancer, however, do not show any of the above genetic abnormalities. Approximately 90% of lung cancers exhibit RB1 mutation/deletion and or KRAS mutations, therefore, the signaling pathways, which regulate multistep tumorigenesis in lung cancer, are important for the treatment of histologic subtypes of lung cancer, which includes NSCLC & SCLC. Equally important was the findings that similar signaling pathways are also shared by other solid tumor types. We have investigated the role of these pathways to target these cancers and develop new strategies to treat lung, brain and related cancers. In addition, our translational studies in other tumor types such as NF2 related malignancies, specifically, Malignant Mesothelioma (MM), in which NF2 related pathway amenable to targeted therapies was identified. Selected examples representing experimental approaches will be discussed to illustrate the critical role of translational research in developing novel therapeutics for the successful and durable responses in some of these cancer types.

SKP2 Activation by Thyroid Hormone Receptor ß2 Bypasses Rb-Dependent Proliferation in Rb-Deficient Cells.

Germline RB1 mutations strongly predispose humans to cone precursor-derived retinoblastomas and strongly predispose mice to pituitary tumors, yet shared cell type-specific circuitry that sensitizes these different cell types to the loss of RB1 has not been defined. Here we show that the cell type-restricted thyroid hormone receptor isoform TRß2 sensitizes to RB1 loss in both settings by antagonizing the widely expressed and tumor-suppressive TRß1. TRß2 promoted expression of the E3 ubiquitin ligase SKP2, a critical factor for RB1-mutant tumors, by enabling EMI1/FBXO5-dependent inhibition of SKP2 degradation. In RB1 wild-type neuroblastoma cells, endogenous Rb or ectopic TRß2 was required to sustain SKP2 expression as well as cell viability and proliferation. These results suggest that in certain contexts, Rb loss enables TRß1-dependent suppression of SKP2 as a safeguard against RB1-deficient tumorigenesis. TRß2 counteracts TRß1, thus disrupting this safeguard and promoting development of RB1-deficient malignancies. Cancer Res; 77(24); 6838-50. ©2017 AACR.

Genomic instability and proliferation/survival pathways in RB1-deficient malignancies.

Genomic instability (GIN) is a hallmark of most cancer cells. However, compared to most human cancer cell types, the retinoblastoma tumor cells show a relatively stable genome. The fundamental basis of this genomic stability has yet to be elucidated, and the role of certain proteins involved in cell cycle regulation may be the key to the development of these specific genotypes. We examined whether thyroid hormone receptor beta 1 and 2 (TRß1 and TRß2), known to regulate tumorigenesis, and PTTG1, a mitotic checkpoint protein, play a role in maintaining genomic stability in retinoblastoma. In order to elucidate the role of these proteins in development of aneuploidy/polyploidy, an indicator of GIN, we first studied comparative GIN in retinoblastomas and multiple RB mutant cancer cell lines using single nucleotide polymorphism (SNP) analysis. We then utilized pLKO lentiviral vectors to selectively modify expression of the targeted cell cycle proteins and interpret their effect on downstream cell cycle proteins and their relative effects on the development of polyploidy in multiple tumor cell lines. The SNP analysis showed that retinoblastomas displayed relatively fewer genomic copy number changes as compared to other RB1-deficient cancer cell lines. Both TRß1 and TRß2 knockdown led to accumulation of E2F1 and PTTG1 and increased GIN as demonstrated by an increase in polyploidy. Downregulation of PTTG1 led to a relative decrease in GIN while upregulation of PTTG1 led to a relative increase in GIN. Knockdown of E2F1 led to a downstream decrease in PTTG1 expression. Rb-knockdown also upregulated E2F1 and PTTG1 leading to increased GIN. We showed that Rb is necessary for PTTG1 inhibition and genomic stability. A relatively stable genome in retinoblastoma tumor cells is maintained by TRß1 and TRß2-mediated PTTG1 inhibition, counteracting Rb-deficiency-related GIN. TRß1, TRß2 and Rb-KD all led to the downstream PTTG1 accumulation, apparently through an activation of E2F1 resulting in extensive genomic instability as seen in other Rb-deficient tumors.

Gain of chromosome 1q portends worse prognosis in multiple myeloma despite novel agent-based induction regimens and autologous transplantation.

We aimed to identify whether the use of autologous hematopoietic cell transplantation (HCT) impacts outcomes for multiple myeloma patients with gains of chromosome 1q (+1q). We retrospectively identified 95 patients, 21% having +1q. For patients with +1q, the overall response rate to induction was 85%, with 40% having ≥ VGPR and 20% achieving a CR, similar to non +1q patients (p = .64). The median PFS from diagnosis with +1q was 2.1 years (95% CI: 1.2-not reached (NR)) vs 4.3 years (95% CI: 3.3 yrs-NR) without +1q (p = .003). Median OS from diagnosis was 4.4 years (95% CI: 2.9-NR) vs not reached, respectively (p = .005). On molecular analysis using the Foundation One Heme assay, the most common mutations seen in +1q patients included TP53 (38%) and KRAS (25%). Overall, gain of 1q portends worse PFS and OS which was not negated by auto HCT. Such patients will likely require additional therapy to improve their survival.

NF2 Loss Promotes Oncogenic RAS-Induced Thyroid Cancers via YAP-Dependent Transactivation of RAS Proteins and Sensitizes Them to MEK Inhibition.

UNLABELLED: Ch22q LOH is preferentially associated with RAS mutations in papillary and in poorly differentiated thyroid cancer (PDTC). The 22q tumor suppressor NF2, encoding merlin, is implicated in this interaction because of its frequent loss of function in human thyroid cancer cell lines. Nf2 deletion or Hras mutation is insufficient for transformation, whereas their combined disruption leads to murine PDTC with increased MAPK signaling. Merlin loss induces RAS signaling in part through inactivation of Hippo, which activates a YAP-TEAD transcriptional program. We find that the three RAS genes are themselves YAP-TEAD1 transcriptional targets, providing a novel mechanism of promotion of RAS-induced tumorigenesis. Moreover, pharmacologic disruption of YAP-TEAD with verteporfin blocks RAS transcription and signaling and inhibits cell growth. The increased MAPK output generated by NF2 loss in RAS-mutant cancers may inform therapeutic strategies, as it generates greater dependency on the MAPK pathway for viability. SIGNIFICANCE: Intensification of mutant RAS signaling through copy-number imbalances is commonly associated with transformation. We show that NF2/merlin inactivation augments mutant RAS signaling by promoting YAP/TEAD-driven transcription of oncogenic and wild-type RAS, resulting in greater MAPK output and increased sensitivity to MEK inhibitors.

Genetic and epigenetic pathways in myelodysplastic syndromes: A brief overview.

Myelodysplastic syndromes (MDS) are a highly heterogenous group of hematopoietic tumors, mainly due to variable clinical features and diverse set of cytogenetic, molecular genetic and epigenetic lesions. The major clinical features of MDS are ineffective hematopoiesis, peripheral cytopenias, and an increased risk of transformation to acute myeloid leukemias, which in turn is most likely determined by specific genetic abnormalities and other presenting hematologic features. The risk of developing MDS is relatively higher in some genetic syndromes such as Fanconi anemia and receipt of chemotherapy and radiation treatment. In recent years a significant progress has occurred and a vast literatures has become available including the spectrum of cytogenetic abnormalities, gene mutations relating to RNA splicing machinery, epigenetic regulation of gene expression and signaling pathways associated with MDS pathogenesis, which have provided opportunities to understand the molecular mechanisms as well as employ targeted therapeutic approaches to treat MDS. The cytogenetic abnormalities detected in MDS varies from a single abnormality to complex karyotype not easily amenable to conventional cytogenetic analysis. In such cases, array based high resolution genomic analysis detected abnormalities, which are diagnostic as well as prognostic. The most common driver gene mutations detected in patients with MDS include RNA splicing (SF3B1,SRSF2,U2F1,ZRSR2), DNA methylation (TET2,DNMT3A,IDH1/IDH2), chromatin modification (ASXL1,EZH2), transcription regulation (RUNX1,BCOR) and DNA repair control p53. A small subset of MDS arise due to deregulation of RAS pathway, mainly due to NRAS/KRAS/NF1 mutations. Identification of these mutations and pathways have provided opportunities for oncologists to target these patients with specific therapies. Several drugs which either target the spliceosome, oncogenic RAS signaling, or hypomethylating agents have been employed to successfully treat MDS patients.

Rb suppresses human cone-precursor-derived retinoblastoma tumours.

Retinoblastoma is a childhood retinal tumour that initiates in response to biallelic RB1 inactivation and loss of functional retinoblastoma (Rb) protein. Although Rb has diverse tumour-suppressor functions and is inactivated in many cancers, germline RB1 mutations predispose to retinoblastoma far more strongly than to other malignancies. This tropism suggests that retinal cell-type-specific circuitry sensitizes to Rb loss, yet the nature of the circuitry and the cell type in which it operates have been unclear. Here we show that post-mitotic human cone precursors are uniquely sensitive to Rb depletion. Rb knockdown induced cone precursor proliferation in prospectively isolated populations and in intact retina. Proliferation followed the induction of E2F-regulated genes, and depended on factors having strong expression in maturing cone precursors and crucial roles in retinoblastoma cell proliferation, including MYCN and MDM2. Proliferation of Rb-depleted cones and retinoblastoma cells also depended on the Rb-related protein p107, SKP2, and a p27 downregulation associated with cone precursor maturation. Moreover, Rb-depleted cone precursors formed tumours in orthotopic xenografts with histological features and protein expression typical of human retinoblastoma. These findings provide a compelling molecular rationale for a cone precursor origin of retinoblastoma. More generally, they demonstrate that cell-type-specific circuitry can collaborate with an initiating oncogenic mutation to enable tumorigenesis.

Recursive partitioning analysis of prognostic variables in newly diagnosed anaplastic oligodendroglial tumors.

BACKGROUND: Anaplastic oligodendroglial tumors are rare, and median survival varies widely. Analysis of 1p19q deletion is performed commonly and is an important prognostic factor. However, age and other clinical variables also carry prognostic value, and it is unclear how to incorporate them into clinical decision making or to combine them for prognostication. METHODS: We compiled a retrospective database of 1013 patients with newly diagnosed anaplastic oligodendrogliomas or oligoastrocytomas and performed a recursive partitioning analysis to generate independent prognostic classes among 587 patients with informative 1p19q status. Variables included for survival classification were age (continuous), history of prior low-grade glioma, 1p19q deletion status, histology (presence or absence of an astrocytic component), tumor lobe, tumor hemisphere, gender, extent of resection, postresection treatment, and performance status at diagnosis. RESULTS: Recursive partitioning analysis identified 5 prognostic groups based on hazard similarity: class I (age <60 y, 1p19q codeleted), class II (age <43 y, not codeleted), class III (age 43-59 y, not codeleted, frontal lobe tumor or age ≥60 y, codeleted), class IV (age 43-59 y, not codeleted, not frontal lobe tumor or age 60-69 y, not codeleted), and class V (age ≥70 y, not codeleted). Survival differences were highly significant (P < .0001), with medians ranging from 9.3 years (95% CI: 8.4-16.0) for class I to 0.6 years (95% CI: 0.5-0.9) for class V. CONCLUSIONS: These 5 distinct classification groups were defined using prognostic factors typically obtained during routine management of patients with anaplastic oligodendroglial tumors. Validation in a prospective clinical trial may better differentiate patients with respect to treatment outcome.

Acute myeloid leukemia arising from a donor derived premalignant hematopoietic clone: A possible mechanism for the origin of leukemia in donor cells.

During recent years, it has become increasingly evident that donor leukemia following allogeneic transplant may be more common then realized in the past. We identified five cases of potential donor leukemia cases during past five years. The precise mechanism of the origin of such leukemias, however, remains poorly defined. In this short communication, we report a well documented case of donor-derived de novo acute myeloid leukemia (AML) that developed fourteen years after allogeneic stem cell transplantation for treatment induced AML for his primary malignancy Immunoblastic lymphoma. This case allows us to postulate a possible mechanism of the origin of donor leukemia. The de novo AML clone contained a distinct cytogenetic abnormality, trisomy 11, which was simultaneously detected in preserved peripheral blood obtained at the time of transplantation as well as in the current bone marrow from an otherwise clinically and phenotypically normal donor. The findings from this unique case, provides insight into the process of leukemogenesis, and suggests that the sequence of events leading to leukemogenesis in this patient involved the senescence/apoptosis of normal donor hematopoietic cells due to telomere shortening resulting in the selective proliferation and transformation of this clone with MLL (mixed-lineage leukemia) gene amplification.

A genome-wide high-resolution array-CGH analysis of cutaneous melanoma and comparison of array-CGH to FISH in diagnostic evaluation.

Benign melanocytic nevi and cutaneous melanomas can be difficult to differentiate by means of routine microscopic analysis. Recent evidence has suggested that cytogenomic analysis may be a useful diagnostic method for evaluation of melanocytic proliferations. We investigated the array-based comparative genomic hybridization (aCGH) platform for DNA copy number analysis of formalin-fixed, paraffin-embedded (FFPE) tissues in melanocytic tumors and compared aCGH analysis with fluorescence in situ hybridization (FISH) assays in diagnosis of melanoma. aCGH findings and FISH results were interpreted independently in a blinded fashion. Positive findings were not noted in any benign nevi at aCGH analysis, whereas substantial unbalanced genomic aberrations were revealed in 92% of melanomas. Positive results were obtained in 72% of melanomas via the four-probe FISH assay (RREB1/MYB/CEP6/CCND1). A few additional FISH studies were performed to verify some aCGH findings of focal amplification of oncogenes and homozygous deletion of tumor suppressor genes. The overall concordance in aberrations detected using the two methods was 90%. Most discrepancies were due to a minor abnormal clone identified via FISH that was below analytical sensitivity of the FFPE aCGH test. Our study demonstrated that copy number analysis of FFPE tumor samples via aCGH is a robust and reliable method in diagnosis of melanoma and that aCGH and FISH tests should be used as complementary methods to improve the accuracy of genetic evaluation of melanocytic tumors.

Initial treatment patterns over time for anaplastic oligodendroglial tumors.

Anaplastic oligodendroglial tumors are rare neoplasms with no standard approach to treatment. We sought to determine patterns of treatment delivered over time and identify clinical correlates of specific strategies using an international retrospective cohort of 1013 patients diagnosed from 1981-2007. Prior to 1990, most patients received radiotherapy (RT) alone as initial postoperative treatment. After 1990, approximately 50% of patients received both RT and chemotherapy (CT) sequentially and/or concurrently. Treatment with RT alone became significantly less common (67% in 1980-1984 vs 5% in 2005-2007, P < .0001). CT alone was more frequently administered in later years (0% in 1980-1984 vs 38% in 2005-2007; P < .0001), especially in patients with 1p19q codeleted tumors (57% of codeleted vs 4% with no deletion in 2005-2007; P < .0001). Temozolomide replaced the combination of procarbazine, lomustine, and vincristine (PCV) among patients who received CT alone or with RT (87% vs 2% in 2005-2007). In the most recent time period, patients with 1p19q codeleted tumors were significantly more likely to receive CT alone (with temozolomide), whereas RT with temozolomide was a significantly more common treatment strategy than either CT or RT alone in cases with no deletion (P < .0001). In a multivariate polytomous logistic regression model, the following were significantly associated with type of treatment delivered: date (5-year interval) of diagnosis (P < .0001), 1p19q codeletion (P < .0001), pure anaplastic oligodendroglioma histology (P < .01), and frontal lobe predominance (P < .05). Limited level 1 evidence is currently available to guide treatment decisions, and ongoing phase III trials will be critical to understanding the optimal therapy.

The checkpoint kinase inhibitor AZD7762 potentiates chemotherapy-induced apoptosis of p53-mutated multiple myeloma cells.

DNA cross-linking agents are frequently used in the treatment of multiple myeloma-generating lesions, which activate checkpoint kinase 1 (Chk1), a critical transducer of the DNA damage response. Chk1 activation promotes cell survival by regulating cell-cycle arrest and DNA repair following genotoxic stress. The ability of AZD7762, an ATP-competitive Chk1/2 inhibitor to increase the efficacy of the DNA-damaging agents bendamustine, melphalan, and doxorubicin was examined using four human myeloma cell lines, KMS-12-BM, KMS-12-PE, RPMI-8226, and U266B1. The in vitro activity of AZD7762 as monotherapy and combined with alkylating agents and the "novel" drug bortezomib was evaluated by studying its effects on cytotoxicity, signaling, and apoptotic pathways. The Chk1/2 inhibitor AZD7762 potentiated the antiproliferative effects of bendamustine, melphalan, and doxorubicin but not bortezomib in multiple myeloma cell lines that were p53-deficient. Increased γH2AX staining in cells treated with bendamustine or melphalan plus AZD7762 indicates a greater degree of DNA damage with combined therapy. Abrogation of the G(2)-M checkpoint by AZD7762 resulted in mitotic catastrophe with ensuing apoptosis evidenced by PARP and caspase-3 cleavage. In summary, the cytotoxic effects of bendamustine, melphalan and doxorubicin on p53-deficient multiple myeloma cell lines were enhanced by the coadministration of AZD7762. These data provide a rationale for testing these combinations in patients with relapsed and/or refractory multiple myeloma.

Will a peripheral blood (PB) sample yield the same diagnostic and prognostic cytogenetic data as the concomitant bone marrow (BM) in myelodysplasia?

In patients with myelodysplastic syndromes (MDS), chromosome anomalies are detected by conventional cytogenetic studies (CCS) and/or interphase fluorescence in situ hybridization (FISH) of bone marrow (BM) samples and provide prognostic and diagnostic information, which can direct therapy. Whether peripheral blood (PB) can be substituted for bone marrow in these cases and can provide the same information remains unknown. Concurrent BM and PB specimens collected from 100 patients with recently diagnosed MDS were studied using both CCS and FISH. While 68% of BM samples showed an abnormal karyotype by CCS, only 31% of PB samples were abnormal by CCS. In 12% of patients, FISH and CCS were discordant due to the inability of the FISH panel to detect all possible abnormalities. However, only one case (1%) had a cryptic abnormality detected by FISH. BM and PB FISH were discordant in 3% of cases, most likely due to the smaller clone size in PB vs. BM. While PB should not be substituted for BM at diagnosis, it is a viable alternative for monitoring patients using the appropriate FISH probe(s).

IDH mutation and neuroglial developmental features define clinically distinct subclasses of lower grade diffuse astrocytic glioma.

PURPOSE: Diffuse gliomas represent the most prevalent class of primary brain tumor. Despite significant recent advances in the understanding of glioblastoma [World Health Organization (WHO) IV], its most malignant subtype, lower grade (WHO II and III) glioma variants remain comparatively understudied, especially in light of their notable clinical heterogeneity. Accordingly, we sought to identify and characterize clinically relevant molecular subclasses of lower grade diffuse astrocytic gliomas. EXPERIMENTAL DESIGN: We conducted multidimensional molecular profiling, including global transcriptional analysis, on 101 lower grade diffuse astrocytic gliomas collected at our own institution and validated our findings using publically available gene expression and copy number data from large independent patient cohorts. RESULTS: We found that IDH mutational status delineated molecularly and clinically distinct glioma subsets, with IDH mutant (IDH mt) tumors exhibiting TP53 mutations, platelet-derived growth factor receptor (PDGFR)A overexpression, and prolonged survival, and IDH wild-type (IDH wt) tumors exhibiting EGFR amplification, PTEN loss, and unfavorable disease outcome. Furthermore, global expression profiling revealed three robust molecular subclasses within lower grade diffuse astrocytic gliomas, two of which were predominantly IDH mt and one almost entirely IDH wt. IDH mt subclasses were distinguished from each other on the basis of TP53 mutations, DNA copy number abnormalities, and links to distinct stages of neurogenesis in the subventricular zone. This latter finding implicates discrete pools of neuroglial progenitors as cells of origin for the different subclasses of IDH mt tumors. CONCLUSION: We have elucidated molecularly distinct subclasses of lower grade diffuse astrocytic glioma that dictate clinical behavior and show fundamental associations with both IDH mutational status and neuroglial developmental stage.

Chromothripsis in a Case of TP53-Deficient Chronic Lymphocytic Leukemia.

We describe genomic findings in a case of CLL with del(17p13.1) by FISH, in which SNP array analysis revealed chromothripsis, a phenomenon by which regions of the cancer genome are shattered and recombined to generate frequent oscillations between two DNA copy number states. The findings illustrate the value of SNP arrays for precise whole genome profiling in CLL and for the detection of alterations that would be overlooked with a standard FISH panel. This second report of chromothripsis in CLL indicates that this phenomenon is a recurrent change in this disease.

International retrospective study of over 1000 adults with anaplastic oligodendroglial tumors.

Treatment for newly diagnosed anaplastic oligodendroglial tumors is controversial. Radiotherapy (RT) alone and in combination with chemotherapy (CT) are the most well studied strategies. However, CT alone is often advocated, especially in cases with 1p19q codeletion. We retrospectively identified 1013 adults diagnosed from 1981-2007 treated initially with RT alone (n = 200), CT + RT (n = 528), CT alone (n = 201), or other strategies (n = 84). Median overall survival (OS) was 6.3 years and time to progression (TTP) was 3.1 years. 1p19q codeletion correlated with longer OS and TTP than no 1p or 19q deletion. In codeleted cases, median TTP was longer following CT + RT (7.2 y) than following CT (3.9 y, P = .003) or RT (2.5 y, P < .001) alone but without improved OS; median TTP was longer following treatment with PCV alone than temozolomide alone (7.6 vs. 3.3 y, P = .019). In cases with no deletion, median TTP was longer following CT + RT (3.1 y) than CT (0.9 y, P = .0124) or RT (1.1 y, P < .0001) alone; OS also favored CT + RT (median 5.0 y) over CT (2.2 y, P = .02) or RT (1.9 y, P < .0001) alone. In codeleted cases, CT alone did not appear to shorten OS in comparison with CT + RT, and PCV appeared to offer longer disease control than temozolomide but without a clear survival advantage. Combined CT + RT led to longer disease control and survival than did CT or RT alone in cases with no 1p19q deletion. Ongoing trials will address these issues prospectively.

MET expression and amplification in patients with localized gastric cancer.

BACKGROUND: MET, the receptor for hepatocyte growth factor, has been proposed as a therapeutic target in gastric cancer. This study assessed the incidence of MET expression and gene amplification in tumors of Western patients with gastric cancer. METHODS: Tumor specimens from patients enrolled on a preoperative chemotherapy study (NCI 5700) were examined for the presence of MET gene amplification by FISH, MET mRNA expression by quantitative PCR, MET overexpression by immunohistochemistry (IHC), and for evidence of MET pathway activation by phospho-MET (p-MET) IHC. RESULTS: Although high levels of MET protein and mRNA were commonly encountered (in 63% and 50% of resected tumor specimens, respectively), none of these tumors had MET gene amplification by FISH, and only 6.6% had evidence of MET tyrosine kinase activity by p-MET IHC. CONCLUSIONS: In this cohort of patients with localized gastric cancer, the presence of high MET protein and RNA expression does not correlate with MET gene amplification or pathway activation, as evidenced by the absence of amplification by FISH and negative p-MET IHC analysis. IMPACT: This article shows a lack of MET amplification and pathway activation in a cohort of 38 patients with localized gastric cancer, suggesting that MET-driven gastric cancers are relatively rare in Western patients.

Genetic abnormalities in non-Hodgkin's lymphoma as revealed by conventional and molecular cytogenetics methods of analyses.

Malignant non-Hodgkin's lymphoma (NHL) is a heterogeneous group of tumors, the histological classification of which based on morphologic evaluation alone is not always possible. Various technological advances in cytogenetics combined with molecular approaches have greatly enhanced our ability to identify genetic abnormalities in any given tumor type. The genetic abnormalities identified with the combination of these methods of analysis have resulted in various histological subtypes of NHL being linked with specific genetic abnormalities. Such a classification based on specific abnormalities has lead to the realization that the same abnormalities associated with initiation, transformation, and progression of the disease have also served as markers of diagnosis, prognosis, and predisposition to a given tumor type, and some abnormalities also served as markers for therapeutic targets. Results of such studies in NHL have not only identified the subsets of various histological types based on specific abnormalities, but, as is evident from recent literature, also set the stage for further evaluation using high-resolution array comparative genomic hybridization (CGH) and expression profiling.