Loss Of Rb Research Articles

Elemental and radiogenic isotope perspective on formation and transformation of cratonic lower crust: Central Slave craton (Canada)

Kimberlite-borne granulite xenoliths provide rare insights into the age, chemical composition and tectonothermal evolution of the otherwise largely inaccessible deep cratonic crust. The formation and transformation of the lower continental crust (LCC) beneath the central Slave craton (Canada) is here illuminated using whole-rock trace-element and Sr-Nd isotope compositions of nine metabasaltic (MBG), one gabbroic (MGG) and two metasedimentary/hybrid (MSG) granulite xenoliths. On the one hand, published sulphide Re-Os and a few zircon U-Pb data indicate that at least a portion of the LCC beneath the central Slave craton has a Palaeoarchaean origin (∼3.3 Ga), which apparently coincides with a period of juvenile crust and deep lithospheric mantle formation during plume impingement beneath the pre-existing cratonic nucleus. On the other hand, enrichment in Li, Sr, LREE, Pb and Th, but relative depletion in Ti, Hf and HREE, suggest formation of (picro)basaltic protoliths by partial melting of a subduction-modified garnet-bearing source, Crystallisation in the crust after fractionation of plagioclase is inidicated by their Sr and Eu negative anomalies, which are complementary to the positive anomalies in the MGG. Samarium-Nd isotopes in MBG and MGG show large scatter, but fall on Neo- or Mesoarchaean age arrays. These elemental systematics are suggested to fingerprint deserpentinisation fluids plus small amounts of sedimentary melt as the main contaminants of the mantle source, supporting the operation of at least regional and transient subduction at 3.3 Ga. Evidence for quasi-coeval plume impingement and subduction beneath the central Slave craton in the Mesoarchaean is reconcilable in a dynamic regime where vertical tectonics, though waning, was still active and plate interactions became increasingly important. Unradiogenic 87Sr/86Sr (down to 0.7017) is consistent with significant loss of Rb and probably other heat-producing elements (K, Th, U) plus H2O during Neoarchaean metamorphism, which helped to enhance LCC viscosity and stabilise the cratonic lithosphere.

Whole-Genome and Transcriptional Analysis of Treatment-Emergent Small-Cell Neuroendocrine Prostate Cancer Demonstrates Intraclass Heterogeneity.

Therapeutic resistance in metastatic castration-resistant prostate cancer (mCRPC) can be accompanied by treatment-emergent small-cell neuroendocrine carcinoma (t-SCNC), a morphologically distinct subtype. We performed integrative whole-genome and -transcriptome analysis of mCRPC tumor biopsies including paired biopsies after progression, and multiple samples from the same individual. t-SCNC was significantly less likely to have amplification of AR or an intergenic AR-enhancer locus, and demonstrated lower expression of AR and its downstream transcriptional targets. Genomic and transcriptional hallmarks of t-SCNC included biallelic loss of RB1, elevated expression levels of CDKN2A and E2F1, and loss of expression of the AR and AR-responsive genes including TMPRSS2 and NKX3-1. We identified three tumors that converted from adenocarcinoma to t-SCNC and demonstrate spatial and temporal intrapatient heterogeneity of metastatic tumors harboring adenocarcinoma, t-SCNC, or mixed expression phenotypes, with implications for treatment strategies in which dual targeting of adenocarcinoma and t-SCNC phenotypes may be necessary. IMPLICATIONS: The t-SCNC phenotype is characterized by lack of AR enhancer gain and loss of RB1 function, and demonstrates both interindividual and intraindividual heterogeneity.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/17/6/1235/F1.large.jpg.

Microarray testing as an efficient tool to redefine hyperdiploid paediatric B-cell precursor acute lymphoblastic leukaemia patients

The aim of our study was to characterize genetic alterations in a cohort of paediatric patients with B-cell progenitors (BCP-ALL) and a hyperdiploid karyotype. In our study, we analysed 55 childhood hyperdiploid BCP-ALL patients using single nucleotide polymorphism (SNP) microarray testing. The group consisted mostly of patients with the modal number of chromosomes between 54 and 58 (34 cases). Within this group, Trisomy 4 and Trisomy 10 (30 cases) were the most frequent cases. Additionally, a total of 93 structural abnormalities mainly affecting chromosomes 1, 6, 9, 12, and 17 as well as 68 copy number alterations (CNAs) were identified. The microarray testing revealed a loss of ETV6, IKZF1, CDKN2A/CDKN2B, PAX5, and RB1. Moreover, chromosomal abnormalities resulting in the loss of heterozygosity (LOH) were also observed. Currently, patients with hyperdiploidy constitute a genetically heterogeneous group, and therefore, it is insufficient to rely only on banding cytogenetic analysis for the identification of hyperdiploid karyotype. Microarray testing has been proven an effective and satisfactory tool for the analysis of molecular karyotypes and to redefine the prognostic criteria in hyperdiploid patients.

Intraductal carcinoma of the prostate in the absence of high-grade invasive carcinoma represents a molecularly distinct type of in situ carcinoma enriched with oncogenic driver mutations.

Intraductal carcinoma of the prostate (IDC-P) most often appears associated with high-grade invasive prostate carcinoma (PCa), where it is believed to represent retrograde spread. However, IDC-P rarely occurs as an isolated finding at radical prostatectomy or with concurrent low-grade (Grade Group 1) invasive carcinoma. We hypothesized that isolated IDC-P (iIDC-P) in these unusual cases may represent a distinct in situ lesion and molecularly profiled 15 cases. iIDC-P was characterized by copy number alteration (CNA) profiling and targeted next generation sequencing in cases with sufficient tissue (n = 7). Immunohistochemistry for PTEN and ERG was performed on the total cohort (n = 15), where areas of iIDC-P and associated invasive disease were evaluated separately (n = 9). By copy number profiling, iIDC-P alterations were similar to those previously described in high-grade invasive PCa (PTEN, RB1, and CHD1 loss; MYC gain). However, in four cases, targeted sequencing revealed a striking number of activating oncogenic driver mutations in MAPK and PI3K pathway genes, which are extraordinarily rare in conventional PCa. In addition, pathogenic mutations in DNA repair genes were found in two cases of iIDC-P (BRCA2, CHEK2, CDK12) and other known PCa-associated mutations (FOXA1, SPOP) in two cases. Overall, ERG was expressed in 7% (1/15) of the iIDC-P lesions and PTEN was lost in 53% (8/15). Discordance for ERG or PTEN status between IDC-P and the low-grade PCa was observed in five of nine cases, with intact PTEN in the invasive tumor and PTEN loss in IDC-P in four. Despite a CNA profile similar to conventional PCa, iIDC-P is enriched with potentially targetable oncogenic driver mutations in MAPK/PI3K genes. Based on PTEN and ERG status, iIDC-P is not likely a precursor to the associated low-grade invasive PCa, but represents a molecularly unique in situ tumor of unclear clinical significance. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Comparison of four DLL3 antibodies performance in high grade neuroendocrine lung tumor samples and cell cultures

BackgroundSmall cell lung cancer (SCLC) is usually diagnosed in the advanced stage. It has a very poor prognosis, with no advancements in therapy in the last few decades. A recent phase 1 clinical study, using an antibody-drug conjugate directed against DLL3, showed promising results. A prerequisite for this therapy is an immunohistochemical test for DLL3 expression. The antibody used in the clinical trial was bound to a specific platform, which is not available in all pathology laboratories. In this study, the expression of DLL3 was analyzed using different DLL3 antibodies in high-grade neuroendocrine tumors of the lung and cell cultures. Additionally, correlation of DLL3 expression with Rb1 loss and TP53 mutation was evaluated.MethodsThe study cohort consisted of surgically resected cases, 24 SCLC and 29 large cell neuroendocrine carcinoma (LCNEC), from which tissue microarrays (TMAs) were constructed. The validation cohort included 46 SCLC samples, mostly small biopsies. Additionally, well-characterized SCLC cell lines were used. Immunohistochemical analysis was performed using four different DLL3 antibodies, as well as TP53 and Rb1 antibodies. Expression was evaluated microscopically and manually scored.ResultsThe comparison of all DLL3 antibodies showed poor results for the overall agreement, as well as positive and negative agreement. Differences were observed regardless of the applied cut-off values and the tumor type. The antibody used in the clinical trial was the only which always positively stained the tumor cells obtained from cell cultures with known DLL3 expression and was negative on cells that did not express DLL3. There was no correlation between p53 and DLL3 expression in SCLC and LCNEC. RB1 loss in SCLC showed statistical significant correlation with the DLL3 positivity (p = 0.037), while no correlation was found in LCNEC.ConclusionThe DLL3 antibody used in the clinical trial demonstrated superiority in the detection of DLL3 expression. Cell cultures, which can be used for DLL3 antibodies as positive and negative probes, were established. Evidence of DLL3 expression in high proportions of patients with LCNEC might provide basis for studies of new therapy options in this group of patients.

Clinical and genomic hallmarks of low PSA secretors in metastatic castration-resistant prostate cancer (mCRPC).

5051 Background: Metastatic disease burden out of proportion to low serum PSA level is frequently used as a clinical surrogate for the diagnosis of treatment-emergent small cell neuroendocrine prostate cancer (t-SCNC), although many t-SCNC patients (pts) have normal or elevated PSA levels. The clinical and genomic characteristics of mCRPC pts who are Low PSA Secretors have not been previously described. Methods: Eligible mCRPC patients (pts) underwent image-guided needle biopsy. Formalin-fixed paraffin embedded tissue was evaluated with targeted next-generation DNA sequencing. Fresh frozen tissue from the same metastatic tumor underwent RNA-seq. A validated AR transcriptional signature was applied. Low PSA Secretors were defined as pts with PSA < 5 ng/mL plus ≥ 6 metastases on conventional imaging at the time of tumor biopsy. Clinical and genomic characteristics were compared between low PSA Secretors and all other pts. Results: Of 89 evaluable pts, 9 (10%) were identified as Low PSA Secretors. There was no difference between Low PSA Secretors and all other pts in: serum PSA at diagnosis, frequency of Gleason ≥ 8 adenocarcinoma at diagnosis, and serum level of LDH, alkaline phosphatase, or hemoglobin at the time of biopsy. Lung and/or liver metastases were more common in low PSA secretors (67% vs. 33%, p = 0.04). There was no difference in serum level of LDH, alkaline phosphatase, or hemoglobin. Tumor biopsies from Low PSA Secretors were more likely to fall within a previously defined t-SCNC transcriptional cluster (80% vs. 6%, p < 0.001). RB1 loss or inactivating mutations appeared to be enriched in Low PSA Secretors (40% vs. 12%, p = 0.09); there was no difference in frequency of TP53 alterations between subgroups. AR transcriptional signature scores were lower in the Low PSA Secretor group (median score -3.63 vs. 0.66, p < 0.001). Conclusions: Low serum PSA levels in relation to metastatic tumor burden may be a reliable surrogate for the detection of mCRPC that harbors the transcriptional and genomic hallmarks of t-SCNC. Validation studies are warranted.

Leptomeningeal spread of glioma in the molecular era.

e13537 Background: Leptomeningeal disease (LMD) is an uncommon event in patients with gliomas which is estimated to occur in 4% of cases and is associated with poor outcome. Genomic alterations in gliomas that precipitate LMD have not been characterized. Methods: We performed a retrospective chart review of patients with gliomas (grade II-IV)- for whom we have genomic testing data at The Ohio State University. We identified patients who developed LMD. LMD was defined as leptomeningeal enhancement as seen on brain or spine imaging. Patients with negative cerebrospinal fluid (CSF) cytology were not excluded if imaging was convincing of LMD. Genomic testing was performed through Foundation One. Results: 129 charts of glioma patients were reviewed. We identified 6 patients with LMD. One is still alive. All patients were diagnosed histologically as glioblastoma. One patient had Lynch syndrome. All patients were men with a median age at diagnosis of 44 (range 20-65). All tumors appeared to have extended into the ventricles on brain imaging. 4 tumors had evidence of subependymal spread prior (range 45-93 days) or at the time of LMD enhancement. 4 patients had available CSF studies: 2 of whom had evidence of atypical or malignant cells. CSF protein was elevated in all four samples (range 113-492 mg/dL). CSF glucose was low in all four samples (range < 10-46 mg/dL). 5 tumor samples were IDH wild type whereas 1 had IDH1 R132H mutation. MGMT promoter was methylated in 3 tumors and unmethylated in 3. The genomic alterations varied among samples and included EGFR V765M mutation (N = 1), PDGFR amplification (N = 1), PDGFRA Y849C subclonal mutation (N = 1), CDKN2A/B loss (N = 3), TP53 mutations (N = 4) and TERT promoter mutations (N = 3). No case had EGFR amplification or EGFRvIII mutation. Other alterations observed included DNMT3A mutation, KIT, MYC and MDM2 amplifications and RB1 losses. PD-L1 expression ranged from 5-40%. One intracranial sample had a 20% PD-L1 expression with LMD from this tumor exhibiting 40% PD-L1 expression. Of the five deceased patients (all IDH WT), median overall survival (OS) was 355 days (range 184-557). Median OS after LMD diagnosis was 84 days (range 58-225). Conclusions: In this series, 4.5% of glioma patients developed LMD. Samples were associated with a variety of genomic alterations but without a specific predictor of leptomeningeal involvement. This is possibly due to the small number of cases. Physical proximity of the tumor to the ventricles appears to be a potential risk factor. Larger studies of LMD in gliomas are warranted to identify potential genetic drivers, if any exist.

Genomic predictors of benefit of docetaxel (D) and next-generation hormonal therapy (NHT) in metastatic castration resistant prostate cancer (mCRPC).

5018 Background: Predictive genomic biomarkers in mCRPC remain elusive. Prior studies suggest that tumor suppressor (TS) loss is prognostic, and may result in less benefit from NHT, but no impact on D efficacy. We assessed genomic predictors of differential benefit of androgen receptor-targeted therapy and chemotherapy for mCRPC. Methods: Patients with mCRPC and targeted exome sequencing of biopsies obtained after metastatic diagnosis were identified (n=109). Patients with pure small cell histology (n=6) were excluded. Time from NHT or D start to clinical/radiographic progression (time to treatment failure, TTTF) was estimated by Kaplan-Meier method, with censoring at next therapy or last follow-up for non-progressors. Results: 80.1% of patients had bone and/or lymph node-only metastases at mCRPC diagnosis. In total, 87/103 (84.5%) and 61/103 (59.2%) received NHT and D for mCRPC, respectively. Median overall survival was 4.5 years from first mCRPC. The frequency and predictive association of selected recurrently-altered genes are detailed in the table. PTEN alterations (alts) were associated with worse TTTF on NHT, but not D, and a similar trend was observed with BRCA2. Biallelic RB1 loss was strongly predictive, conferring significantly shorter TTTF on both NHT and D. A score based on presence of tumor PTEN alt (1) and/or biallelic RB1 alt (1) was predictive of TTTF on NHT (median TTTF of score 0 vs 1 vs 2: 14.7 vs 12 vs 3.8 months; log rank p=0.003). Conclusions: The presence of single or compound PTEN and RB1 alts predict poorer outcomes with NHT for mCRPC. Chemotherapy may be a preferred therapeutic strategy for this patient population. [Table: see text]

Genomic profiling in ovarian cancer retreated with platinum based chemotherapy presented homologous recombination deficiency and copy number imbalances of CCNE1 and RB1 genes

BackgroundOvarian carcinomas presenting homologous recombination deficiency (HRD), which is observed in about 50% of cases, are more sensitive to platinum and PARP inhibitor therapies. Although platinum resistant disease has a low chance to be responsive to platinum-based chemotherapy, a set of patients is retreated with platinum and some of them are responsive. In this study, we evaluated copy number alterations, HR gene mutations and HR deficiency scores in ovarian cancer patients with prolonged platinum sensitivity.MethodsIn this retrospective study (2005 to 2014), we selected 31 patients with platinum resistant ovarian cancer retreated with platinum therapy. Copy number alterations and HR scores were evaluated using the OncoScan® FFPE platform in 15 cases. The mutational profile of 24 genes was investigated by targeted-NGS.ResultsThe median values of the four HRD scores were higher in responders (LOH = 15, LST = 28, tAI = 33, CS = 84) compared with non-responders (LOH = 7.5, LST = 17.5, tAI = 23, CS = 47). Patients with high LOH, LST, tAI and CS scores had better response rates, although these differences were not statistically significant. Response rate to platinum retreatment was 22% in patients with CCNE1 gains and 83.5% in patients with no CCNE1 gains (p = 0.041). Furthermore, response rate was 54.5% in patients with RB1 loss and 25% in patients without RB1 loss (p = 0.569). Patients with CCNE1 gains showed a worse progression free survival (PFS = 11.1 months vs 3.7 months; p = 0.008) and a shorter overall survival (OS = 39.3 months vs 7.1 months; p = 0.007) in comparison with patients with no CCNE1 gains. Patients with RB1 loss had better PFS (9.0 months vs 2.6 months; p = 0.093) and OS (27.4 months vs 3.6 months; p = 0.025) compared with cases with no RB1 loss. Four tumor samples were BRCA mutated and tumor mutations were not associated with response to treatment.ConclusionsHR deficiency was found in 60% of our cases and HRD medium values were higher in responders than in non-responders. Despite the small number of patients tested, CCNE1 gain and RB1 loss discriminate patients with tumors extremely sensitive to platinum retreatment.

Altered Rb, p16, and p53 expression is specific for porocarcinoma relative to poroma.

Porocarcinomas are rare aggressive carcinomas that harbor tumor suppressor mutations and must be distinguished from benign entities such as poromas. To determine whether altered expression of these genes was diagnostically informative, we examined p53, Rb, and p16 staining patterns in 15 poromas and 16 porocarcinomas. Poromas consistently displayed diffuse strong expression of Rb in all but one case that displayed focal loss (1/15, 7%), and no evidence of aberrancy in p53 or p16. Porocarcinomas displayed diffuse or focal loss of Rb expression in 9/16 (56%) cases, diffuse loss or overexpression of p53 in 8/15 (53%), and diffuse loss or overexpression of p16 in 6/14 (43%). Diffuse aberrancy in p53 and Rb expression correlated with tumor mutations in TP53 and RB1, respectively, whereas focal Rb loss was associated with wild type RB1. Diffuse p16 overexpression correlated with Rb loss rather than CDKN2A mutation. For porocarcinomas with all three stains evaluable, 10/13 (77%) displayed aberrancy in at least one marker. Our findings suggest that altered p53, p16, and/or Rb expression is relatively specific to porocarcinoma in comparison with poroma. Technical limitations of this panel, including possible focal Rb loss, must be kept in mind, especially in limited samples.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Loss of the Retinoblastoma Protein Modulates Glucose Metabolism in Lung Cancer

Dysregulated metabolism is a hallmark of cancer cells and is driven in part by specific genetic alterations in various oncogenes or tumor suppressors. The retinoblastoma protein (Rb) is a tumor suppressor that was initially characterized in regulating cell cycle progression but recent studies have highlighted a functional role for Rb in controlling cellular metabolism. As such, we aim to characterize the metabolic phenotype caused by genetic loss of Rb in a mouse model of lung cancer. We have employed the Kras+/LSLG12D/Rb1lox/lox and Kras+/LSLG12D/Rb1+/+ transgenic model to study the metabolic effects of Rb1 loss in lung cancer. Cre‐exposed Kras+/LSLG12D/Rb1lox/lox mice exhibit increased tumor burden as well as decreased overall survival compared to Kras+/LSLG12D/Rb1+/+ mice. Our tracer studies using bolus dosing of 13C‐glucose revealed an increase in glucose carbon incorporation into select glycolytic intermediates. Consistent with this result, Rb1 depleted tumors expressed higher hexokinase 2 and glucose transporter 1. Additionally, compared to Rb1‐proficient tumors, Rb1 null tumors exhibit decreased one and three carbon labeled aspartate, which is indicative of pyruvate carboxylase (PC) activity. However, Rb1−/− tumors were found to express higher PC compared to Rb1+/+ tumors, suggesting these tumors may utilize an alternative nutrient source for TCA anaplerosis other than glucose. Taken together, these data have identified several metabolic genes under Rb control, and suggest Rb may regulate differential nutrient utilization in lung tumors.Support or Funding InformationNCI RO1CA166327, American Cancer Society RSG 13‐139‐01This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Distinct Clinical Features of Plasma Cell Myeloma Patients Exhibiting Dysmorphic Plasma Cells: Association with More Plasma Cells at Diagnosis.

Dysmorphic plasma cells are occasionally found in bone marrow (BM) aspirates of plasma cell myeloma (PCM) patients. We retrospectively analyzed the incidences of significant dysmorphic plasma cells (SDPC) presentations and their associations with clinical features in PCM patients. Total 91 PCM patients diagnosed from January 2013 to December 2017 at author's institution were enrolled. SDPC presentation was determined as ≥ 5% (SDPC5) or ≥ 10% (SDPC10) among total PC and clinical features of PCM patients were compared with respect to SDPC presentation status. Incidence of SDPC5/SDPC10 presentation was 39.6%/18.7%. Patients with SDPC5/SDPC10 showed significantly more BM PC (P = 0.004/0.020) and higher incidences of CKS1B gains (P = 0.022/0.001) and RB1 loss (P = 0.032 for SDPC10 only) at diagnosis than those without SDPC5/SDPC10. Patients with SDPC5/SDPC10 also showed significantly greater absolute BM PC (P = 0.007/0.034 and 0.047/0.049 for 1st and 2nd follow-up, respectively) and serum M-protein (P = 0.041/0.044 and 0.039/0.049 for 1st and 2nd follow-up, respectively) reductions after chemotherapy than those without SDPC5/SDPC10. SDPC5/SDPC10 presentation was confirmed as an independent predictor of BM PC ≥ 37.7% [hazard ratio (HR) 4.649/2.613, P = 0.005/0.039]. Our present study demonstrated that SDPC presentation would be an independent predictor of more BM PC at diagnosis in PCM patients. Associations between SDPC presentation and higher incidence of CKS1B gains and RB1 loss, greater PC/serum monoclonal protein reductions after chemotherapy were also identified. Association between SDPC presentation and favorable treatment response should be evaluated in more comprehensive study.

Expression of MT4-MMP, EGFR, and RB in Triple-Negative Breast Cancer Strongly Sensitizes Tumors to Erlotinib and Palbociclib Combination Therapy.

Here, we investigated the clinical relevance of an unprecedented combination of three biomarkers in triple-negative breast cancer (TNBC), both in human samples and in patient-derived xenografts of TNBC (PDX-TNBC): EGFR, its recently identified partner (MT4-MMP), and retinoblastoma protein (RB).Experimental Design: IHC analyses were conducted on human and PDX-TNBC samples to evaluate the production of the three biomarkers. The sensitivity of cancer cells expressing or not MT4-MMP to anti-EGFR (erlotinib) or anti-CDK4/6 inhibitor (palbociclib) was evaluated in vitro in 2D and 3D proliferation assays and in vivo using xenografts and PDX-TNBC displaying different RB, MT4-MMP, and EGFR status after single (erlotinib or palbociclib) or combined (erlotinib + palbociclib) treatments. EGFR and MT4-MMP were coexpressed in >70% of TNBC samples and PDX-TNBC, among which approximately 60% maintained RB expression. Notably, approximately 50% of all TNBC and PDX-TNBC expressed the three biomarkers. Single erlotinib and palbociclib treatments drastically reduced the in vitro proliferation of cells expressing EGFR and MT4-MMP when compared with control cells. Both TNBC xenografts and PDX expressing MT4-MMP, EGFR, and RB, but not PDX-TNBC with RB loss, were sensitive to erlotinib and palbociclib with an additive effect of combination therapy. Moreover, this combination was efficient in another PDX-TNBC expressing the three biomarkers and resistant to erlotinib alone. We defined a new association of three biomarkers (MT4-MMP/EGFR/RB) expressed together in 50% of TNBC and demonstrated its usefulness to predict the TNBC response to anti-EGFR and anti-CDK4/6 drugs used in single or combined therapy.

Genomic drivers of poor prognosis and enzalutamide resistance in metastatic castration-resistant prostate cancer.

146 Background: Metastatic castration-resistant prostate cancer (mCRPC) is the lethal form of the disease. Several recent efforts have identified genomic alterations in mCRPC, but the clinical implications of these alterations have not been fully elucidated. We conducted a prospective cohort study (n = 101) using whole genome sequencing (WGS) to analyze the association between key driver gene alterations and overall survival. We also performed whole-transcriptome RNA sequencing (RNA-seq) analyses to identify potential mechanisms of enzalutamide resistance in mCRPC. Methods: Metastasis biopsies were obtained in 101 mCRPC patients as part of the multi-institutional West Coast Prostate Cancer Dream Team project. Samples underwent WGS and RNA-seq. The resulting mutation, copy number, and structural variant calls were integrated to determine functional copy number status of candidate genes for downstream clinical analyses. We performed univariate and multivariable analyses to assess the prognostic significance of candidate genomic events with respect to overall survival. To nominate and investigate genomic pathways associated with enzalutamide resistance, we performed expression-based gene set enrichment analysis followed by cross-sectional enrichment and survival analyses related to the top nominated pathway. Results: RB1 loss was associated with poor overall survival (median 14.1 vs. 42.0 months, p < 0.001). When we compared enzalutamide resistant versus naïve samples using gene set enrichment analysis, we identified the Wnt/beta-catenin pathway as the top differentially expressed pathway in enzalutamide-resistant patients. Furthermore, CTNNB1 (beta-catenin) activating mutations were exclusive to enzalutamide-resistant patients (p = 0.013) and predictive of poor overall survival (median 13.6 vs. 41.7 months, p < 0.001). Conclusions: Impaired survival in mCRPC patients is associated with RB1 loss, identified by integrated genomic analysis of CRPC metastasis biopsies. Among men with mCRPC that was enzalutamide-resistant, the Wnt/beta-catenin pathway is nominated as an important predictive (and potentially therapeutic) pathway.

Molecular updates in adipocytic neoplasms✰.

Adipocytic neoplasms include a diversity of both benign tumors (lipomas) and malignancies (liposarcomas), and each tumor type is characterized by its own unique molecular alterations driving tumorigenesis. Work over the past 30 years has established the diagnostic utility of several of these characteristic molecular alterations (e.g. MDM2 amplification in well- and dedifferentiated liposarcoma, FUS/EWSR1-DDIT3 gene fusions in myxoid liposarcoma, RB1 loss in spindle cell/pleomorphic lipoma). More recent studies have focused on additional molecular alterations which may have therapeutic or prognostic impact. This review will summarize several of the important molecular findings in adipocytic tumors that have been described over the past 10 years.

Updates in spindle cell/pleomorphic lipomas.

Spindle cell/pleomorphic lipomas are benign lipomatous neoplasms that show loss of RB1 and classically harbor components of mature adipose tissue, bland spindled cells and ropy collagen. This review highlights the clinicopathologic features and morphologic spectrum of spindle cell/pleomorphic lipoma, discusses an updated differential diagnosis, and provides a practical diagnostic strategy for spindle cell/pleomorphic lipomas with atypical clinical presentations.

Abstract PD7-12: Inhibition of CDK7 overcomes resistance to CDK4/6 inhibitors in hormone receptor positive breast cancer cells

Abstract Background: Despite the efficacy of the combinations of endocrine therapy (ET) and CDK4/6 inhibitors in the hormone receptor positive (HR+) metastatic breast cancer (BC) setting, the majority of patients eventually acquire resistance to these treatments. The loss of Rb activity is an important mechanism of acquired resistance to CDK4/6 inhibitors. Preclinical and clinical data support the RB1 genetic aberrations, including mutations and deletions with loss of expression, as mechanism of palbociclib (palbo) resistance. To study vulnerabilities and potential treatment targets to overcome resistance to CDK4/6 inhibitors associated with Rb loss we studied a T47D palbo-resistant (T47D PDR) HR+ BC cell model obtained by exposing palbo-sensitive cells to increasing concentrations of palbo. These cells harbour deletion of RB1, and show increased cyclin E1 and decreased ER expression. Methods: To identify genes that are essential for cell growth and genes whose loss enhances cell growth in palbo-resistant cells with loss of Rb, we performed genome-wide CRISPR-Cas9 knockout (KO) screens in T47D palbo-sensitive (T47D PDS) and T47D PDR cells. We searched for significantly essential genes that are targets with available drugs and tested the efficacy of these compounds in T47D PDS and T47D PDR cells. Results: By employing a genome wide CRISPR KO screen, we identified 29 genes that were significantly positively selected in the T47D PDR cells. Among these genes were known tumor suppressor genes, such as TSC2 (β score: 0.89) and PTEN (β score: 0.68). We identified close to 600 genes that were significantly essential (negatively selected) for T47D PDR growth. CDK4, CDK6 and CCND1 were essential in the T47D PDS parental cells but lost essentiality with the acquisition of palbo resistance (FDR> 0.05). CDK7 (β score: -1.21) and CDK2 (β score: -1.67) were among the top ranked essential genes (FDR: 0). CDK7 is a transcriptional CDK and has CDK activating kinase (CAK) activity. The CAK activity includes phosphorylation of CDK2 and CDK9. To follow up on our CRISPR screen results we tested the activity of the selective CDK7 inhibitors SY-1365 (currently in phase 1 clinical development) and THZ1 in the T47D PDS and T47D PDR cells. We saw dose dependent activity in both cell line models. Moreover, the IC50 values were comparable in T47D PDS and T47D PDR cells (THZ1; PDS: 9.93 nM, PDR with Palbo: 32.8 nM, PDR without Palbo: 1.08 nM) (SY1365; PDS: 1.60 nM, PDR with Palbo: 6.70 nM, PDR without Palbo: 1.87 nM). Combination studies of SY-1365 and fulvestrant in T47D PDR showed synergistic activity at lower concentrations. Conclusions: In a model of palbo resistance with loss of Rb we found that cyclin D1, CDK4 and CDK6 are not essential, suggesting cross-resistance to other CDK4/6 inhibitors in this setting. We identified CDK7 as a significant essential gene and potential therapeutic target. We validated these findings with the selective CDK7 inhibitors, THZ1 and SY-1365. Our results offer a new therapeutic strategy for the treatment of palbo-resistant HR+ BC. Citation Format: Guarducci C, Nardone A, Feiglin A, Migliaccio I, Malorni L, Bonechi M, Benelli M, Di Leo A, Hodgson G, Shapiro G, Brown M, Jeselsohn R. Inhibition of CDK7 overcomes resistance to CDK4/6 inhibitors in hormone receptor positive breast cancer cells [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD7-12.

Abstract PD9-02: Evolutionary analysis of 462 serial metastatic biopsies from 208 patients with estrogen receptor-positive (ER+) metastatic breast cancer (MBC) using whole exome sequencing (WES)

Abstract Background: While great strides have been made in the treatment of ER+ MBC, therapeutic resistance is nearly universal. The genomic evolution of ER+ breast cancer in the metastatic setting under the selective pressure of multiple lines of therapies is not well understood. To address this, we analyzed the clonal dynamics of serial metastatic samples (mets) to evaluate how tumors evolve and to identify acquired resistance mechanisms. Methods: We performed WES on 462 clinically annotated samples from 208 patients (pts) with ER+ MBC, including 67 primary tumor biopsies, 229 metastatic biopsies and 160 blood samples (cfDNA). Pts with multiple mets included cases with temporally concordant metastatic tumor and blood samples (48 pts) and cases with serial mets obtained over the course of treatment in the metastatic setting (69 pts). Treatments given between the serial mets included CDK4/6 inhibitors (23 pts), and selective estrogen receptor degraders (19 pts), among others. Results: In the temporally-concordant mets, we found that cfDNA mutations (muts) largely overlap with muts found in tumor biopsies, capturing >85% of clonal tumor muts. However, we observed a higher level of heterogeneity in cfDNA compared to biopsies (p.value< 1.05e-19, Welch test) and a subset of high-confidence muts that were only detected in cfDNA, including in clinically important genes such as ESR1, PIK3CA, KRAS, and ERBB2. Analysis of serial mets was used to elucidate the evolutionary dynamics within the metastatic setting under the selective pressure of treatment. The median duration between mets was 112 days and the median number of inter-biopsy unique treatments was two. Most tumors continued to evolve within the metastatic setting, with 50 out of 69 pts (72%) acquiring a meaningful sub-clone (50% increase in relative cancer cell fraction) and 31 out of 69 (45%) acquiring muts in known cancer genes, including a subset acquiring a plausible resistance alteration such as alterations that dysregulate ER (5 out of 69 pts, 7%; ESR1 mut, FOXA1 amplification (amp), NCOR1 bi-allelic deletion (del)), ERBB (4%; ERBB2 amp, ERBB3 mut), RAS (4%; KRAS mut, NRAS amp, NF1 del), FGF/FGFR (12%; FGFR2 mut, FGFR1/2 amp, FGF3 amp), and cell cycle (13%; RB1 del, CDK4 amp, AURKA amp, CDKN2A del). Finally, in pts who had multiple mets, we observed several cases of evolutionary convergence toward equivalent resistance mechanisms including convergent RB1 loss as a mechanism of resistance to a CDK4/6 inhibitor and convergent BRCA2 reversion following resistance to a PARP inhibitor. Conclusions: This study demonstrates that ER+ MBC continues to evolve under the selective pressure of treatments in the metastatic setting. These findings elucidate the challenge of studying high complexity and heavily treated tumors, while also highlighting some commonalities in the evolutionary trajectories selected by these treatments. The multiplicity of clinically relevant genomic alterations acquired in these advanced stages highlights the need for serial biopsies and the potential to inform post-progression therapeutic choices through targeting the acquired dependencies in post-progression tumors. Citation Format: Cohen O, Buendia-Buendia J, Wander S, Nayar U, Mao P, Waks A, Kim D, Freeman S, Adalsteinsson V, Helvie K, Livitz D, Rosebrock D, Leshchiner I, Dellostritto L, Garrido-Castro A, Jain E, Periyasamy S, Mackichan C, Lloyd M, Marini L, Krop I, Garraway L, Getz G, Winer E, Lin N, Wagle N. Evolutionary analysis of 462 serial metastatic biopsies from 208 patients with estrogen receptor-positive (ER+) metastatic breast cancer (MBC) using whole exome sequencing (WES) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD9-02.

Cells Lacking the RB1 Tumor Suppressor Gene Are Hyperdependent on Aurora B Kinase for Survival.

Small cell lung cancer (SCLC) accounts for 15% of lung cancers and is almost always linked to inactivating RB1 and TP53 mutations. SCLC frequently responds, albeit briefly, to chemotherapy. The canonical function of the RB1 gene product RB1 is to repress the E2F transcription factor family. RB1 also plays both E2F-dependent and E2F-independent mitotic roles. We performed a synthetic lethal CRISPR/Cas9 screen in an RB1 -/- SCLC cell line that conditionally expresses RB1 to identify dependencies that are caused by RB1 loss and discovered that RB1 -/- SCLC cell lines are hyperdependent on multiple proteins linked to chromosomal segregation, including Aurora B kinase. Moreover, we show that an Aurora B kinase inhibitor is efficacious in multiple preclinical SCLC models at concentrations that are well tolerated in mice. These results suggest that RB1 loss is a predictive biomarker for sensitivity to Aurora B kinase inhibitors in SCLC and perhaps other RB1 -/- cancers. SIGNIFICANCE: SCLC is rarely associated with actionable protooncogene mutations. We did a CRISPR/Cas9-based screen that showed that RB1 -/- SCLC are hyperdependent on AURKB, likely because both genes control mitotic fidelity, and confirmed that Aurora B kinase inhibitors are efficacious against RB1 -/- SCLC tumors in mice at nontoxic doses.See related commentary by Dick and Li, p. 169.This article is highlighted in the In This Issue feature, p. 151.