Ribonucleotide Reductase M1 Research Articles

Rapid immunohistochemical analysis of pancreatic cytology from endoscopic ultrasound-guided fine-needle aspirates: A prospective clinical trial.

400 Background: Acquisition of pancreatic cancer (PC) tissue specimens from endoscopic ultrasound-guided fine needle aspirates (EUS-FNA) is crucial to investigational pancreatic cancer trials seeking to utilize molecular profile directed therapy. Methods: In an ongoing prospective clinical trial we have utilized molecular profiling of EUS-FNA specimens from patients with resectable and borderline resectable pancreatic cancers. Cytologic specimens were evaluated for six biomarkers to guide the choice of neoadjuvant therapy: secreted protein acid rich in cysteine (SPARC), thymidylate synthase (TYMS), ribonucleotide reductase M1 (RRM1), human equilibrative nucleoside transporter 1 (ENT1), excision repair cross-complementing 1 (ERCC), and topoisomerase 1 (TOPO). Final immunohistochemical (IHC) interpretation was scored by a single pathologist using both staining intensity and percent immunochemically reactive cells. Results: The trial has enrolled 99 patients to date; 47 (47%) resectable patients and 52 (52%) borderline resectable patients. No patient experienced a EUS-FNA related complication. IHC profiling was reported in a median of 5 business days (IQR:3). Of the 99 patient samples, 73 (74%) had adequate cellularity for IHC profiling and this was not affected by stage of disease (n = 35, resectable; n = 38 borderline resectable; p = 0.82). Analysis of SPARC expression was limited to specimens with adequate stromal cells for analysis (n = 50, 51%). Among the 73 patients with adequate tissue for profiling, expression profiling was interpreted to be favorable for the following therapeutic agents: nab-paclitaxel, (SPARC, n = 35, 48%), 5-fluorouracil (TYMS, n = 68, 93%), gemcitabine (RRM1, n = 34, 47%; ENT1, n = 38, 52%), platinum agents (ERCC, n = 30, 41%), and irinotecan (TOPO, n = 62, 85%). Conclusions: The use of EUS-FNA specimens for molecular diagnostics is feasible and IHC analysis was possible in 74% of patient specimens, with preservation of stromal components in over 50%. Further refinement of molecular techniques may expand the breadth of analysis which may be performed, to include quantitative polymerase chain reaction and genetic sequencing Clinical trial information: NCIT01726582.

MicroRNA-101-3p reverses gemcitabine resistance by inhibition of ribonucleotide reductase M1 in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDA) is among the most lethal malignancies and resistance to chemotherapy prevents the therapeutic outcome. MicroRNAs provide a novel therapeutic strategy. Here, the established and primary human PDA cell lines PANC-1, AsPC-1, MIA-PaCa2, AsanPaCa, BxPC-3 and three gemcitabine-resistant subclones were examined. A gene expression profiling revealed that the ribonucleotide reductase M1 (RRM1) was upregulated in gemcitabine-resistant cells, which was confirmed by qRT-PCR, Western blot analysis and immunostaining. Inhibition of RRM1 by lipotransfection of siRNA reduced its expression and reversed gemcitabine resistance. The expression of RRM1 correlated to gemcitabine resistance in vitro and was higher in malignant patient pancreas tissue compared to non-malignant pancreas tissue. By microRNA expression profiling, we identified microRNA-101-3p as top-downregulated candidate. Lipotransfection of microRNA-101-3p mimics inhibited the expression of RRM1, reduced the luciferase activity of its 3'UTR and sensitized for gemcitabine-induced cytotoxicity. These results underline the relevance of microRNA-101-3p-driven regulation of RRM1 in drug resistance and suggest the co-delivery of microRNA-101-3p and gemcitabine for more effective therapy outcome.

Inhibition of hepatitis B virus replication by targeting ribonucleotide reductase M2 protein

Chronic hepatitis B virus (HBV) infection is a key factor for hepatocellular carcinoma worldwide. Ribonucleotide reductase (RR) regulates the deoxyribonucleoside triphosphates biosynthesis and serves as a target for anti-cancer therapy. Here, we demonstrate that RR is essential for HBV replication and the viral covalently-closed-circular DNA (cccDNA) synthesis in host liver cells. By performing computer-assisted virtual screening against the crystal structure of RR small subunit M2 (RRM2), osalmid, was identified as a potential RRM2-targeting compound. Osalmid was shown to be 10-fold more active in inhibiting RR activity than hydroxyurea, and significantly inhibited HBV DNA and cccDNA synthesis in HepG2.2.15 cells. In contrast, hydroxyurea and the RR large subunit (RRM1)-inhibitory drug gemcitabine showed little selective activity against HBV replication. In addition, osalmid also was shown to possess potent activity against a 3TC-resistant HBV strain, suggesting utility in treating drug-resistant HBV infections. Interestingly, osalmid showed synergistic effects with lamivudine (3TC) in vitro and in vivo without significant toxicity, and was shown to inhibit RR activity in vivo, thus verifying its in vivo function. Furthermore, 4-cyclopropyl-2-fluoro-N-(4-hydroxyphenyl) benzamide (YZ51), a novel derivative of osalmid, showed higher efficacy than osalmid with more potent RR inhibitory activity. These results suggest that RRM2 might be targeted for HBV inhibition, and the RRM2-targeting compound osalmid and its derivative YZ51 could be a novel class of anti-HBV candidates with potential use for hepatitis B and HBV-related HCC treatment.

Analysis of ERCC1, BRCA1, RRM1 and TUBB3 as predictors of prognosis in patients with non-small cell lung cancer who received cisplatin-based adjuvant chemotherapy: A prospective study.

Although adjuvant platinum-based chemotherapy has been demonstrated to improve survival in patients with completely resected non-small cell lung cancer (NSCLC), individualized approaches to therapy are urgently required to improve the treatment efficacy and reduce unnecessary toxicity. It was hypothesized in the present study that the protein levels of excision repair cross-complementation group 1 (ERCC1), breast cancer 1 (BRCA1), ribonucleotide reductase M1 (RRM1) and class III β-tubulin (TUBB3) may influence the therapeutic effect of adjuvant cisplatin-based chemotherapy. The expression of ERCC1, BRCA1, RRM1 and TUBB3 in tissues obtained from 84 patients with NSCLC was analyzed in the present non-interventional study by immunohistochemistry prior to adjuvant chemotherapy. All patients received adjuvant cisplatin-based chemotherapy. The primary endpoint in the present study was disease free survival (DFS). Out of the 84 tumors, the expression of ERCC1, BRCA1, RRM1 and TUBB3 was identified in 46 (55%), 11 (13%), 73 (87%) and 76 (90%) tissues, respectively. A beneficial response to adjuvant cisplatin-based chemotherapy in DFS was associated with the absence of the expression of ERCC1 [hazard ratio (HR), 2.166; 95% confidence interval (CI), 1.049-4.474; P=0.037] and BRCA1 (HR, 2.419; 95% CI, 1.127-5.193; P=0.023), but not with the expression status of RRM1 (HR, 0.568; 95% CI, 0.234-1.379; P=0.212) or TUBB3 (HR, 1.874; 95% CI, 0.448-7.842; P=0.39). In addition, patients lacking the expression of ERCC1 and BRCA1 benefited more from adjuvant cisplatin-based chemotherapy compared with patients that expressed either ERCC1 or BRCA1 (HR, 3.102; 95% CI, 1.343-7.163; P=0.008). The expression of ERCC1 and BRCA1 was significantly associated with the DFS time in patients with NSCLC treated with adjuvant cisplatin-based chemotherapy, respectively. The combination of the ERCC1 and BRCA1 expression levels may be a promising prognostic prediction for adjuvant cisplatin-based chemotherapy.

Targeting Ribonucleotide Reductase M2 and NF-κB Activation with Didox to Circumvent Tamoxifen Resistance in Breast Cancer.

Tamoxifen is widely used as an adjuvant therapy for patients with estrogen receptor (ERα)-positive tumors. However, the clinical benefit is often limited because of the emergence of drug resistance. In this study, overexpression of ribonucleotide reductase M2 (RRM2) in MCF-7 breast cancer cells resulted in a reduction in the effectiveness of tamoxifen, through downregulation of ERα66 and upregulation of the 36-kDa variant of ER (ERα36). We identified that NF-κB, HIF1α, and MAPK/JNK are the major pathways that are affected by RRM2 overexpression and result in increased NF-κB activity and increased protein levels of EGFR, HER2, IKKs, Bcl-2, RelB, and p50. RRM2-overexpressing cells also exhibited higher migratory and invasive properties. Through time-lapse microscopy and protein profiling studies of tamoxifen-treated MCF-7 and T-47D cells, we have identified that RRM2, along with other key proteins, is altered during the emergence of acquired tamoxifen resistance. Inhibition of RRM2 using siRRM2 or the ribonucleotide reductase (RR) inhibitor didox not only eradicated and effectively prevented the emergence of tamoxifen-resistant populations but also led to the reversal of many of the proteins altered during the process of acquired tamoxifen resistance. Because didox also appears to be a potent inhibitor of NF-κB activation, combining didox with tamoxifen treatment cooperatively reverses ER-α alterations and inhibits NF-κB activation. Finally, inhibition of RRM2 by didox reversed tamoxifen-resistant in vivo tumor growth and decreased in vitro migratory and invasive properties, revealing a beneficial effect of combination therapy that includes RRM2 inhibition to delay or abrogate tamoxifen resistance.

ATPS-84HYDROXYUREA SENSITIZES PATIENT-DERIVED GLIOBLASTOMA TUMORS TO TEMOZOLOMIDE IRRESPECTIVE OF MGMT STATUS

Glioblastoma (GBM) is the most common and most aggressive primary malignant brain tumor. Standard-of-care treatment involves radiation, chemotherapy (temozolomide; TMZ), and maximal surgical resection of the tumor. The 5-year survival rate of patients with GBM is <10%, a colossal failure that has been partially attributed to intrinsic and/or acquired resistance to TMZ through MGMT tumor status. Through drug screening, we identified hydroxyurea (HU), an FDA-approved drug, to sensitize GBM cells to TMZ. HU synergized with TMZ in both newly diagnosed GBM with different MGMT status as well as recurrent, TMZ-resistant, tumors in culture as well as in different quantitative intracranial in vivo models. HU acts specifically on the S-phase of the cell cycle by inhibiting the enzyme ribonucleotide reductase M2. Knockdown of this enzyme using RNA interference and other known inhibitors exerted a similar effect to HU in combination with TMZ both in vitro and in vivo. Although HU has been previously evaluated in malignant gliomas in combination with radiation or cytotoxic chemotherapy, and has shown limited efficacy, it was never evaluated in combination with TMZ. In summary, we demonstrate preclinical efficacy of ribonucleotide reductase inhibition using hydroxyurea in combination with TMZ for the treatment of glioblastoma, which warrant further evaluation in a clinical setting.

Cytidine Deaminase as a Molecular Predictor of Gemcitabine Response in Patients with Biliary Tract Cancer

Objective: Gemcitabine-based chemotherapy is regarded as the standard treatment for biliary tract cancer (BTC). Potential biomarkers for gemcitabine response include the activities of cytidine deaminase (CDA), human equilibrative nucleoside transporter 1 (hENT1), deoxycytidine kinase (DCK), and ribonucleotide reductase M1 (RRM1). Here, we investigated whether single nucleotide polymorphisms (SNPs) in their encoding genes were associated with the efficacy of gemcitabine chemotherapy in treating BTC. Methods: We retrospectively evaluated 11 SNPs in the CDA, hENT1, DCK, human concentrative nucleoside transporter 3 (hCNT3), and RRM1 genes in 80 patients with unresectable, metastatic, or recurrent BTC who were treated with gemcitabine plus cisplatin. Results: After the results were adjusted for clinical predictors, the variant allele of rs1048977 in the CDA gene was associated with tumor response in a dominant model (OR, 0.23; 95% CI, 0.06-0.93; p = 0.039). No significant association was detected between the 11 SNPs and grade 3/4 toxicity. Conclusions: Our findings suggest that the polymorphism of CDA may be a potential predictive marker for the efficacy of gemcitabine-based chemotherapy in patients with BTC.

Abstract 1961: Anti-endocrine therapy in breast cancerresistance can be circumvented by targeting ribonucleotide reductase M2 usingthe pharmaceutical agent Didox

Abstract Breast Cancers (BC) that are sensitive to anti-endocrine therapies such as tamoxifen develop resistance despite their initial favorable response. To gain mechanistic insight on endocrine resistance, gene expression profiling was performed to determine the transcripts that are differentially expressed post-tamoxifen therapy under conditions of prolonged drug treatment and development of resistance. This resistance outcome is the result of the cancer cells invoking survival functions that are up-regulated leading to enhanced cell proliferation and cell motility networks. These networks were validated in a larger panel of breast cancer cells. Here, we report the activation of Growth factor signaling and IKK-βsignaling, NFκB signaling, in BC cells upon acquired resistance to ER blockade by anti-endocrine therapy. Moreover, ribonucleotide reductase (RRM2) was revealed as a key contributor to acquired tamoxifen resistance. An RNA interference screen demonstrated that suppression of RRM2 by the RR inhibitor Didox results in down-regulation of Her2 and EGFR, modulates ER-α signaling, alters NFκB activity and expression, and decreases anti-apoptotic pathways. Indeed, concomitant treatment of tamoxifen and Didox causes blockade of ER and RRM2 induced prolonged ER inhibition and severely impairs the growth of resistant tumor cells. Collectively, these results identify distinct mechanisms that mediate secondary resistance to anti-endocrine therapies, all of which reactivate RR signaling. These observations provide a rational strategy to overcome the multifaceted clonal heterogeneity that emerges when tumors are treated with targeted agents. We propose that Didox and an anti-hormonal therapy should be tested in BC patients who become refractory to anti-endocrine therapies. Citation Format: Khyati N. Shah, Howard L. Elford, Jesika S. Faridi. Anti-endocrine therapy in breast cancerresistance can be circumvented by targeting ribonucleotide reductase M2 usingthe pharmaceutical agent Didox. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1961. doi:10.1158/1538-7445.AM2015-1961

RNA expression of genes involved in cytarabine metabolism and transport predicts cytarabine response in acute myeloid leukemia.

Variation in terms of outcome and toxic side effects of treatment exists among acute myeloid leukemia (AML) patients on chemotherapy with cytarabine (Ara-C) and daunorubicin (Dnr). Candidate Ara-C metabolizing gene expression in primary AML cells is proposed to account for this variation. Ex vivo Ara-C sensitivity was determined in primary AML samples using MTT assay. mRNA expression of candidate Ara-C metabolizing genes were evaluated by RQPCR analysis. Global gene expression profiling was carried out for identifying differentially expressed genes between exvivo Ara-C sensitive and resistant samples. Wide interindividual variations in ex vivo Ara-C cytotoxicity were observed among samples from patients with AML and were stratified into sensitive, intermediately sensitive and resistant, based on IC50 values obtained by MTT assay. RNA expression of deoxycytidine kinase (DCK), human equilibrative nucleoside transporter-1 (ENT1) and ribonucleotide reductase M1 (RRM1) were significantly higher and cytidine deaminase (CDA) was significantly lower in ex vivo Ara-C sensitive samples. Higher DCK and RRM1 expression in AML patient's blast correlated with better DFS. Ara-C resistance index (RI), a mathematically derived quotient was proposed based on candidate gene expression pattern. Ara-C ex vivo sensitive samples were found to have significantly lower RI compared with resistant as well as samples from patients presenting with relapse. Patients with low RI supposedly highly sensitive to Ara-C were found to have higher incidence of induction death (p = 0.002; RR: 4.35 [95% CI: 1.69-11.22]). Global gene expression profiling undertaken to find out additional contributors of Ara-C resistance identified many apoptosis as well as metabolic pathway genes to be differentially expressed between Ara-C resistant and sensitive samples. This study highlights the importance of evaluating expression of candidate Ara-C metabolizing genes in predicting ex vivo drug response as well as treatment outcome. RI could be a predictor of ex vivo Ara-C response irrespective of cytogenetic and molecular risk groups and a potential biomarker for AML treatment outcome and toxicity. Original submitted 22 December 2014; Revision submitted 9 April 2015.

Fasting cycles potentiate the efficacy of gemcitabine treatment in in vitro and in vivo pancreatic cancer models.

Pancreatic cancer (PC) is ranked as the fourth leading cause of cancer-related deaths worldwide. Despite recent advances in treatment options, a modest impact on the outcome of the disease is observed so far. Short-term fasting cycles have been shown to potentiate the efficacy of chemotherapy against glioma. The aim of this study was to assess the effect of fasting cycles on the efficacy of gemcitabine, a standard treatment for PC patients, in vitro and in an in vivo pancreatic cancer mouse xenograft model. BxPC-3, MiaPaca-2 and Panc-1 cells were cultured in standard and fasting mimicking culturing condition to evaluate the effects of gemcitabine. Pancreatic cancer xenograft mice were subjected to 24h starvation prior to gemcitabine injection to assess the tumor volume and weight as compared to mice fed ad libitum. Fasted pancreatic cancer cells showed increased levels of equilibrative nucleoside transporter (hENT1), the transporter of gemcitabine across the cell membrane, and decreased ribonucleotide reductase M1 (RRM1) levels as compared to those cultured in standard medium. Gemcitabine was more effective in inducing cell death on fasted cells as compared to controls. Consistently, xenograft pancreatic cancer mice subjected to fasting cycles prior to gemcitabine injection displayed a decrease of more than 40% in tumor growth. Fasting cycles enhance gemcitabine effect in vitro and in the in vivo PC xenograft mouse model. These results suggest that restrictive dietary interventions could enhance the efficacy of existing cancer treatments in pancreatic cancer patients.

Advanced squamous lung carcinoma in a patient experiencing long-term survival following repeated responses to gemcitabine and cisplatin chemotherapy: A case report.

The cisplatin and gemcitabine regimen is one of the most effective regimens against advanced non-small-cell lung cancer. However, tumors that are initially sensitive to chemotherapy treatment may acquire drug resistance. Excision repair cross complementation 1 gene (ERCC1) is involved in the repair of DNA damage caused by cisplatin, and ribonucleotide reductase M1 subunit (RRM1) is associated with gemcitabine resistance in tumor cells. The current study reports the case of a patient with advanced squamous cell lung carcinoma exhibiting low ERCC1 and RRM1 expression levels, who experienced long-term survival following repeated responses to gemcitabine and cisplatin chemotherapy. This case indicates that selected patients may benefit from multiple courses of gemcitabine and cisplatin chemotherapy, and the sustained clinical benefits suggest that further investigation into individualized therapy is merited.

MP47-20 IDENTIFICATION OF ACTIONABLE TARGETS IN CHROMOPHOBE RENAL CELL CARCINOMA DETECTED BY MULTIPLATFORM MOLECULAR ANALYSIS

You have accessJournal of UrologyKidney Cancer: Basic Research II1 Apr 2015MP47-20 IDENTIFICATION OF ACTIONABLE TARGETS IN CHROMOPHOBE RENAL CELL CARCINOMA DETECTED BY MULTIPLATFORM MOLECULAR ANALYSIS Philip Abbosh, Sherri Millis, Nancy Doll, Adam Hauben, Sandeep Reddy, Daniel Geynisman, and Robert Uzzo Philip AbboshPhilip Abbosh More articles by this author , Sherri MillisSherri Millis More articles by this author , Nancy DollNancy Doll More articles by this author , Adam HaubenAdam Hauben More articles by this author , Sandeep ReddySandeep Reddy More articles by this author , Daniel GeynismanDaniel Geynisman More articles by this author , and Robert UzzoRobert Uzzo More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.1540AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Chromophobe renal cell carcinoma (chRCC) is a rare kidney cancer subtype that infrequently metastasizes or recurs after definitive local therapy. Limited biomarker data exists to prognosticate outcome or predict response to therapy. We explored the utility of a CLIA-certified biomarker panel which uses immunohistochemistry (IHC) and targeted next generation sequencing (tNGS) to assess presence of druggable targets with the intent of developing targeted therapy protocols in patients with (chRCC). Because of the non-aggressive nature of small chRCCs, we excluded T1a tumors. METHODS Cases were identified from 65,000+ tumors submitted for commercial testing at Caris Life Sciences. All tissues were internally reviewed by a dedicated genitourinary pathologist to confirm diagnosis. Markers were analyzed on tumor tissue removed during partial or radical nephrectomy. IHC was performed on 19 proteins. FISH was performed on EGFR, HER2, and TOP2A. tNGS was performed on 47 genes. RESULTS Twelve patients with chRCC underwent biomarker panel testing. Five patients had metastatic disease. Median age was 54 years old; 50% of patients were male. One patient had a T1b tumor, two patients had T2 tumors, 8 patients had T3 tumors, and one had a T4 tumor. Thymidylate synthase (TS), ribonucleotide reductase M1 (RRM1), and O-6-methylguanine-DNA methyltransferase (MGMT) expression was absent in 12/12, 11/12 and 11/12 tumors, respectively. PTEN expression was absent in 7/12 of tumors. cKIT and PDGFR was expressed in 6/9 and 0/4 tumors, respectively. Lastly, point mutations in APC and TP53 were detected in 1/7 and 3/7 tumors, respectively. No mutations were identified in the other 45 genes tested and no changes in copy number were identified. CONCLUSIONS Biomarker analysis showed that advanced chRCC might be amenable to chemotherapy with 5-fluorouracil, gemcitabine, or temozolamide because of absent TS, RRM1, and MGMT expression, respectively. Such rationale has been used successfully in targeted treatment of other cancers. In addition, sunitinib or imatinib but not sorafenib might be preferred tyrosine kinase inhibitors, as cKIT but not PDGFR was detected. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e559 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Philip Abbosh More articles by this author Sherri Millis More articles by this author Nancy Doll More articles by this author Adam Hauben More articles by this author Sandeep Reddy More articles by this author Daniel Geynisman More articles by this author Robert Uzzo More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Ribonucleotide reductase M2 is a promising molecular target for the treatment of oral squamous cell carcinoma.

In our previous study, ribonucleotide reductase M2 (RRM2) was identified as a cancer-related gene commonly overexpressed in human oral squamous cell carcinoma (OSCC) cell lines. Herein, we attempted to determine whether targeting RRM2 may be a plausible therapeutic approach for the treatment of patients with OSCC. First, we examined the expression levels of RRM2 in human OSCC cell lines and tissues. Overexpression of RRM2 in OSCC was confirmed by western blot analysis. Subsequently, we investigated the effects of a synthetic small interfering RNA specific for RRM2 and gemcitabine (GEM), an inhibitor of RRM2 enzymatic activity, on the growth of human OSCC cell lines and primary cultured cells. Targeting RRM2 by RNA interference almost completely suppressed the expression of RRM2 and markedly suppressed the growth of both types of cells by >54.8%. GEM also reduced the growth rate of these cells by >83.0%. Finally, we evaluated the antitumor effects of GEM, cisplatin (CDDP), 5-fluorouracil (5-FU), and docetaxel (DOC) against OSCC cells using the collagen gel droplet embedded culture drug sensitivity test. OSCC cells were more sensitive to GEM and DOC than to CDDP and 5-FU, regardless of the expression level of RRM2 mRNA. These results suggested that RRM2 supported the growth of human OSCC cells and that targeting of RRM2, e.g., via GEM treatment, may be a promising therapeutic strategy for OSCC.

Identification of actionable targets in chromophobe renal cell carcinoma detected by multiplatform molecular analysis.

451 Background: Chromophobe renal cell carcinoma (chRCC) is a rare kidney cancer subtype that infrequently metastasizes or recurs after definitive local therapy. Limited biomarker data exists to prognosticate outcome or predict response to therapy. We explored the utility of multiplatform tumor profiling, which uses immunohistochemistry (IHC) and targeted next generation sequencing (tNGS) to identify druggable targets, with the intent of developing targeted therapy protocols in patients with (chRCC). Because of the non-aggressive nature of small chRCCs, we excluded T1a tumors. Methods: Cases were identified from 65,000+ tumors submitted for commercial testing at Caris Life Sciences. All tissues were internally reviewed by a dedicated genitourinary pathologist to confirm diagnosis. Markers were analyzed on tumor tissue removed during partial or radical nephrectomy. IHC was performed on 19 proteins. FISH was performed on EGFR, HER2, and TOP2A. tNGS was performed on 47 genes. Results: Twelve patients with chRCC were profiled. Five patients had metastatic disease. Median age was 54 years old; 50% of patients were male. One patient had a T1b tumor, two patients had T2 tumors, 8 patients had T3 tumors, and one had a T4 tumor. Thymidylate synthase (TS), ribonucleotide reductase M1 (RRM1), and O-6-methylguanine-DNA methyltransferase (MGMT) expression was absent in 12/12, 11/12 and 11/12 tumors, respectively. PTEN expression was absent in 7/12 of tumors. cKIT and PDGFR was expressed in 6/9 and 0/4 tumors, respectively. Lastly, point mutations in APC and TP53 were detected in 1/7 and 3/7 tumors, respectively. No mutations were identified in the other 45 genes tested and no changes in copy number were identified. Conclusions: Biomarker analysis showed that advanced chRCC might be amenable to chemotherapy with 5-fluorouracil, gemcitabine, or temozolamide because of absent TS, RRM1, and MGMT expression, respectively. Such rationale has been used successfully in targeted treatment of other cancers. In addition, sunitinib or imatinib but not sorafenib might be preferred tyrosine kinase inhibitors, as cKIT but not PDGFR was detected.

Importance of excision repair cross-complementation group 1 and ribonucleotide reductase M1 as prognostic biomarkers in malignant pleural mesothelioma treated with platinum-based induction chemotherapy followed by surgery

Survival and response to platinum-based induction chemotherapy are heterogeneous among patients with malignant pleural mesothelioma. The aim of the present study was to assess the prognostic role of DNA repair markers, such as excision repair cross-complementation group 1 and ribonucleotide reductase M1, in multimodally treated patients with malignant pleural mesothelioma. Tumor tissue of a malignant pleural mesothelioma cohort (n = 107) treated with platinum/gemcitabine (n = 46) or platinum/pemetrexed (n = 61) induction chemotherapy followed by extrapleural pneumonectomy was assembled on a tissue microarray. Immunohistochemical expression of excision repair cross-complementation group 1 (nuclear) and ribonucleotide reductase M1 (nuclear and cytoplasmic) was assessed for its prognostic impact (association with overall survival or freedom from recurrence). Patients with high nuclear ribonucleotide reductase M1 expression before chemotherapy showed significantly longer freedom from recurrence (P = .03). When specifically analyzed in the subgroup of patients receiving platinum/gemcitabine followed by extrapleural pneumonectomy, high nuclear ribonucleotide reductase M1 was associated with prolonged freedom from recurrence (P = .03) and overall survival (P = .02). Low excision repair cross-complementation group 1 expression in prechemotherapy tumor tissues was associated with significantly longer freedom from recurrence (P = .04). Nuclear ribonucleotide reductase M1 and excision repair cross-complementation group 1 were independent prognosticators of freedom from recurrence in addition to pT stage in multivariate analysis. In the present study, nuclear ribonucleotide reductase M1 and excision repair cross-complementation group 1 expression were identified as independent prognosticators for freedom from recurrence of malignant pleural mesothelioma in patients undergoing induction chemotherapy followed by extrapleural pneumonectomy.

Biomarker expression and druggable gene alterations for development of an appropriate therapeutic protocol for pulmonary adenosquamous carcinoma.

Pulmonary adenosquamous carcinoma (ASC) is more aggressive than adenocarcinoma (AC) and squamous cell carcinoma (SCC). The genetic features and biomarkers of ASC are not well known. Here, we attempted to identify potential therapeutic markers for ASC. Surgically resected ASC samples from 65 patients were analysed. We examined the expression of β III-tubulin, thymidylate synthase, breast cancer susceptibility gene 1 and ribonucleotide reductase M1 (RRM1); identified mutations in epidermal growth factor receptor (EGFR), KRAS, BRAF and HER2; and detected ALK, ROS1 and RET rearrangements. Gene amplification and expression of EGFR, human epidermal growth factor receptor 2 (HER2), fibroblast growth factor receptor-1 and MET were also examined. β III-Tubulin showed the highest expression (P = 0.002), and its expression was more frequent in the AC than in the SCC component (P = 0.013). RRM1 expression was more frequent in the SCC component (P = 0.046). EGFR and KRAS mutations were detected in both components (21.5 and 10.9%, respectively). ALK and ROS1 rearrangements and MET amplification were detected in both components in one (1.5%) case. In ASC, drug response-specific gene alterations could occur in both AC and SCC components, suggesting that patients with confirmed or suspected ASC should undergo further testing for driver gene analyses.

Therapeutic approach guided by genetic alteration: Use of MTOR inhibitor in renal medullary carcinoma with loss of PTEN expression

Renal Medullary Cancer (RMC) is a rare and aggressive type of renal cell cancer that presents predominantly in patients with sickle cell hemoglobinopathies, and is typically metastatic at the time of presentation. Although platinum based chemotherapeutic regimens have recently emerged as the best option for producing a clinically significant response as reported in various case series, the response is far from satisfactory, as most RMC patients still succumb to their disease within a year of diagnosis. There is currently no standard of care for treatment of this disease. We report, to our knowledge, the first case of RMC where in molecular characterization of the tumor was used to guide therapy. In our patient, molecular analysis identified a decreased expression of Ribonucleotide Reductase M1(RRM1) and phosphatase and tensin homolog (PTEN). Based on these results of PTEN deficiency, we started our patient on everolimus (an MTOR inhibitor) maintenance after treating him with an induction chemotherapy regimen of Paclitaxel-Cisplatin-Gemcitabine (PCG). His tumor responded to induction therapy and he went into complete remission and remained in remission for 7 months. He is now alive about 14 months from his diagnosis and is asymptomatic with minimal disease. The rarity of RMC makes it very difficult to do any meaningful clinical trials in this group of patients. The overall prognosis for RMC remains very poor and knowledge about driver mutations may help in guiding therapy to improve survival in this select group of patients, where there is dearth of available therapies.

The genotype of ribonucleotidereductase M1 -269C > A is associated with the response to platinum-based chemotherapy and as a prognostic biomarker in advanced nonsmall cell lung cancer.

Genetic polymorphisms of ribonucleotidereductase M1 (RRM1) was a DNA repair gene, which may affect patients' response to platinum-based chemotherapy or gemcitabine-based chemotherapy. We retrospectively assessed whether single nucleotide polymorphisms (SNPs) of RRM1 can be used to predict overall survival (OS), progression free survival and response in nonsmall cell lung cancer (NSCLC) patients treated with platinum-based regimens as first-line chemotherapy. The genotypes of four tagSNPs (RRM1 -316C > A, RRM1 -269C > A, RRM1 -702G > A and RRM1 -585T > G) were determined by SNaPshot detection technology and sequencing approaches in 184 advanced NSCLC patients by using peripheral blood. The overall response rate for 178 patients was 40.2% and the disease control rate was 90.2%. In patients who had ever smoked, a significant correlation was observed between the genotype of RRM1 -269C > A and response (P = 0.046). There was a significant difference in response according to the genotype of RRM1 -702G > A (P = 0.043). Using Log-rank test, we found that patients with the allelotype (CC) of RRM1 -269C > A had a shorter OS (P = 0.006) than the allelotype (CA + AA). The genotype of RRM1 -269C > A was significantly associated with platinum-based chemotherapy sensitivity in smoking patients and can be used to predict OS in advanced NSCLC patients who received platinum-based chemotherapy or gemcitabine-based chemotherapy. And the genotype of RRM1 -702G > A can serve as a biomarker for chemotherapy sensitivity in advanced NSCLC patients.

Molecular Profiling of Refractory Adrenocortical Cancers and Predictive Biomarkers to Therapy.

PURPOSECurrent first-line chemotherapy for patients with metastatic adrenocortical cancer (ACC) includes doxorubicin, etoposide, cisplatin, and mitotane with a reported response rate of only 23.2%. New therapeutic leads for patients with refractory tumors are needed; there is no standard second-line treatment.METHODSSamples from 135 ACC tumors were analyzed by immunohistochemistry, in situ hybridization (FISH or CISH), and/or gene sequencing at a single commercial reference laboratory (Caris Life Sciences) to identify markers associated with drug sensitivity and resistance.RESULTSOverexpression of proteins related to demonstrated chemotherapy sensitivity or resistance included topoisomerase 1, progesterone receptor, and topoisomerase 2-alpha in 46%, 63%, and 42% of cases, respectively. Loss of excision repair cross-complementary group 1 (ERCC1), phosophatase and tensin homolog, O(6)-methylguanine-methyltransferase, and ribonucleotide reductase M1 (RRM1) was identified in 56%, 59%, 71%, and 58% of cases, respectively. Other aberrations included overexpression of programmed death-ligand 1 or programmed cell death protein 1 tumor-infiltrating lymphocytes in >40% of cases. In all, 35% of cases had a mutation in the canonical Wnt signaling pathway (either CTNNB1 or APC) and 48% had a mutation in TP53. No other genomic alterations were identified.CONCLUSIONBiomarker alterations in ACC may be used to direct therapies, including recommendations for and potential resistance of some patients to traditional chemotherapies, which may explain the low response rate in the unselected population. Limited outcomes data support the use of mitotane and platinum therapies for patients with low levels of the proteins RRM1 and ERCC1.

Expression of Ribonucleotide Reductase Subunit-2 and Thymidylate Synthase Correlates with Poor Prognosis in Patients with Resected Stages I-III Non-Small Cell Lung Cancer.

Biomarkers can help to identify patients with early-stages or locally advanced non-small cell lung cancer (NSCLC) who have high risk of relapse and poor prognosis. To correlate the expression of seven biomarkers involved in DNA synthesis and repair and in cell division with clinical outcome, we consecutively collected 82 tumour tissues from radically resected NSCLC patients. The following biomarkers were investigated using IHC and qRT-PCR: excision repair cross-complementation group 1 (ERCC1), breast cancer 1 (BRCA1), ribonucleotide reductase subunits M1 and M2 (RRM1 and RRM2), subunit p53R2, thymidylate synthase (TS), and class III beta-tubulin (TUBB3). Gene expression levels were also validated in an available NSCLC microarray dataset. Multivariate analysis identified the protein overexpression of RRM2 and TS as independent prognostic factors of shorter overall survival (OS). Kaplan-Meier analysis showed a trend in shorter OS for patients with RRM2, TS, and ERCC1, BRCA1 overexpressed tumours. For all of the biomarkers except TUBB3, the OS trends relative to the gene expression levels were in agreement with those relative to the protein expression levels. The NSCLC microarray dataset showed RRM2 and TS as biomarkers significantly associated with OS. This study suggests that high expression levels of RRM2 and TS might be negative prognostic factors for resected NSCLC patients.