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PDF file - 45KB, Radiation sensitivity after FANCD2 knockdown.

Oncoprint of all somatic alterations detected on MSK-IMPACT

PDF file - 147KB, Changes in mTOR signaling and FANCD2 levels in treatment groups.

Tables S1-S6

supplemental legend

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a 5-year survival rate Experimental Design: Peripheral blood was collected from 73 patients presenting with previously untreated metastatic PDAC. Extensive immunologic profiling was conducted to assess relationships between OS and the level of soluble plasma biomarkers or detailed immune cell phenotypes as measured by flow cytometry.Results: Higher baseline levels of the immunosuppressive cytokines IL6 and IL10 were strongly associated with poorer OS (P = 0.008 and 0.026, respectively; HR = 1.16 and 1.28, respectively), whereas higher levels of the monocyte chemoattractant MCP-1 were associated with significantly longer OS (P = 0.045; HR = 0.69). Patients with a greater proportion of antigen-experienced T cells (CD45RO+) had longer OS (CD4 P = 0.032; CD8 P = 0.036; HR = 0.36 and 0.61, respectively). Although greater expression of the T-cell checkpoint molecule CTLA-4 on CD8+ T cells was associated with significantly shorter OS (P = 0.020; HR = 1.53), the TIM3 molecule had a positive association with survival when expressed on CD4+ T cells (P = 0.046; HR = 0.62).Conclusions: These data support the hypothesis that baseline immune status predicts PDAC disease course and overall patient survival. To our knowledge, this work represents the largest cohort and most comprehensive immune profiling of treatment-naïve metastatic PDAC patients to date. Clin Cancer Res; 22(10); 2565–74. ©2015 AACR.

PDF file - 64KB

Table S1. Antibodies used for flow cytometry Table S2. Monte Carlo cross-validation of continuous measure markers. Table S3. Monte Carlo cross-validation of dichotomous measure markers. Table S4. Pancreatic cancer patient circulating immune cell frequencies Figure S1. Overall survival does not predict plasma MCP-1 levels

Purpose: Alveolar rhabdomyosarcoma that harbors the PAX3–FOXO1 fusion gene (t-ARMS) is a common and lethal subtype of this childhood malignancy. Improvement in clinical outcomes in this disease is predicated upon the identification of novel therapeutic targets.Experimental Design: Robust mouse models were used for in vivo analysis, and molecular studies were performed on xenografts treated in parallel. Two independent patient sets (n = 101 and 124) of clinically annotated tumor specimens were used for analysis of FANCD2 levels and its association with clinical and molecular characteristics and outcomes.Results: Our xenograft studies reveal a selective suppression of FANCD2 by m-TOR kinase inhibition and radiosensitization of the t-ARMS line only. In the initial patient set, we show that FANCD2 transcript levels are prognostic in univariate analysis, and are significantly associated with metastatic disease and that the copresence of the translocation and high expression of FANCD2 is independently prognostic. We also demonstrate a significant and nonrandom enrichment of mTOR-associated genes that correlate with FANCD2 gene expression within the t-ARMS samples, but not within other cases. In the second patient set, we show that on a protein level, FANCD2 expression correlates with PAX3–FOXO1 fusion gene and is strongly associated with phospho-P70S6K expression in cases with the fusion gene.Conclusions: Our data demonstrate that FANCD2 may have a significant role in the radiation resistance and virulence of t-ARMS. Indirectly targeting this DNA repair protein, through mTOR inhibition, may represent a novel and selective treatment strategy. Clin Cancer Res; 20(14); 3884–95. ©2014 AACR.

PDF file - 83KB, Skin toxicity scores during radiation treatments.

Purpose:Gemcitabine and albumin-bound paclitaxel (ABP) exhibit synergistic antitumor efficacy, with ABP serving to increase the intratumoral gemcitabine concentration. Both drugs are active in squamous cell lung cancers (SQCLC) and are conventional partners for carboplatin. We hypothesized that combining gemcitabine and ABP would enhance the antitumor activity in patients with advanced SQCLCs.Patients and Methods:This was a Simon two-stage, open-label, single-arm, multicenter phase II study that enrolled patients between August 1, 2015 and June 1, 2018. We enrolled 37 patients with chemotherapy-naïve, PD-L1 low/unknown advanced stage IV SQCLC. Patients were administered weekly intravenous gemcitabine (1,000 mg/m2) plus ABP (100 mg/m2) in a 3-week on, 1-week off schedule during stage I and a 2-week on, 1-week off schedule in stage II. The primary endpoint was best objective response rate (ORR). Next-generation sequencing by MSK-IMPACT was used to calculate tumor mutation burden and genome doubling and assess somatic variants for correlations with efficacy.Results:Thirty-two patients were evaluable for response assessment. The study satisfied its primary endpoint, with confirmed partial responses in 18 of 32 patients and a complete response in 1 patient [ORR 59%; 95% confidence interval (CI), 42%–74%]. Median progression-free survival (PFS), a secondary endpoint, was 7.5 (95% CI, 6.7–10.5) months. There were no unexpected toxicities.Conclusions:Gemcitabine plus ABP was a safe, tolerable, and effective first-line therapy for patients with chemotherapy-naïve SQCLCs, with an ORR and median PFS substantially higher than carboplatin doublet regimens and efficacy comparable with carboplatin plus taxane plus pembrolizumab.

PDF file - 149KB, Table 1. Treatment schema. Table 2. Radiation enhancement and calculations. Table 3. Demographics and univariate analysis. Table 4. mTOR associated genes in GESA.

The recommended treatment for patients with unresectable stage 3 non-small cell lung cancer (NSCLC) is definitive chemoradiation followed by 1 year of maintenance durvalumab. Our objective was to assess the rate of maintenance durvalumab use after chemoradiation.Analyses were conducted in both open claims (IQVIA pharmacy and medical claims data) and adjudicated closed claims (IQVIA PharMetrics Plus Health Plan Claims Database). Patients with a lung cancer diagnosis between November 2017 and November 2020 who received definitive chemoradiation were included.Of the 5802 NSCLC patients included in the open claims source, 1794 (31%) received durvalumab, 1403 (24%) received maintenance chemotherapy, and 2605 (45%) did not receive any maintenance therapy. Of the 239 NSCLC patients included in the closed claims source, 127 (53%) received durvalumab, 40 (17%) received maintenance chemotherapy, and 72 (30%) did not receive any maintenance therapy. The most common maintenance chemotherapy agents patients received were carboplatin, pemetrexed, and paclitaxel.The rate of durvalumab utilization was overall low in both the open and closed claims data sources (31% and 53%, respectively). It remains unknown what percent of eligible patients end up receiving durvalumab, as our analysis was unable to filter out patients who were unfit for durvalumab or if they had progression after chemoradiation. Future efforts are needed to increase maintenance durvalumab utilization and to determine how best to manage patients who are unfit for durvalumab.

The tumor suppressor protein p53 is a key regulator of cell cycle control, apoptosis, and genomic stability in response to various cellular stresses. Loss of function of p53 causes an inability by the cell to regulate these important processes and results in uncontrolled cell proliferation, which leads to tumor growth. Mutations in the TP53 gene are commonly associated with cancers, and for this reason TP53/p53 is one of the most important genes in cancer research. PRIMA-1 targets mutated p53 protein and has been shown to restore its wild type function and to induce apoptosis in human tumor cells, but the underlying mechanisms behind it are still not well understood. Micro RNAs (miRNAs) are small, noncoding RNA molecules that act as gene expression regulators. The involvement of miRNA in restoring tumor suppressor function of mutant p53 by Prima-1 remains unclear. The aim of this research is to investigate the associations between expression of miRNA and the PRIMA-1 anticancer treatment. We have developed a lung tumor model in transgenic mice in which the human mutant p53(273H) is expressed in a lung specific manner, under the control of the surfactant protein C promoter. These mice developed lung adenocarcinomas at an age range of 12-15 months. A micro-CT scanner was used on the mice to screen for lung tumors. To evaluate feasibility and preliminary activity of PRIMA-1 on the murine spontaneous lung tumor, we treated 4 lung tumor bearing mice via intraperitoneal (i.p.) injection with PRIMA-1 at a dose of 100 mg/kg in 0.2 ml PBS every other day for two weeks. A control group containing 3 tumor bearing mice was treated with PBS only. After two weeks of treatment, all mice were then sacrificed for tissue collection. Total RNA was isolated with Trizol method from the frozen lung tumor tissues and matching lung tissues. miRNA array was carried out with a NanoString counter. miRNA expression level was compared between the treated and control groups. After comparing 599 miRNA expressions in lung tumor tissue between PRIMA-1 treated and non-treated mice, 26 miRNAs were changed at least 3-fold. From these 26, 23 of were decreased and only 3 were increased. Of particular interest was that 9 miRNAs changed at least 6 folds post Prima-1 treatment as comparing to control group. These were miR-194, miR-1937a, 1937b, miR-34c, miR-192, miR-1949, miR-2135, miR-3472, miR-712, and miR-1931. Because PRIMA-1 restores the tumor suppression function of mutant p53 and to induce cell cycle arrest or apoptosis in tumor cells containing mutant p53, PRIMA-1 may act as an anti-cancer drug through regulating miRNAs. Because the great majority of the miRNA expression levels among the miRNA with bigger expression changes in PRIMA-1 treated group comparing to the non-treated controls are reduced, it implies that these miRNAs may promote tumor development. Mir-194, for example, has been reported to have tumor suppressor function in lung tissue. Citation Format: Javier A. Feito, Abeer Almiman, Li Gao, Shirley Tang, Kathleen Dotts, Miguel Villalona-Calero, Wenrui Duan. miRNA expression of spontaneous non-small cell lung cancers in p53 mutant transgenic mice following exposure to PRIMA-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3546.

Purpose: Biliary cancers (BCs) respond poorly to chemotherapy. Lapatinib is a dual inhibitor of epidermal growth factor receptor (EGFR) and HER2/neu, both implicated in cholangiocarcinogenesis. This trial was designed to determine the safety and efficacy of lapatinib in BC. Methods: A Fleming phase II design with a single stage of 25 patients was used. The dose of lapatinib was 1,500 mg/day administered orally in 28-day cycles. Tumor and blood specimens were analyzed for expression of HER2/neu and EGFR.Results:Nine patients with BC enrolled in this study. The study was terminated early because of futility. The most common toxicities were nausea and fatigue (78%) and diarrhea (67%). No responses were observed. Of 8 evaluable patients, 4 (50%) had stable disease. Median progression-free survival was 2.6 months (95% CI 1.6–4.4) and median overall survival was 5.1 months (95% CI 2.0–16.5). No somatic mutations in EGFR (exons 18–21) or HER2/neu were found. We did not find evidence of HER2 overexpression. Conclusions:Lapatinib is well tolerated but failed to show activity as a single agent in treating patients with BC. Despite the small patient population, our study is consistent with previous findings, suggesting that targeting HER2/neu does not appear to be an effective therapy for BC.

Rad51c is a tumor suppressor gene and known for its function in homologous recombination and DNA repair in early and late stages of HR (Homologous Recombination). Studies from breast and ovarian cancer patients reveled a bi-allelic homozygous germline missense mutation in the codon 773 and 258 of Rad51C gene. At the cellular level the effects include chromosomal instability (increased chromosomal breakage) associated with hypersensitivity to DNA damaging agents, and defective DNA damage repair. Mutations, which include base insertions and missense, have been reported in the Rad51C gene. Alternatively spliced transcriptomes have been shown to play a different or antagonistic biological role compared to the full length wild type mRNA. Some of the important diseases caused by cis acting or trans acting protein splicing factors include cystic fibrosis, dementia, premature aging and cancer. In addition, promoter methylation has been associated with gene silencing, while DNA methylation at both intronic and exonic regions are shown to correlate with isoform-specific transcription by alternative splicing or by utilizing alternate promoters. DNA methylation at the cytosine residues (5-methylcytosine) of the CpG di-nucleotides is carried out by DNA methyltransferases (DNMT) and is generally considered to be a repressive epigenetic modification. By RT-PCR, we identified splice variants of Rad51C gene that include variant-1 (without exon-7), variant-2 (without exon-6, 7) and variant-3 (without exon-7, 8) in colorectal tumors. Of the 38 colorectal tumors, 18 contained variant 1, 12 contained variant 2, 14 contained variant 3, and eight had no expression of any of the variants. Bisulfate DNA sequencing and Methylation Specific PCR (MS-PCR) showed promoter methylation of Rad51C in tumor cells. 5-Azacytidine treatment of LS-174T cells caused a 14 fold increase in variant 1, a 4.8 fold increase for variant 3 and 3.4 fold for variant 2 compared to no treatment. Real time PCR analysis of 9 pair-matched colorectal tumors and non-tumors showed that variant 1 was overexpressed in tumors comparing to matched non-tumors. Expression of Rad51C variants in these tumors was associated with microsatellite stability and with maintenance of functionality of the fanconi anemia repair pathway. In vitro transient overexpression of Rad51C variant-1 in colorectal tumor cells over the wild type Rad51C caused a 1.8 fold increase in the cell proliferation as analyzed by BrdU immunofluorescence staining and FACS. Given that the Rad51C splice variant-1 is overexpressed in colorectal tumors and has a role in promoting cell proliferation, further investigation regarding its potential role in oncogenesis in colorectal tumor cells is warranted. Citation Format: Arjun Kalvala, Li Gao, Kathleen Dotts, Fernando Ochoa Cortes, Brittany Barnwell, Greg Otterson, Miguel Villalona Calero, Wenrui Duan. Potential oncogenic function of Rad51C splice variant in colorectal tumors. [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 3866. doi:10.1158/1538-7445.AM2015-3866

Background Several studies showed that gain-of-function somatic mutations affecting the catalytic domain of EGFR in non-small cell lung carcinomas were associated with response to gefitinib and erlotinib, both EGFR-tyrosine kinase inhibitors. In addition, 4% of non-small cell lung carcinomas were shown to have ERBB2 mutations in the kinase domain. In our study, we sought to determine if similar respective gain-of-function EGFR and ERBB2 mutations were present in hepatoma and/or biliary cancers. Methods We extracted genomic DNA from 40 hepatoma (18) and biliary cancers (22) samples, and 44 adenocarcinomas of the lung, this latter as a positive control for mutation detection. We subjected those samples to PCR-based semi-automated double stranded nucleotide sequencing targeting exons 18–21 of EGFR and ERBB2. All samples were tested against matched normal DNA. Results We found 11% of hepatoma, but no biliary cancers, harbored a novel ERBB2 H878Y mutation in the activating domain. Conclusion These newly described mutations may play a role in predicting response to EGFR-targeted therapy in hepatoma and their role should be explored in prospective studies.

Fanconi anemia (FA) is a rare inherited chromosomal instability genetic disorder characterized by congenital musculoskeletal abnormalities, bone marrow failure and cancer susceptibility. At the genetic level, monoubiquitination of FANCD2 and I by an FA core complex (FANCA, B, C, E, F, G M and L) is impaired. Monoubiquitinated FANCD2 and I co-localize with DNA damage response proteins such as Rad51C (FANCO) to mediate homologous recombination (HR) repair. Defects in Rad51C have been documented as the cause of FA complementation group O (FANCO) disorder.In cells the effects include chromosomal instability, hypersensitivity to DNA damaging agents and defective DNA damage repair. Germline mutations for Rad51C gene in patients have been reported in breast and ovarian cancers. Although germline mutations for Rad51C gene are known to cause an FA like phenotype, somatic mutations for Rad51C in colon or other tumors have not been previously described. We evaluated 40 formalin fixed paraffin embedded (FFPE) colorectal adenocarcinomas by an immunofluorescence based triple staining method (FATSI) that we have developed to assess for potential somatic functional deficiency of the FA pathway. Thirteen of the 40 (33%) FFPE samples had been noted to lack FANCD2 foci formation. Among the 40 tumors, we analyzed 31 available frozen tumors that included all 13 foci negative tumors and matching non-tumor tissues for mutations in Rad51C coding region. The tumor and non-tumor tissue samples were procured following an IRB protocol of the Ohio State University. RNA and DNA were isolated from the fresh frozen tissue samples with TRIzol reagent. Direct sequencing of PCR amplicons was performed. Two of the 31 tumor DNA samples contained point mutation at the codon 319 (from CGA to TGA, a stop codon) which technically results in a truncated FANCO protein. The Rad51C protein domain shows amino acid 125 to132 (exon 2 to 5) is important for exerting single stranded DNA dependent ATPase activity, a functional nuclear localization signal located from aa 366 to 370 (exon-9). The mutation substitutes arginine at codon 319 to a premature stop codon terminating the protein abruptly, without producing a full length Rad51C protein. Thus the altered protein is unable to localize in the nucleus. Of interest, both tumors with RAD51C mutations lacked formation of FANCD2 foci by FATSI staining. In both cases, the mutation was absent from the matched non-tumor tissues, suggesting a somatic mutation. The underlining molecular alterations that cause deficiency in FANCD2 foci formation in other 11 foci negative tumors are still under investigation currently. Given that the FA pathway plays essential role in response to DNA interstrand cross-link damage agent, and that cancers with defective FA pathway are expected to be more sensitive to cross-link based therapy, our findings have the potential significance of identifying a subpopulation among colorectal cancer patients specially susceptible to these type of treatments. Citation Format: Arjun Kalvala, Li Gao, Brittany Barnwel, Greg Otterson, Miguel A Villalona Calero, Wenrui Duan. Mutations in Rad51C in colon tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4221. doi:10.1158/1538-7445.AM2013-4221