Search

Purpose: Non-small-cell lung cancer (NSCLC) accounts for the majority of lung cancer and is the most common cause of cancer death in industrialized countries. Epigenetic modifications are observed universally during the tumorigenesis of lung cancer. The development of epigenetic-modulating agents utilizing the synergism between hypomethylating agents and histone deacetylase (HDAC) inhibitors provides a novel therapeutic approach in treating NSCLC.Methods: We performed a phase I trial combining 5-aza-2'-deoxycytidine (decitabine) and valproic acid (VPA), in patients with advanced stage NSCLC. Patients were treated with escalating doses of decitabine (5-15 mg/m(2)) IV for 10 days in combination with VPA (10-20 mg/kg/day) PO on days 5-21 of a 28-day cycle. Pharmacokinetic and pharmacodynamic analysis included decitabine pharmacokinetics and fetal hemoglobin expression.Results: Eight patients were accrued to this phase I study. All patients had advanced NSCLC and had received prior chemotherapy. Eastern Cooperative Oncology Group performance status was 0-2. Major toxicities included myelosuppression and neurotoxicity. Dose-limiting toxicity was seen in two patients suffering grade 3 neurotoxicity during cycle one including disorientation, lethargy, memory loss, and ataxia at dose level 1. One patient had grade 3 neutropenia at the de-escalated dose. No objective response was observed, and stable disease was seen in one patient. Fetal hemoglobin levels increased after cycle one in all seven patients with evaluable results.Conclusions: We observed that decitabine and valproic acid are an effective combination in reactivating hypermethylated genes as demonstrated by re-expressing fetal hemoglobin. This combination in patients with advanced stage IV NSCLC, however, is limited by unacceptable neurological toxicity at a relatively low dosage. Combining hypomethylating agents with alternative HDAC inhibitors that lack the toxicity of VPA should be explored further. [ABSTRACT FROM AUTHOR]

We described previously a human natural killer (NK) cell population that upregulates PD-L1 expression upon recognizing and reacting to tumor cells or exposure to a combination of IL12, IL18, and IL15. Here, to investigate the safety and efficacy of tumor-reactive and cytokine-activated (TRACK) NK cells, human NK cells from umbilical cord blood were expanded, transduced with a retroviral vector encoding soluble (s) IL15, and further cytokine activated to induce PD-L1 expression. Our results show cryopreserved and thawed sIL15_TRACK NK cells had significantly improved cytotoxicity against non-small cell lung cancer (NSCLC) in vitro when compared with non-transduced (NT) NK cells, PD-L1+ NK cells lacking sIL15 expression (NT_TRACK NK), or NK cells expressing sIL15 without further cytokine activation (sIL15 NK cells). Intravenous injection of sIL15_TRACK NK cells into immunodeficient mice with NSCLC significantly slowed tumor growth and improved survival when compared with NT NK and sIL15 NK cells. The addition of the anti-PD-L1 atezolizumab further improved control of NSCLC growth by sIL15_TRACK NK cells in vivo. Moreover, a dose-dependent efficacy was assessed for sIL15_TRACK NK cells without observed toxicity. These experiments indicate that the administration of frozen, off-the-shelf allogeneic sIL15_TRACK NK cells is safe in preclinical models of human NSCLC and has potent antitumor activity without and with the administration of atezolizumab. A phase I clinical trial modeled after this preclinical study using sIL15_TRACK NK cells alone or with atezolizumab for relapsed or refractory NSCLC is currently underway (NCT05334329)., (©2024 American Association for Cancer Research.)

Recent data suggest that patients with advanced cancer who participate in biomarker/genomically informed early-stage clinical trials experience clinical benefit. While most early-stage clinical trials are conducted in major academic centers, the majority of cancer patients in the United States are treated in community practices. Here, we describe ongoing efforts at the City of Hope Cancer Center to integrate our network community oncology clinical practices into our academic, centralized biomarker/genomic-driven, early-stage clinical trial program to build an understanding of the approaches that provide the benefits of early-stage clinical trial participation to community patients. Our efforts include three key initiatives: the development of a virtual "Refractory Disease" phase 1 trial matching televideo clinic, the construction of infrastructure to support the expansion of phase 1 clinical trials to a distant regional clinical satellite hub, and the implementation of an enterprise-wide precision medicine, germline, and somatic testing program. Our work at City of Hope may serve as an example to facilitate similar efforts at other institutions.

Background: Prior meta-analysis of stem-cell transplantation trials for renal-cell carcinoma observed that clinical outcomes vary by subjects' order of entry, specifically their quartile of accrual. We test this hypothesis using meta-analysis of individual patient data from diverse Phase II trials conducted by an oncology consortium., Methods: Eligible were all Phase II trials in hematologic or solid tumors opened and closed by California Cancer Consortium during 2005-2020. Excluded were trials closed in first quartile or currently embargoed pending publication and subjects ineligible per protocol or untreated on study. The primary risk factor was entry by quartile of planned sample size. As a cross-protocol endpoint, primary outcome was time to discontinuation of intervention. One-stage meta-analysis used a shared frailty model with trial as random effect. As covariates, stepwise selection retained tumor type, obesity, their interaction, calendar year, entry at least 3 years post-diagnosis, and performance status but rejected age, sex, randomized design, and class of drug., Results: Twenty trials (including 8 terminated early, 2 not published) included n = 923 subjects. Most (90.6%) subjects discontinued intervention, usually for disease progression or toxicity. Independently of covariates, risk of discontinuation increased (p < 0.0001) with each quartile of entry (Hazards Ratio 1.13, 95% CI 1.06-1.22), culminating at Quartile 4 (HR 1.46, 1.36-1.57). The 95% prediction interval for the Hazards Ratio in future trials was (1.04-1.24). Progression-free survival similarly worsened by quartile of entry., Conclusion: In Phase II trials, clinical outcome worsens with quartile of entry. This finding merits independent replication, and the cause of this phenomenon merits investigation., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: M.A. Villalona-Calero: Owns stock (Moderna Inc.); Participates in a Speakers Bureau (Amgen Inc.). C.E. Behrendt, E.M. Newman, P.H. Frankel: No potential conflicts to declare., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Tyrosine kinase inhibitor (TKI) therapy is the recommended first-line treatment for metastatic non-small-cell lung cancer (NSCLC) positive for epidermal growth factor receptor (EGFR) gene mutation. However, most individuals treated with TKI therapy for EGFR-mutant NSCLC will develop tumor resistance to TKI therapy. Therapeutic strategies to overcome TKI resistance are the topic of several ongoing clinical trials. One potential strategy, which has been explored in numerous trials, is the treatment of progressive sites of disease with stereotactic body radiation treatment (SBRT) or stereotactic radiosurgery (SRS). We sought to review the literature pertaining to the use of local ablative radiation therapy in the setting of acquired resistance to TKI therapy and to discuss stereotactic radiation therapy as a strategy to overcome TKI resistance.

Background: Given the observed antitumor activity of immune-checkpoint-inhibitors in patients with mismatch-repair deficient (MSI-H) tumors, we hypothesized that deficiency in homologous-recombination-repair (HRR) can also influence susceptibility., Methods: Patients with disease progression on standard of care and for whom pembrolizumab had no FDA approved indication received pembrolizumab. Patients with MSI-H tumors were excluded. Objectives included immune-related objective response rate (iORR), progression-free survival (PFS) and 20-weeks-PFS. Pembrolizumab was given every 3 weeks and scans performed every six. We evaluated a triple-stain (FANCD2foci/DAPI/Ki67) functional assay of the Fanconi Anemia (FA) pathway: FATSI, in treated patients' archived tumors. The two-stage sample size of 20/39 patients evaluated an expected iORR≥20% in the whole population vs. the null hypothesis of an iORR≤5%, based on an assumed iORR≥40% in patients with functional FA deficiency, and < 10% in patients with intact HRR. An expansion cohort of MSI stable endometrial cancer (MS-EC) followed. Exploratory stool microbiome analyses in selected patients were performed., Results: Fifty-two patients (45F,7M;50-evaluable) were enrolled. For the 39 in the two-stage cohort, response evaluation showed 2CR,5PR,11SD,21PD (iORR-18%). FATSI tumor analyses showed 29 competent (+) and 10 deficient (-). 2PR,9SD,17PD,1NE occurred among the FATSI+ (iORR-7%) and 2CR,3PR,2SD,3PD among the FATSI(-) patients (iORR-50%). mPFS and 20w-PFS were 43 days and 21% in FATSI+, versus 202 days and 70% in FATSI(-) patients. One PR occurred in the MS-EC expansion cohort., Conclusions: Pembrolizumab has meaningful antitumor activity in malignancies with no current FDA approved indications and FA functional deficiency. The results support further evaluation of FATSI as a discriminatory biomarker for population-selected studies., (© 2022. The Author(s).)

Background: Somatic genomic testing is recommended by numerous expert guidelines to inform targeted therapy treatment for patients with advanced nonsquamous non-small cell lung cancer (aNSCLC). The NILE study was a prospective observational study that demonstrated noninferiority of cell-free circulating tumor DNA (cfDNA)-based tumor genotyping compared to tissue-based genotyping to find targetable genomic alterations in patients with newly diagnosed nonsquamous aNSCLC. As the cohort has matured, clinical outcomes data can now be analyzed., Methods: This prospective, multicenter North American study enrolled patients with previously untreated nonsquamous aNSCLC who had standard of care (SOC) tissue genotyping performed and concurrent comprehensive cfDNA analysis (Guardant360). Patients with targetable genomic alterations, as defined by NCCN guidelines, who were treated with physician's choice of therapy had objective response rates, disease control rate, and time to treatment collected and compared to published outcomes., Results: Among 282 patients, 89 (31.6%) had an actionable biomarker, as defined by NCCN, detected by tissue (21.3%) and/or cfDNA (27.3%) analysis. Sixty-one (68.5%) of these were treated with an FDA-approved targeted therapy guided by somatic genotyping results (EGFR, ALK, ROS1). Thirty-three patients were eligible for clinical response evaluation and demonstrated an objective response rate of 58% and disease control rate of 94%. Twenty-five (76%) and 17 (52%) achieved a durable response > 6 months and 12 months, respectively. The time to treatment (TtT) was significantly faster for cfDNA-informed biomarker detection as compared to tissue genotyping (18 vs. 31 days, respectively; P = .0008)., Conclusions: cfDNA detects guideline-recommended biomarkers at a rate similar to tissue genotyping, and therapeutic outcomes based on plasma-based comprehensive genomic profiling are comparable to published targeted therapy outcomes with tissue profiling, even in community-based centers., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selective killing of cancer cells underlines its anticancer potential. However, poor tolerability and resistance underscores the need to identify cancer-selective TRAIL-sensitizing agents. Apigenin, a dietary flavonoid, sensitizes lung cancer cell lines to TRAIL. It remains unknown, however, whether apigenin sensitizes primary lung cancer cells to TRAIL and its underlying mechanisms. Here we show that apigenin reprograms alternative splicing of key TRAIL/death-inducing-signaling-complex (DISC) components: TRAIL Death Receptor 5 (DR5) and cellular-FLICE-inhibitory-protein (c-FLIP) by interacting with the RNA-binding proteins hnRNPA2 and MSI2, resulting in increased DR5 and decreased c-FLIP S protein levels, enhancing TRAIL-induced apoptosis of primary lung cancer cells. In addition, apigenin directly bound heat shock protein 70 (Hsp70), promoting TRAIL/DISC assembly and triggering apoptosis. Our findings reveal that apigenin directs alternative splicing and inhibits Hsp70 enhancing TRAIL anticancer activity. These findings underscore impactful synergies between diet and cancer treatments opening new avenues for improved cancer treatments.

The Fanconi Anemia (FA) pathway is essential for human cells to maintain genomic integrity following DNA damage. This pathway is involved in repairing damaged DNA through homologous recombination. Cancers with a defective FA pathway are expected to be more sensitive to cross-link based therapy or PARP inhibitors. To evaluate downstream effectors of the FA pathway, we studied the expression of 734 different micro RNAs (miRNA) using NanoString nCounter miRNA array in two FA defective lung cancer cells and matched control cells, along with two lung tumors and matched non-tumor tissue samples that were deficient in the FA pathway. Selected miRNA expression was validated with real-time PCR analysis. Among 734 different miRNAs, a cluster of microRNAs were found to be up-regulated including an important cancer related micro RNA, miR-200C. MiRNA-200C has been reported as a negative regulator of epithelial-mesenchymal transition (EMT) and inhibits cell migration and invasion by promoting the upregulation of E-cadherin through targeting ZEB1 and ZEB2 transcription factors. miRNA-200C was increased in the FA defective lung cancers as compared to controls. AmpliSeq analysis showed significant reduction in ZEB1 and ZEB2 mRNA expression. Our findings indicate the miRNA-200C potentially play a very important role in FA pathway downstream regulation.

Background: Lung cancer is the number one cancer killer worldwide. A major drawback in the lung cancer treatment field is the lack of realistic mouse models that replicate the complexity of human malignancy and immune contexture within the tumor microenvironment. Such models are urgently needed. Mutations of the tumor protein p53 are among the most common alterations in human lung cancers., Methods: Previously, we developed a line of lung cancer mouse model where mutant human TP53-273H is expressed in a lung specific manner in FVB/N background. To investigate whether the human TP53 mutant has a similar oncogenic potential when it is expressed in another strain of mouse, we crossed the FVB/N-SPC-TP53-273H mice to A/J strain and created A/J-SPC-TP53-273H transgenic mice. We then compared lung tumor formation between A/J-SPC-TP53-273H and FVB/N-SPC-TP53-273H., Results: We found the TP53-273H mutant gene has a similar oncogenic potential in lung tumor formation in both mice strains, although A/J strain mice have been found to be a highly susceptible strain in terms of carcinogen-induced lung cancer. Both transgenic lines survived more than 18 months and developed age related lung adenocarcinomas. With micro CT imaging, we found the FVB-SPC-TP53-273H mice survived more than 8 weeks after initial detection of lung cancer, providing a sufficient window for evaluating new anti-cancer agents., Conclusions: Oncogenic potential of the most common genetic mutation, TP53-273H, in human lung cancer is unique when it is expressed in different strains of mice. Our mouse models are useful tools for testing novel immune checkpoint inhibitors or other therapeutic strategies in the treatment of lung cancer.

Background: Poly(ADP-ribose) polymerase inhibitors (PARPis) are U.S. Food and Drug Administration (FDA) approved for treatment of BRCA-mutated metastatic breast cancer. Furthermore, the BROCADE studies demonstrated benefit of adding an oral PARPi, veliparib, to carboplatin and paclitaxel in patients with metastatic breast cancer harboring BRCA mutation. Given multiple possible dosing schedules and the potential benefit of this regimen for patients with defective DNA repair beyond BRCA, we sought to find the recommended phase II dose (RP2D) and schedule of veliparib in combination with carboplatin in patients with advanced breast cancer, either triple-negative (TNBC) or hormone receptor (HR)-positive, human epidermal growth receptor 2 (HER2) negative with defective Fanconi anemia (FA) DNA-repair pathway based on FA triple staining immunofluorescence assay., Materials and Methods: Patients received escalating doses of veliparib on a 7-, 14-, or 21-day schedule with carboplatin every 3 weeks. Patients underwent [18]fluoro-3'-deoxythymidine ( 18 FLT) positron emission tomography (PET) imaging., Results: Forty-four patients (39 TNBC, 5 HR positive/HER2 negative with a defective FA pathway) received a median of 5 cycles (range 1-36). Observed dose-limiting toxicities were grade (G) 4 thrombocytopenia (n = 4), G4 neutropenia (n = 1), and G3 akathisia (n = 1). Common grade 3-4 toxicities included thrombocytopenia, lymphopenia, neutropenia, anemia, and fatigue. Of the 43 patients evaluable for response, 18.6% achieved partial response and 48.8% had stable disease. Median progression-free survival was 18.3 weeks. RP2D of veliparib was established at 250 mg twice daily on days 1-21 along with carboplatin at area under the curve 5. Patients with partial response had a significant drop in maximum standard uptake value (SUV max ) of target lesions between baseline and early in cycle 1 based on 18 FLT-PET (day 7-21; p trend  = .006)., Conclusion: The combination of continuous dosing of veliparib and every-3-week carboplatin demonstrated activity and an acceptable toxicity profile. Decrease in SUV max on 18 FLT-PET scan during the first cycle of this therapy can identify patients who are likely to have a response., Implications for Practice: The BROCADE studies suggest that breast cancer patients with BRCA mutation benefit from addition of veliparib to carboplatin plus paclitaxel. This study demonstrates that a higher dose of veliparib is tolerable and active in combination with carboplatin alone. With growing interest in imaging-based early response assessment, the authors demonstrate that decrease in [18]fluoro-3'-deoxythymidine positron emission tomography (FLT-PET) SUV max during cycle 1 of therapy is associated with response. Collectively, this study established a safety profile of veliparib and carboplatin in advanced breast cancer while also providing additional data on the potential for FLT-PET imaging modality in monitoring therapy response., (© 2020 The Authors. The Oncologist published by Wiley Periodicals LLC on behalf of AlphaMed Press.)

TP53 and K-ras mutations are two of the major genetic alterations in human nonsmall cell lung cancers. The association between these two genes during lung tumorigenesis is unknown. We evaluated the potential of two common Type I (273H, contact) and Type II (175H, conformational) TP53 mutations to induce lung tumors in transgenic mice, as well as K-ras status, and other driver mutations in these tumors. Among 516 (138 nontransgenic, 207 SPC-TP53-273H, 171 SPC-TP53-175H) mice analyzed, 91 tumors, all adenocarcinomas, were observed. Type II mutants developed tumors more frequently (as compared to nontransgenics, p = 0.0003; and Type I, p = 0.010), and had an earlier tumor onset compared to Type I (p = 0.012). K-ras mutations occurred in 21 of 50 (42%) of murine lung tumors sequenced. For both the nontransgenic and the SPC-TP53-273H transgenics, tumor K-ras codon 12-13 mutations occurred after 13 months with a peak incidence at 16-18 months. However, for the SPC-TP53-175H transgenics, K-ras codon 12-13 mutations were observed as early as 6 months, with a peak incidence between the ages of 10-12 months. Codons 12-13 transversion mutations were the predominant changes in the SPC-TP53-175H transgenics, whereas codon 61 transition mutations were more common in the SPC-TP53-273H transgenics. The observation of accelerated tumor onset, early appearance and high frequency of K-ras codon 12-13 mutations in the Type II TP53-175H mice suggests an enhanced oncogenic function of conformational TP53 mutations, and gains in early genetic instability for tumors containing these mutations compared to contact mutations., (© 2019 UICC.)

Purpose: Complete and timely tissue genotyping is challenging, leading to significant numbers of patients with newly diagnosed metastatic non-small cell lung cancer (mNSCLC) being undergenotyped for all eight genomic biomarkers recommended by professional guidelines. We aimed to demonstrate noninferiority of comprehensive cell-free DNA (cfDNA) relative to physician discretion standard-of-care (SOC) tissue genotyping to identify guideline-recommended biomarkers in patients with mNSCLC., Patients and Methods: Prospectively enrolled patients with previously untreated mNSCLC undergoing physician discretion SOC tissue genotyping submitted a pretreatment blood sample for comprehensive cfDNA analysis (Guardant360)., Results: Among 282 patients, physician discretion SOC tissue genotyping identified a guideline-recommended biomarker in 60 patients versus 77 cfDNA identified patients (21.3% vs. 27.3%; P < 0.0001 for noninferiority). In tissue-positive patients, the biomarker was identified alone (12/60) or concordant with cfDNA (48/60), an 80% cfDNA clinical sensitivity for any guideline-recommended biomarker. For FDA-approved targets ( EGFR, ALK, ROS1, BRAF ) concordance was >98.2% with 100% positive predictive value for cfDNA versus tissue (34/34 EGFR-, ALK-, or BRAF -positive patients). Utilizing cfDNA, in addition to tissue, increased detection by 48%, from 60 to 89 patients, including those with negative, not assessed, or insufficient tissue results. cfDNA median turnaround time was significantly faster than tissue (9 vs. 15 days; P < 0.0001). Guideline-complete genotyping was significantly more likely (268 vs. 51; P < 0.0001)., Conclusions: In the largest cfDNA study in previously untreated mNSCLC, a validated comprehensive cfDNA test identifies guideline-recommended biomarkers at a rate at least as high as SOC tissue genotyping, with high tissue concordance, more rapidly and completely than tissue-based genotyping. See related commentary by Meador and Oxnard, p. 4583 ., (©2019 American Association for Cancer Research.)

Background: The risk factors for immune-related adverse events (irAEs) remain undefined. Recently, a correlation between irAEs and clinical benefit was suggested. We examined the risk factors for irAEs and their effect on survival in patients with non-small-cell lung cancer (NSCLC) who had received immunotherapy., Patients and Methods: We performed a retrospective review of patients with NSCLC treated with single-agent immunotherapy at our institution. irAEs were determined by treating physician diagnosis. A landmark analysis was performed at 3 months using log-rank tests and the Bonferroni method., Results: irAEs occurred in 27 of 91 patients (30%). The median overall survival (OS) for patients with irAEs was longer than that for patients without (24.3 vs. 5.3 months; hazard ratio, 2.75; 95% confidence interval, 1.54-4.92; P < .001). However, a landmark analysis of patients after 3 months of treatment revealed no difference in OS between patients with and without irAEs. No increased risk of pneumonitis was seen in patients with previous thoracic radiotherapy, although these patients had shorter survival (4.2 vs. 9.7 months; P = .004). Radiotherapy after the initiation of immunotherapy (n = 15) did not increase the risk of irAEs or pneumonitis; however, these patients had improved OS (17.3 vs. 6.0 months; P = .016)., Conclusion: The development of irAEs did not significantly correlate with survival when controlling for the duration of therapy in a landmark analysis. We found no increased risk of pneumonitis or irAEs in patients who had received radiotherapy. Radiotherapy before immunotherapy was associated with shorter survival, and radiotherapy after immunotherapy was associated with improved survival., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

With the advent of precision medicine, medical oncology is undergoing a transcendental change. These molecular studies have allowed us to learn about potential targeted therapies for patients with advanced cancers. Perhaps the best-known example of success in precision medicine is chronic myeloid leukemia and its response to tyrosine kinase inhibitors targeting the BCR-ABL kinase. Since that original discovery, the role of molecular therapeutics has expanded, and it now presents us with treatment options for common malignancies and rare atypical tumors. In this article, we present a case of a 61-year-old female with a recurrent pulmonary inflammatory myofibroblastic tumor. Subsequent molecular studies revealed an ALK rearrangement. The significance of this alteration in this tumor type and its therapeutic implications are discussed herein., Key Points: This case exemplifies the heterogeneous behavior of inflammatory myofibroblastic tumors (IMTs) and the current role of targeted therapy in the therapeutic armamentarium of neoplastic processes.As evidenced by the different mutations found in IMTs, it is of great importance to perform next-generation sequencing in uncommon neoplasms.These studies can find different potential targets and therapeutic options for patients devoid of standard effective therapies., Competing Interests: Disclosures of potential conflicts of interest may be found at the end of this article., (© AlphaMed Press 2018.)

Background: Fusion proteins have unique oncogenic properties and their identification can be useful either as diagnostic or therapeutic targets. Next generation sequencing data have previously shown a fusion gene formed between Rad51C and ATXN7 genes in the MCF7 breast cancer cell line. However, the existence of this fusion gene in colorectal patient tumor tissues is largely still unknown., Methods: We evaluated for the presence of Rad51C-ATXN7 fusion gene in colorectal tumors and cells by RT-PCR, PCR, Topo TA cloning, Real time PCR, immunoprecipitation and immunoblotting techniques., Results: We identified two forms of fusion mRNAs between Rad51C and ATXN7 in the colorectal tumors, including a Variant 1 (fusion transcript between Rad51C exons 1-7 and ATXN7 exons 6-13), and a Variant 2 (between Rad51C exons 1-6 and ATXN7 exons 6-13). In silico analysis showed that the Variant 1 produces a truncated protein, whereas the Variant 2 was predicted to produce a fusion protein with molecular weight of 110 KDa. Immunoprecipitation and Western blot analysis further showed a 110 KDa protein in colorectal tumors. 5-Azacytidine treatment of LS-174 T cells caused a 3.51-fold increase in expression of the fusion gene (Variant 2) as compared to no treatment controls evaluated by real time PCR., Conclusion: In conclusion we found a fusion gene between DNA repair gene Rad51C and neuro-cerebral ataxia Ataxin-7 gene in colorectal tumors. The in-frame fusion transcript of Variant 2 results in a fusion protein with molecular weight of 110 KDa. In addition, we found that expression of fusion gene is associated with functional impairment of Fanconi Anemia (FA) DNA repair pathway in colorectal tumors. The expression of Rad51C-ATXN7 in tumors warrants further investigation, as it suggests the potential of the fusion gene in treatment and predictive value in colorectal cancers.

Pelareorep causes oncolysis in tumor cells with activated Ras. We hypothesized that pelareorep would have efficacy and immunomodulatory activity in metastatic pancreatic adenocarcinoma (MPA) when combined with carboplatin and paclitaxel. A randomized phase 2 study (NCT01280058) was conducted in treatment-naive patients with MPA randomized to two treatment arms: paclitaxel/carboplatin + pelareorep (Arm A, n = 36 evaluable patients) versus paclitaxel/carboplatin (Arm B, n = 37 evaluable patients). There was no difference in progression-free survival (PFS) between the arms (Arm A PFS = 4.9 months, Arm B PFS = 5.2 months, P = 0.6), and Kirsten rat sarcoma viral oncogene (KRAS) status did not impact outcome. Quality-adjusted Time without Symptoms or Toxicity analysis revealed that the majority of PFS time was without toxicity or progression (4.3 months). Patient immunophenotype appeared important, as soluble immune biomarkers were associated with treatment outcome (fractalkine, interleukin (IL)-6, IL-8, regulated on activation, normal T cell expressed and secreted (RANTES), and vascular endothelial growth factor (VEGF)). Increased circulating T and natural killer (NK)-cell subsets were also significantly associated with treatment outcome. Addition of pelareorep was associated with higher levels of 14 proinflammatory plasma cytokines/chemokines and cells with an immunosuppressive phenotype (Tregs, cytotoxic T lymphocyte associated protein 4 (CTLA4)(+) T cells). Overall, pelareorep was safe but does not improve PFS when administered with carboplatin/paclitaxel, regardless of KRAS mutational status. Immunologic studies suggest that chemotherapy backbone improves immune reconstitution and that targeting remaining immunosuppressive mediators may improve oncolytic virotherapy.

Competing Interests: AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST Disclosures provided by the authors are available with this article at www.jco.org.

Background: The type 3 Dearing reovirus (Reolysin) is a naturally occurring virus that preferentially infects and causes oncolysis in tumor cells with a Ras-activated pathway. It induces host immunity and cell cycle arrest and acts synergistically with cytotoxic agents., Methods: This study evaluated Reolysin combined with paclitaxel and carboplatin in patients with metastatic/recurrent KRAS-mutated or epidermal growth factor receptor (EGFR)-mutated/amplified non-small cell lung cancer., Results: Thirty-seven patients were treated. Molecular alterations included 20 KRAS mutations, 10 EGFR amplifications, 3 EGFR mutations, and 4 BRAF-V600E mutations. In total, 242 cycles (median, 4; range, 1-47) were completed. The initial doses were area under the curve (AUC) 6 mg/mL/min for carboplatin, 200 mg/m(2) for paclitaxel on day 1, and 3 × 10(10) 50% tissue culture infective dose for Reolysin on days 1 to 5 of each 21-day cycle. Because of diarrhea and febrile neutropenia (in the first 2 patients), subsequent doses were reduced to 175 mg/m(2) for paclitaxel and AUC 5 mg/mL/min for carboplatin. Toxicities included fatigue, diarrhea, nausea/vomiting, neutropenia, arthralgia/myalgia, anorexia, and electrolyte abnormalities. Response Evaluation Criteria in Solid Tumors 1.0 responses included the following: partial response for 11 patients, stable disease (SD) for 20 patients, progressive disease for 4 patients, and not evaluable for 2 patients (objective response rate, 31%; 90% 1-sided lower confidence interval, 21%). Four SD patients had >40% positron emission tomography standardized uptake value reductions. The median progression-free survival, median overall survival, and 12-month overall survival rate were 4 months, 13.1 months, and 57%, respectively. Seven patients were alive after a median follow-up of 34.2 months; they included 2 patients without disease progression at 37 and 50 months., Conclusions: Reolysin in combination with paclitaxel and carboplatin was well tolerated. The observed response rate suggests a benefit of the reovirus for chemotherapy. A follow-up randomized study is recommended. The proportion of patients surviving longer than 2 years (30%) suggests a second/third-line treatment effect or possibly the triggering of an immune response after tumor reovirus infiltration., (© 2015 American Cancer Society.)

Background: BRCA germline mutations are being targeted for development of PARP inhibitors. BRCA genes collaborate with several others in the Fanconi Anemia (FA) pathway. We screened cancer patients' tumors for FA functional defects then aimed to establish the safety/feasibility of administering PARP inhibitors as monotherapy and combined with a DNA-breaking agent., Methods: Patients underwent FA functional screening for the presence (or lack) of tumor FancD2 nuclear foci formation on their archival tumor material, utilizing a newly developed method (Fanconi Anemia triple-stain immunofluorescence [FATSI]), performed in a Clinical Laboratory Improvement Amendments-certified laboratory. FATSI-negative patients were selected for enrollment in a two-arm dose escalation trial of veliparib, or veliparib/mitomycin-C (MMC)., Results: One hundred eighty-five of 643 (28.7%) screened patients were FATSI-negative. Sixty-one received veliparib or veliparib/MMC through 14 dose levels. Moderate/severe toxicities included fatigue (DLT at veliparib 400mg BID), diarrhea, and thrombocytopenia. Recommended doses are 300mg BID veliparib and veliparib 200mg BID for 21 days following 10mg/m(2) MMC every 28 days. Six antitumor responses occurred, five in the combination arm (3 breast, 1 ovarian, 1 endometrial [uterine], and 1 non-small cell lung cancer). Two patients have received 36 and 60 cycles to date. BRCA germline analysis among 51 patients revealed five deleterious mutations while a targeted FA sequencing gene panel showed missense/nonsense mutations in 29 of 49 FATSI-negative tumor specimens., Conclusions: FATSI screening showed that a substantial number of patients' tumors have FA functional deficiency, which led to germline alterations in several patients' tumors. Veliparib alone or with MMC was safely administered to these patients and produced clinical benefit in some. However, a better understanding of resistance mechanisms in this setting is needed., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Unlabelled: In this phase II trial, carboplatin with nanoparticle albumin-bound (nab)-paclitaxel as first-line therapy for advanced non-small-cell lung cancer (NSCLC) was evaluated. Most patients had squamous cell histology. Tumor-associated stromal caveolin-1 (Cav-1) expression was correlated with improved response rate and survival in NSCLC patients who received nab-paclitaxel in this phase II trial. These results suggest Cav-1 might serve as a potential biomarker in this patient population., Background: The combination of bevacizumab with platinum-based chemotherapy results in greater response rate (RR) and overall survival (OS) in advanced non-small-cell lung cancer (NSCLC). Bevacizumab is contraindicated in patients with squamous histology or hemoptysis. Nanoparticle albumin-bound (nab)-paclitaxel is a novel formulation of paclitaxel with greater dose tolerance and improved efficacy. We hypothesized that nab-paclitaxel and carboplatin would be superior to alternative doublets in advanced NSCLC patients ineligible for bevacizumab., Patients and Methods: We conducted a single-arm phase II trial (NCT00729612) with carboplatin and nab-paclitaxel on day 1 of a 21-day cycle to evaluate RR (primary end point), safety, toxicity, and OS. Eligibility included: squamous histology, hemoptysis, or ongoing anticoagulation. Correlative studies included immunohistochemistry for secreted protein acid rich in cysteine (SPARC) and caveolin-1 (Cav-1)., Results: Sixty-three patients were enrolled. Most patients had squamous cell carcinoma (n = 48); other reasons for eligibility included hemoptysis (n = 11) and anticoagulation (n = 2). Toxicity Grade ≥ 3/4 included neuropathy, cytopenias, and fatigue. RR was 38% (24 partial response/0 complete response); 20 patients had stable disease (32%). Median progression-free survival was 5 months and median OS was 9.7 months. Immunohistochemistry for SPARC and Cav-1 was performed in 38 and 37 patients respectively. Although no association was found for SPARC expression in tumor or stroma with RR or OS, we found that higher Cav-1 levels in tumor-associated stroma was associated with improved RR and OS., Conclusion: Carboplatin and nab-paclitaxel every 21 days demonstrated promising efficacy with tolerable toxicity in NSCLC patients ineligible for bevacizumab therapy. Further analysis and validation of Cav-1 and SPARC expression in tumor and stromal compartments as prognostic and/or predictive biomarkers of NSCLC or nab-paclitaxel treatment is warranted., Competing Interests: The authors have stated that they have no conflicts of interest., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Biliary cancers (BC) are rare, chemoresistant and are associated with a poor prognosis. Targeting the Akt pathway is of significance in BC. We hypothesized that the allosteric inhibitor MK-2206 will be active in BC. This was a multi-institutional phase II study of MK-2206 given to patients with advanced, refractory BC. The primary end point was overall response rate. We also characterized pharmacokinetic profiles of MK-2206 in these patients and explored its potential correlation with clinical outcomes. Eight patients were enrolled prior to early termination of the trial. All patients had received prior systemic therapy. The best response observed was stable disease, exceeding 12 weeks in two patients. Toxicities were mild and tolerable. MK-2206 exhibited a pharmacokinetic profile with an apparent slow absorption followed by biphasic elimination in these patients with BC. No significant association was observed between the pharmacokinetic properties of MK-2206 and clinical outcomes. MK-2206 as a single-agent in BC is tolerable with pharmacokinetic properties similar to patients with other solid tumors. No clinical activity was observed in this limited population. Further development of Akt inhibitors may need to focus on combinations with other molecular targeted agents, conventional cytotoxic chemotherapy and prospective patient selection.

Functional alterations in Rad51C are the cause of the Fanconi anemia complementation group O (FANCO) gene disorder. We have identified novel splice variants of Rad51C mRNA in colorectal tumors and cells. The alternatively spliced transcript variants are formed either without exon-7 (variant 1), without exon 6 and 7 (variant 2) or without exon 7 and 8 (variant 3). Real time PCR analysis of nine pair-matched colorectal tumors and non-tumors showed that variant 1 was overexpressed in tumors compared to matched non-tumors. Among 38 colorectal tumor RNA samples analyzed, 18 contained variant 1, 12 contained variant 2, 14 contained variant 3, and eight expressed full length Rad51C exclusively. Bisulfite DNA sequencing 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 2.5 fold increase in WT. Expression of Rad51C variants is associated with FANCD2 foci positive colorectal tumors and is associated with microsatellite stability in those tumors. Further investigation is needed to elucidate differential function of the Rad51C variants to evaluate potential effects in drug resistance and DNA repair.

Purpose: Veliparib, a PARP inhibitor, demonstrated clinical activity in combination with oral cyclophosphamide in patients with BRCA-mutant solid tumors in a phase I trial. To define the relative contribution of PARP inhibition to the observed clinical activity, we conducted a randomized phase II trial to determine the response rate of veliparib in combination with cyclophosphamide compared with cyclophosphamide alone in patients with pretreated BRCA-mutant ovarian cancer or in patients with pretreated primary peritoneal, fallopian tube, or high-grade serous ovarian cancers (HGSOC)., Experimental Design: Adult patients were randomized to receive cyclophosphamide alone (50 mg orally once daily) or with veliparib (60 mg orally once daily) in 21-day cycles. Crossover to the combination was allowed at disease progression., Results: Seventy-five patients were enrolled and 72 were evaluable for response; 38 received cyclophosphamide alone and 37 the combination as their initial treatment regimen. Treatment was well tolerated. One complete response was observed in each arm, with three partial responses (PR) in the combination arm and six PRs in the cyclophosphamide alone arm. Genetic sequence and expression analyses were performed for 211 genes involved in DNA repair; none of the detected genetic alterations were significantly associated with treatment benefit., Conclusion: This is the first trial that evaluated single-agent, low-dose cyclophosphamide in HGSOC, peritoneal, fallopian tube, and BRCA-mutant ovarian cancers. It was well tolerated and clinical activity was observed; the addition of veliparib at 60 mg daily did not improve either the response rate or the median progression-free survival., (©2015 American Association for Cancer Research.)

This study was designed to evaluate the safety, pharmacokinetics, and clinical activity of pazopanib combined with paclitaxel to determine the recommended phase II dose in the first-line setting in patients with advanced solid tumors. Patients were enrolled in a 3+3 dose-escalation design to determine the maximum tolerated regimen (MTR) of once daily pazopanib plus paclitaxel administered every 3 weeks at four dose levels (DL1-4). Safety, pharmacokinetics, pharmacogenetics, and disease assessments were performed. Twenty-eight patients received treatment. One patient at DL1 had dose-limiting toxicity (DLT) of elevated hepatic enzymes. After pazopanib discontinuation, liver enzyme concentrations remained high until a concurrent medication, simvastatin, was discontinued. This patient had the defective CYP2C8*3*3 genotype. At DL2, 1 patient had DLT of elevated hepatic enzymes with rash and 1 patient had DLT of rash. The MTR was paclitaxel 150 mg/m(2) plus pazopanib 800 mg. The most common toxicities were alopecia, fatigue, hypertension, nausea, diarrhea, dysgeusia, neutropenia, myalgia, hair color changes, and peripheral neuropathy. Coadministration of pazopanib and paclitaxel resulted in a 38% increase in systemic exposure to paclitaxel, relative to administration of paclitaxel alone, at the MTR. Of the 28 patients treated with the combination, 10 achieved a partial response and 10 achieved stable disease of ≥12 weeks. Pazopanib 800 mg daily plus paclitaxel 150 mg/m(2) every 3 weeks was the recommended phase II dose, with a manageable safety profile, and with clinical activity in both melanoma and non-small cell lung cancer that suggest further evaluation of this combination is warranted., (©2014 American Association for Cancer Research.)

The Fanconi anemia (FA) pathway is a major mechanism of homologous recombination DNA repair. The functional readout of the pathway is activation through mono-ubiquitination of FANCD2 leading to nuclear foci of repair. We have recently developed an FA triple-staining immunofluorescence based method (FATSI) to evaluate FANCD2 foci formation in formalin fixed paraffin-embedded (FFPE) tumor samples. DNA-repair deficiencies have been considered of interest in lung cancer prevention, given the persistence of damage produced by cigarette smoke in this setting, as well as in treatment, given potential increased efficacy of DNA-damaging drugs. We screened 139 non-small cell lung cancer (NSCLC) FFPE tumors for FANCD2 foci formation by FATSI analysis. Among 104 evaluable tumors, 23 (22%) were FANCD2 foci negative, thus repair deficient. To evaluate and compare novel-targeted agents in the background of FA deficiency, we utilized RNAi technology to render several lung cancer cell lines FANCD2 deficient. Successful FANCD2 knockdown was confirmed by reduction in the FANCD2 protein. Subsequently, we treated the FA defective H1299D2-down and A549D2-down NSCLC cells and their FA competent counterparts (empty vector controls) with the PARP inhibitors veliparib (ABT-888) (5 μM) and BMN673 (0.5 μM), as well as the CHK1 inhibitor Arry-575 at a dose of 0.5 μM. We also treated the FA defective small cell lung cancer cell lines H719D2-down and H792D2-down and their controls with the BCL-2/XL inhibitor ABT-263 at a dose of 2 μM. The treated cells were harvested at 24, 48, and 72 h post treatment. MTT cell viability analysis showed that each agent was more cytotoxic to the FANCD2 knock-down cells. In all tests, the FA defective lung cancer cells had less viable cells as comparing to controls 72 h post treatment. Both MTT and clonogenic analyses comparing the two PARP inhibitors, showed that BMN673 was more potent compared to veliparib. Given that FA pathway plays essential roles in response to DNA damage, our results suggest that a subset of lung cancer patients are likely to be more susceptible to DNA cross-link based therapy, or to treatments in which additional repair mechanisms are targeted. These subjects can be identified through FATSI analysis. Clinical trials to evaluate this therapeutic concept are needed.

Cigarette smoking is one of the most significant public health issues and the most common environmental cause of preventable cancer deaths worldwide. EGFR (Epidermal Growth Factor Receptor)-targeted therapy has been used in the treatment of LC (lung cancer), mainly caused by the carcinogens in cigarette smoke, with variable success. Presence of mutations in the KRAS (Kirsten rat sarcoma viral oncogene homolog) driver oncogene may confer worse prognosis and resistance to treatment for reasons not fully understood. NQO1 (NAD(P)H:quinone oxidoreductase), also known as DT-diaphorase, is a major regulator of oxidative stress and activator of mitomycins, compounds that have been targeted in over 600 pre-clinical trials for treatment of LC. We sequenced KRAS and investigated expression of NQO1 and five clinically relevant proteins (DNMT1, DNMT3a, ERK1/2, c-MET, and survivin) in 108 patients with non-small cell lung carcinoma (NSCLC). NQO1, ERK1/2, DNMT1, and DNMT3a but not c-MET and survivin expression was significantly more frequent in patients with KRAS mutations than those without, suggesting the following: (1) oxidative stress may play an important role in the pathogenesis, worse prognosis, and resistance to treatment reported in NSCLC patients with KRAS mutations, (2) selecting patients based on their KRAS mutational status for future clinical trials may increase success rate, and (3) since oxidation of nucleotides also specifically induces transversion mutations, the high rate of KRAS transversions in lung cancer patients may partly be due to the increased oxidative stress in addition to the known carcinogens in cigarette smoke.

Lung cancer (LC) and colorectal cancer (CRC) are the first and second deadliest types of cancer worldwide. EGFR-based therapy has been used in the treatment of these cancers with variable success. Presence of mutations in the KRAS driver oncogene, possibly induced by environmental factors such as carcinogens in diet and cigarette smoke, may confer worse prognosis and resistance to treatment for reasons not fully understood. Data on possible associations between KRAS mutational status and clinical and metabolic parameters, which may help in clinical management, as well as in identifying risk factors for developing these cancers, are limited in the current literature. We sequenced the KRAS gene and investigated the associations of variations in 108 patients with non-small cell lung carcinoma (NSCLC), the most common form of LC, and in 116 patients with CRC. All of the mutations originated from the guanosine nucleotide and over half of all transversions in NSCLC and CRC were c.34 G>T and c.35 G>T, respectively. c.35 G>A was the most frequent type of transition in both cancers. Excluding smoking, the clinical and metabolic parameters in patients carrying mutant and wild type KRAS were similar except that the CRC patients with transversion mutations were 8.6 years younger than those carrying the transitions (P < 0.01). Dyslipidemia, hypertension, family cancer history, and age of diagnosis older than 60 years were more frequent in NSCLC than CRC (P ≤ 0.04). These results suggest that most of the clinical and metabolic parameters investigated in this study are probably not associated with the more aggressive phenotype and differences in response to EGFR-based treatment previously reported in patients with KRAS mutations. However, the increased rates of abnormal metabolic parameters in patients with NSCLC in comparison to CRC indicate that these parameters may be more important in the management of NSCLC. CRC patients carrying transition mutations are older than those carrying transversions, suggesting that age may determine the type of KRAS mutation in CRC patients.

Molecular selection has led to the successful use of novel tyrosine kinase inhibitors (TKIs) in non-small-cell lung cancers (NSCLCs). For instance, mutations in EGFR and translocations and fusions in ALK render tumor cells sensitive to some TKIs, leading to substantial clinical benefits. Molecular testing such as DNA sequencing or fragment analysis following PCR, and evaluation of copy number and gene positioning by FISH, have been developed and used clinically to identify mutations/fusions. Meanwhile, TKIs to target actionable mutations/fusions in several other oncogenes are being evaluated. High-throughput sequencing can provide therapy-predictive information as well as identify novel targetable genomic alterations. In this article, we report our experience enabling single-gene testing, and our evolution to panel-based next-generation sequencing.

Prospective studies on epidermal growth factor receptor (EGFR) inhibitors in African Americans with non-small cell lung cancer (NSCLC) have not previously been performed. In this phase II randomized study, 55 African Americans with NSCLC received 150 mg/day erlotinib or a body weight-adjusted dose with subsequent escalations to the maximum-allowable dose, 200 mg/day, to achieve rash. Erlotinib and OSI-420 exposures were lower than those observed in previous studies, consistent with CYP3A pharmacogenetics implying higher metabolic activity. Tumor genetics showed only two EGFR mutations, EGFR amplification in 17/47 samples, eight KRAS mutations, and five EML4-ALK translocations. Although absence of rash was associated with shorter time to progression (TTP), disease-control rate, TTP, and 1-year survival were not different between the two dose groups, indicating the dose-to-rash strategy failed to increase clinical benefit. Low incidence of toxicity and low erlotinib exposure suggest standardized and maximum-allowable dosing may be suboptimal in African Americans.

Racial and ethnic disparities in the pathogenesis of common malignancies and outcomes from treatment remain a major health concern. Factors attributed to these disparities include differences in lifestyle, environment, genetics, and tumor biology. As we strive to personalize cancer therapy, it will be imperative that we understand the relative contributions of each factor so that we may apply this knowledge in choosing the best treatment for each individual, regardless of his or her racial or ethnic heritage.

Targeted cancer therapies often induce "outlier" responses in molecularly defined patient subsets. One patient with advanced-stage lung adenocarcinoma, who was treated with oral sorafenib, demonstrated a near-complete clinical and radiographic remission for 5 years. Whole-genome sequencing and RNA sequencing of primary tumor and normal samples from this patient identified a somatic mutation, ARAF S214C, present in the cancer genome and expressed at high levels. Additional mutations affecting this residue of ARAF and a nearby residue in the related kinase RAF1 were demonstrated across 1% of an independent cohort of lung adenocarcinoma cases. The ARAF mutations were shown to transform immortalized human airway epithelial cells in a sorafenib-sensitive manner. These results suggest that mutant ARAF is an oncogenic driver in lung adenocarcinoma and an indicator of sorafenib response.

Background: Second-line chemotherapy for advanced non-small cell lung cancer (NSCLC) improves survival modestly but new strategies are needed. This trial was designed to evaluate an antivascular endothelial growth factor strategy with or without standard chemotherapy in previously treated NSCLC., Methods: Patients with stage IIIB/IV NSCLC with performance status 0 to 1 progressive after first-line chemotherapy were eligible for randomization to pemetrexed, sunitinib, or the combination. Patients were stratified by performance status, stage, and sex. Primary objective was 18-week progression-free survival (PFS) rate; secondary objectives included response, overall survival (OS), and toxicity. Target accrual was 225. The study was terminated early because of decreasing accrual rates., Results: Between April 2008 and September 2011, 130 patients were registered and randomized; of this, 125 patients were treated. Baseline characteristics in the three arms were well balanced. Toxicity was higher in the sunitinib-containing arms. The 18-week PFS rate in the pemetrexed, sunitinib, and combination arms was 54% (95% confidence interval [CI], 40-71), 37% (95% CI, 25-54), and 48% (95% CI, 35-66), respectively (p = 0.25). Median PFS in the pemetrexed, sunitinib, and combination arms in months was 4.9 (2.1-8.8), 3.3 (2.3-4.2), and 3.7 (2.5-5.8), respectively (p = 0.18). There was an overall statistically significant difference in OS between the three arms: median OS in months was 10.5 (8.3-20.2) for pemetrexed, 8.0 (6.8-13.5) for sunitinib, and 6.7 (4.1-10.1) for the combination (p = 0.03)., Conclusion: Pemetrexed had a superior toxicity profile to either sunitinib or the combination of pemetrexed and sunitinib. The 18-week PFS rate was not significantly different between the arms. OS was significantly better with pemetrexed alone compared with the two sunitinib-containing arms, with the doublet performing worst for OS.

A major mechanism of DNA repair related to homologous recombination is the Fanconi anemia (FA) pathway. FA genes collaborate with BRCA genes to form foci of DNA repair on chromatin after DNA damage or during the S phase of the cell cycle. Our goal was to develop a method capable of evaluating the functional status of the pathway in patients' tumor tissue, which could also be practically incorporated into large-scale screening. To develop this method, we first used Western immunoblot to detect FANCD2 protein monoubiquitination in fresh tumor specimens of patients with ovarian cancer undergoing surgery and stained formalin-fixed paraffin-embedded tumor tissue simultaneously with 4',6-diamidino-2-phenylindole, FANCD2, and Ki67 antibodies, eventually extending this method to other solid tumors. This triple stain permitted evaluation of the presence, or lack thereof, of FANCD2 subnuclear repair foci in proliferating cells by immunofluorescence microscopy. Overall, we evaluated 156 formalin-fixed paraffin-embedded tumor samples using the FA triple-staining immunofluorescence method. The ratios of FANCD2 foci-negative tumors in ovarian, lung, and breast tumor samples were 21%, 20%, and 29.4%, respectively. Our studies have led to the development of a suitable method for screening, capable of identifying tumors with somatic functional defects in the FA pathway. The use of paraffin-embedded tissues renders the reported method suitable for large-scale screening to select patients for treatment with DNA interstrand crosslinking agents, poly ADP-ribose polymerase inhibitors, or their combination., (Copyright © 2013 Mosby, Inc. All rights reserved.)

A combinatorial library of 6 × 10 6 cyclic peptides was synthesized in the one bead-two compound format, with each bead displaying a unique cyclic peptide on its surface and a linear peptide encoding tag in its interior. Screening of the library against K-Ras identified compounds that bound K-Ras with submicromolar affinity and disrupted its interaction with effector proteins.

Introduction: This phase II single-arm trial of docetaxel and capecitabine in previously untreated non-small cell lung cancer (NSCLC) patients was designed to evaluate response rate of this regimen based on promising efficacy data from phase II testing in pre-treated NSCLC patients. The trial also evaluated the correlation between peripheral blood dihydropyrimidine dehydrogenase (DPD) expression and efficacy/toxicity., Methods: Patients with advanced NSCLC (metastatic, including malignant pleural effusion) without prior chemotherapy were enrolled. Baseline DPD screening was performed; patients with baseline DPD level < 0.07 nmol/min/mg protein were considered ineligible for the study. Treatment included a 28-day cycle of docetaxel 36 mg/m(2) on days 1, 8, 15 and capecitabine 1250 mg/m(2)/day in divided doses on days 5-18. Overall response rate (RR) was the primary endpoint with a target RR of 50%. Correlative studies included evaluation of DPD activity levels in peripheral blood and correlation with clinical responses., Results: Twenty-eight patients received 86 cycles of treatment (median 3 cycles) and were evaluable for response. The RR was 18% (5 patients); RR did not meet the pre-specified efficacy endpoint and the trial was stopped. 14 patients had stable disease (SD - 50%) and 4 patients had SD >12 weeks. Median time to progression was 3.3 months (95% CI 1.5-4.6 months). Median overall survival was 10.5 months (95% CI: 3.2-15 months). Main toxicities included fatigue, stomatitis and leukopenia. DPD levels ranged from 0.06 to 0.26 nmol/min/mg. The majority of responders (4/5) had DPD levels ≤0.1 nmol/min/mg. Most of the responders (4/5) experienced grade 3 toxicities including leukopenia, dehydration, fatigue, and diarrhea. None of the patients (0/4) with higher DPD levels (> 0.2 nmol/min/mg) had a response., Conclusion: The response rate for the regimen did not demonstrate sufficient activity and further study of this regimen in this setting is not indicated. Interestingly, the results suggest that low DPD expression may be associated with response to capecitabine but also with increased toxicity., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Decitabine (DAC) is used for treatment of patients with myelodysplastic syndromes and acute myeloid leukemia (AML). Following cellular uptake, DAC is activated to DAC-triphosphate (TP) and incorporated into DNA. Once incorporated into the DNA, DAC-TP binds and inactivates DNA methyltransferases (DNMTs), thereby leading to hypomethylation and re-expression of epigenetically silenced tumor suppressor genes and ultimately antileukemia activity. However, direct evidence of in vivo DAC-TP occurrence in DAC-treated patients has been difficult to demonstrate due to a lack of suitable validated analytical methodology. Thus, we developed and validated a nonradioactive sensitive and specific LC-MS/MS assay for quantification of DAC-TP. The assay is linear from 50 to 1,000 nM and from 1 to 10 μM and has a lower limit of quantitation of 50 nM and a coefficient of variation for both within- and between-day precision <20%. Following DAC treatment, we detected DAC-TP in parental and DAC-resistant AML cells (in vitro) and bone marrow (BM) and spleen of normal and leukemic mice (in vivo). Downregulation of DNMTs and correlation of DAC-TP concentration with proteins involved in mechanisms of DAC resistance were also demonstrated. The clinical applicability of this method was proven by measuring DAC-TP level in BM and blood mononuclear cells from DAC-treated AML patients. Higher levels are seemingly associated with clinical response. Monitoring the DAC-TP intracellular level may serve as a novel pharmacological endpoint for designing more effective DAC-based regimens.

Purpose: Erlotinib is clinically effective in patients with non-small-cell lung cancer (NSCLC) who have adenocarcinoma, are never or limited former smokers, or have EGFR mutant tumors. We investigated the efficacy of erlotinib alone or in combination with chemotherapy in patients with these characteristics., Patients and Methods: Patients with advanced NSCLC (adenocarcinoma) who were epidermal growth factor receptor tyrosine kinase inhibitor and chemotherapy naive never or light former smokers (smokers of > 100 cigarettes and ≤ 10 pack years and quit ≥ 1 year ago) were randomly assigned to continuous erlotinib or in combination with carboplatin and paclitaxel (ECP) for six cycles followed by erlotinib alone. The primary end point was progression-free survival (PFS). Tissue collection was mandatory., Results: PFS was similar (5.0 v 6.6 months; P = .1988) in patients randomly assigned to erlotinib alone (arm A; n = 81) or to ECP (arm B; n = 100). EGFR mutation analysis was possible in 91% (164 of 181) of patients, and EGFR mutations were detected in 40% (51 of 128) of never smokers and in 42% (15 of 36) of light former smokers. In arm A, response rate (70% v 9%), PFS (14.1 v 2.6 months), and overall survival (OS; 31.3 v 18.1 month) favored EGFR-mutant patients. In arm B, response rate (73% v 30%), PFS (17.2 v 4.8 months), and OS (38.1 v 14.4 months) favored EGFR-mutant patients. Incidence of grades 3 to 4 hematologic (2% v 49%; P < .001) and nonhematologic (24% v 52%; P < .001) toxicity was greater in patients treated with ECP., Conclusion: Erlotinib and erlotinib plus chemotherapy have similar efficacy in clinically selected populations of patients with advanced NSCLC. EGFR mutations identify patients most likely to benefit.

Purpose: First-in-man study of KOS-1584, a second generation epothilone., Methods: Patients with advanced solid malignancies received KOS-1584 every 3 weeks until disease progression. Using a modified Fibonacci dose escalation scheme, one patient was enrolled at each dose level until the first instance of grade 2 toxicity. Thereafter, a standard 3 + 3 design was utilized., Results: Sixty-six patients in 14 cohorts were dosed from 0.8 to 48 mg/m(2). Diarrhea, arthralgias, and encephalopathy were dose-limiting toxicities (DLTs) at doses ≥36 mg/m(2). At the recommended phase II dose (RP2D), the most common adverse effects were peripheral neuropathy (low grade), fatigue, arthralgias/myalgias, and diarrhea (31, 6%). The incidence of neutropenia was low. The overall clearance, volume of distribution, and half-life of KOS-1584 were 11 ± 6.17 L/h/m(2), 327 ± 161 L/m(2), and 21.9 ± 8.75 h, respectively. The half-life for the seco-metabolite (KOS-1891) was 29.6 ± 13.8 h. KOS-1584 exhibited linear pharmacokinetics. A dose-dependent increase in microtubulin bundle formation was observed at doses ≥27 mg/m(2). Two patients achieved partial responses and 24 patients had stable disease (SD)., Conclusions: The RP2D of KOS-1584 is 36 mg/m(2). The lack of severe neurologic toxicity, diarrhea, neutropenia, or hypersensitivity reactions; favorable pharmacokinetic profile; and early evidence of activity support further evaluation.

Purpose: Biliary cancers (BCs) carry a poor prognosis, but targeting the RAS/RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal-related kinase (ERK) pathway is of significance. Selumetinib is an inhibitor of MEK1/2, so this trial was designed to determine the safety and efficacy of selumetinib in BC., Patients and Methods: This was a multi-institutional phase II study of selumetinib at 100 mg given orally twice per day to patients with advanced BC. The primary end point was response rate. All patients were required to provide tissue before enrolling. The levels of phosphorylated ERK (pERK) and AKT (pAKT) were assessed by immunohistochemistry. Tumors were genotyped for the presence of BRAF- and/or RAS-activating mutations., Results: Twenty-eight eligible patients with a median age of 55.6 years were enrolled. Thirty-nine percent of patients had received one prior systemic therapy. Three patients (12%) had a confirmed objective response. Another 17 patients (68%) experienced stable disease (SD), 14 of whom (56%) experienced prolonged SD (> 16 weeks). Patients gained an average nonfluid weight of 8.6 pounds. Median progression-free survival was 3.7 months (95% CI, 3.5 to 4.9) and median overall survival was 9.8 months (95% CI, 5.97 to not available). Toxicities were mild, with rash (90%) and xerostomia (54%) being most frequent. Only one patient experienced grade 4 toxicity (fatigue). All patients had tissue available for analysis. No BRAF V600E mutations were found. Two patients with short-lived SD had KRAS mutations. Absence of pERK staining was associated with lack of response., Conclusion: Selumetinib displays interesting activity and acceptable tolerability in patients with metastatic BC. Our results warrant further evaluation of selumetinib in patients with metastatic BC.

Clinical trials and correlative laboratory research are increasingly reliant upon archived paraffin-embedded samples. Therefore, the proper processing of biological samples is an important step to sample preservation and for downstream analyses like the detection of a wide variety of targets including micro RNA, DNA and proteins. This paper analyzed the question whether routine fixation of cells and tissues in 10% buffered formalin is optimal for in situ and solution phase analyses by comparing this fixative to a variety of cross linking and alcohol (denaturing) fixatives. We examined the ability of nine commonly used fixative regimens to preserve cell morphology and DNA/RNA/protein quality for these applications. Epstein-Barr virus (EBV) and bovine papillomavirus (BPV)-infected tissues and cells were used as our model systems. Our evaluation showed that the optimal fixative in cell preparations for molecular hybridization techniques was "gentle" fixative with a cross-linker such as paraformaldehyde or a short incubation in 10% buffered formalin. The optimal fixatives for tissue were either paraformaldehyde or low concentration of formalin (5% of formalin). Methanol was the best of the non cross-linking fixatives for in situ hybridization and immunohistochemistry. For PCR-based detection of DNA or RNA, some denaturing fixatives like acetone and methanol as well as "gentle" cross-linking fixatives like paraformaldehyde out-performed other fixatives. Long term fixation was not proposed for DNA/RNA-based assays. The typical long-term fixation of cells and tissues in 10% buffered formalin is not optimal for combined analyses by in situ hybridization, immunohistochemistry, or--if one does not have unfixed tissues--solution phase PCR. Rather, we recommend short term less intense cross linking fixation if one wishes to use the same cells/tissue for in situ hybridization, immunohistochemistry, and solution phase PCR., (Published by Elsevier Inc.)

Purpose: In preclinical models, non-cytotoxic suramin (concentrations <50 μM) potentiates the activity of multiple chemotherapeutic agents. The present study evaluated the safety and tolerability of suramin in combination with docetaxel or gemcitabine in previously chemotherapy-treated patients with advanced non-small cell lung cancer., Methods: Patients received suramin intravenously in combination with either docetaxel on day 1 or gemcitabine on days 1 and 8, of each 21-day treatment cycle. After 3 cycles, patients with partial response (PR) or better continued on the same combination, whereas patients with stable disease (SD) or worse crossed-over to the other combination. Pharmacokinetic analyses were performed before and after each treatment., Results: Eighteen patients received a total of 79 courses (37 suramin plus docetaxel, 42 suramin plus gemcitabine). The dose-limiting toxicity (DLT) was febrile neutropenia, observed in three of six patients treated with suramin and docetaxel 75 mg/m(2). No DLTs were observed with suramin plus docetaxel 56 mg/m(2) or suramin plus gemcitabine 1,250 mg/m(2). Common adverse events included neutropenia, thrombocytopenia, anemia, fatigue, nausea, vomiting, skin rash, hyperglycemia, and electrolyte abnormalities. The target plasma suramin concentration range of 10-50 μM was achieved in 90% of treatments. Discernable antitumor activity was noted in 11 patients (2 PR, 9 SD)., Conclusions: Non-cytotoxic suramin, in combination with docetaxel 56 mg/m(2) or gemcitabine 1,250 mg/m(2), was reasonably well-tolerated with a manageable toxicity profile. Target plasma concentrations were correctly predicted by our previously described dosing nomogram. The observed preliminary evidence of antitumor activity encourages evaluation of this strategy in efficacy trials.