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Background: The benefit of chemotherapy combined with immunotherapy in EGFR-mutant lung adenocarcinoma (LUAD) patients whose tumor developed resistance to EGFR tyrosine kinase inhibitors (TKIs) is not thoroughly investigated. The goal of this retrospective cohort study is to assess the clinical efficiency of immunotherapy alone or in combination with chemotherapy in a real-world setting. Methods: This retrospective cohort study enrolled LUAD patients with EGFR sensitive mutations whose tumor had acquired resistance to EGFR TKIs and received systemic treatment with chemotherapy (chemo; n = 84), chemotherapy combined with immunotherapy (chemoIO; n = 30), chemotherapy plus bevacizumab with or without IO (withBev; n = 42), and IO monotherapy (IO-mono; n = 22). Clinical progression-free survival (PFS) and overall survival (OS) were evaluated. Associations of clinical characteristics with outcomes were assessed using univariable and multi-covariate Cox Proportional Hazards regression models. Results: A total of 178 patients (median age = 63.3; 57.9% females) with a median follow-up time of 42.0 (Interquartile range: 22.9–67.8) months were enrolled. There was no significant difference in PFS between chemoIO vs. chemo groups (5.3 vs. 4.8 months, p = 0.8). Compared to the chemo group, patients who received withBev therapy trended towards better PFS (6.1 months vs. 4.8; p = 0.3; HR 0.79; 95% CI: 0.52–1.20), while patients treated with IO-mono had inferior PFS (2.2 months; p = 0.001; HR 2.22; 95% CI: 1.37–3.59). Furthermore, PD-L1 level was not associated with PFS benefit in the chemoIO group. Patients with EGFR-mutant LUAD with high PD-L1 (≥50%) had shorter PFS (5.8 months) than non-EGFR/ALK LUAD patients who received chemoIO (12.8 months, p = 0.002; HR 0.22; 95% CI: 0.08–0.56) as first-line treatment. Chemotherapy-based therapy rendered similar benefit to patients with either EGFR exon19 deletion vs. L858R in the LUAD. Conclusions: This retrospective analysis revealed that immunotherapy provided limited additional benefit to chemotherapy in TKI-refractory EGFR-mutant LUAD. Chemotherapy alone or combined with bevacizumab remain good choices for patients with actionable EGFR mutations.

Aim:Vestibular schwannomas (VS) are benign slow-growing tumours treated either with microsurgery or stereotactic radiosurgery (SRS) or both. The aim of this study was to correlate the outcome factors—tumour control and adverse factors—facial nerve function and hearing loss with patient and treatment factors.Materials and methods:A retrospective review of the records of 98 patients with 99 VS treated from June 2007 to June 2014, all patients receiving Linear Accelerator (LINAC)-based SRS.Results:Median follow-up period was 5·6 years (range: 1–12 years). The response to treatment was stable disease in 37 (37·4%), regression in 46 (46·5%), asymptomatic minimal progression in 9 (9·1%) and symptomatic progression in 5 (5%) and unknown in 2 (2%) patients. There was no evidence of SRS induced tissue damage on magnetic resonance scans for any. Hearing preservation rate after SRS was 92%. The patients who developed worsening of facial function were predominantly in the cohort that had prior surgery.Findings:SRS is an effective modality to treat VS lesser than 3 cm in size. Tumour control rate was 95% with a median follow-up period of 5·6 years. The complication rates were 8% each for facial function worsening and worsening of hearing. Prior surgery was a statistically significant factor that affected facial nerve function deterioration.

Aim of the study:To assess the relapse-free survival (RFS) and the factors influencing local recurrence in patients with desmoid fibromatosis (DF) treated at our centre and to determine the role of post-operative radiotherapy (RT) in improving local control.Methods:A retrospective analysis of 51 patients treated for DF from January 2004 to December 2013 was undertaken. The RFS was calculated using the Kaplan–Meier curve. Univariate analysis was done to assess correlation with tumour size, site, the extent of surgery, margin status and adjuvant RT with RFS.Results:The median age was 28 years with a male:female ratio of 1:3. The most common location of the tumour was anterior abdominal wall (47%). The median tumour size was 10 cm. Wide local excision was done in most patients. Complete resection with negative margin was achieved in eight patients. Post-operative RT was indicated for 43 patients of whom 19 received RT. At a median follow-up of 37 months, RFS in the complete resection with margin negative group was 100%. RFS for the patients with positive or close margins who received RT was 79% and for those who did not receive RT, it was 87%.Conclusions:Complete excision with negative margins gives the best local control in DF. The benefit of post-operative RT could not be ascertained.

e21169 Background: The benefit of adding immunotherapy to chemotherapy in EGFR-mutant lung adenocarcinoma (LUAD) with acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) is not fully investigated. This study aimed to explore the outcomes of immunotherapy combinations in such population in a real-world setting. Methods: This retrospective analysis included patients with EGFR-mutant LUAD who progressed on EGFR TKIs and received subsequent therapy with chemotherapy (chemo; n = 84), chemotherapy with immunotherapy (chemoIO; n = 30), chemotherapy plus bevacizumab with or without IO (withBev; n = 42), and IO monotherapy (IO-mono; n = 22). Clinical progression-free survival (cPFS) from the start of the therapy to discontinuation was assessed. Associations of clinical characteristics with cPFS were evaluated using univariable and adjusted Cox regression models. Results: A total of 178 patients (mean age = 62.6; 57.9% females) with a median cPFS (mcPFS) of 4.9 (95% CI: 4.4-5.4) months with 162 (91%) progression events. There was no difference in cPFS between chemoIO vs chemo groups (5.3 months vs 4.8, P = 0.8). Compared to the chemo group, patients who were treated withBev trended towards better PFS (6.1 months vs 4.8; P = 0.3; HR 0.79; 95% CI: 0.52-1.20;), while patients with IO-mono had inferior PFS (2.2 months; P = 0.001; HR 2.22; 95% CI: 1.37-3.59). Patients who were administered one-line of TKI prior to subsequent therapy acquired better PFS when compared to those with multiple prior TKIs (5.2 months vs 4.5; P = 0.011; HR 0.66; 95% CI: 0.48-0.91). The significance remained the same after adjusted by other clinicopathological variables including post-TKI treatment strategies and metastatic status (P = 0.005). Furthermore, among the chemoIO group, patients with PD-L1 expression had better PFS (n = 15, 7.7 months vs 4.5, P = 0.04; HR 0.36; 95% CI: 0.13-0.96) than those without PD-L1 expression (n = 6). However, PD-L1 was not associated with PFS among the patients treated with chemoIO plus bevacizumab. Brain or liver metastases were associated with worse PFS. Conclusions: Immunotherapy adds limited benefit when combined with chemotherapy in TKI-refractory EGFR-mutant LUAD. Chemotherapy +/- bevacizumab are reasonable treatment options for these patients.

AimTo compare the dosimetric outcomes of linear accelerator-based stereotactic radiotherapy (SRT) techniques—static conformal field (SCF), static conformal arc (SCA) and dynamic conformal arc (DCA), for treating pituitary adenoma and craniopharyngioma.Materials and methodsComputer image sets of 20 patients with pituitary adenoma or craniopharyngioma and treated with post-operative SRT were selected for this study. For each dataset, three SRT plans, with SCF, SCA and DCA techniques were generated using Brain LAB, iPlan RT V.4.5.3, TPS software. The conformity index (CI), homogeneity index (HI), quality of coverage of the target, dose–volume histograms for the target and organs at risk (OARs) and the time taken to deliver treatment was compared across three sets of plan.ResultsThere were 12 patients with pituitary adenoma and eight with craniopharyngioma. The CI and HI were comparable across three techniques. The quality of coverage was superior in DCA technique. OARs were better spared in SCF and DCA techniques. Time taken to deliver treatment was least in SCF technique.ConclusionsThe linac-based SRT techniques SCF, SCA and DCA are efficient in delivering highly conformal and homogenous dose to the target in pituitary adenoma and craniopharyngioma. Among these three techniques, SCF and DCA had acceptable quality of coverage. The dose received by OARs was least in the SCF technique.

AimIndeterminate pulmonary nodules incidentally detected during radiological imaging completed for radiotherapy planning always creates dilemma for the oncologist. The purpose of this study is to evaluate the clinical significance of pulmonary nodules incidentally detected in patients undergoing locoregional radiotherapy for breast cancer and present a retrospective analysis of the natural progression of such nodules.MethodsA retrospective review of computed tomography scans of breast cancer patients who underwent radiotherapy over a period of 3 years to screen out patients with indeterminate lung nodules was undertaken. This was correlated with the patient and tumour characteristics and the status of the disease at last follow-up.ResultsOf the 132 patients reviewed 28 had indeterminate lung nodules. Of the 28 patients, four had progressive lung nodules on follow-up. Subgroup analyses did not show any significant correlation.Discussion and conclusionOne fifth of patients may present with incidentally detected lung nodules. Multiple nodules, ER negative status and locally advanced breast cancer may point to a higher risk of these nodules progressing to metastatic cancer. There is no indication to stop locoregional therapy in the presence of indeterminate nodules, but close follow-up of high-risk group is recommended.

Oncolytic viruses are an innovative therapeutic strategy for cancer, wherein viral replication and cytotoxicity are selective for tumor cells. Here we show the efficacy of systemically administered oncolytic viruses for the treatment of spontaneously arising tumors, specifically the use of oncolytic herpes simplex viruses (HSV) administered i.v. to treat spontaneously developing primary and metastatic prostate cancer in the transgenic TRAMP mouse, which recapitulates human prostate cancer progression. Four administrations of systemically delivered NV1023 virus, an HSV-1/HSV-2 oncolytic recombinant, to TRAMP mice at 12 or 18 weeks of age (presence of prostate adenocarcinoma or metastatic disease, respectively) inhibited primary tumor growth and metastases to lymph nodes. Expression of interleukin 12 (IL-12) from NV1042 virus, a derivative of NV1023, was additionally effective, significantly reducing the frequency of development of prostate cancer and lung metastases, even when the mice were treated after the onset of metastasis at 18 weeks of age. NV1042-infected cells, as detected by 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside staining for Lac Z expressed by the virus, were present in prostate tumors 1 week after the final virus injection and viral DNA was detected at 2 weeks after final virus injection by real-time PCR in primary and metastatic tumors but not in liver or blood. No toxicity was observed in any of the treated mice. The efficacy of the IL-12–expressing NV1042 virus in this aggressive prostate cancer model using a clinically relevant treatment paradigm merits its consideration for clinical studies. [Cancer Res 2007;67(19):9371–9]

Oncolytic herpes simplex virus vectors are a promising strategy for cancer therapy, as direct cytotoxic agents, inducers of antitumor immune responses, and as expressers of anticancer genes. Progress is dependent upon representative preclinical models to evaluate therapy. In this study, two families of oncolytic herpes simplex virus vectors (G207 and NV1020 series) that have been in clinical trials were examined for the treatment of breast cancer, using the C3(1)/T-Ag transgenic mouse model. Female mice spontaneously develop mammary carcinomas, and the C3(1)/T-Ag–derived tumor cell line M6c forms implantable tumors. Both in vitro and in vivo, G47Δ, derived from G207 by deletion of ICP47 and the US11 promoter, was more efficacious than G207. Whereas NV1023, derived from NV1020 by deletion of ICP47 and insertion of LacZ, was as cytotoxic to M6c cells in vitro as G47Δ, it did not inhibit the growth of s.c. M6c tumors but did extend the survival of intracerebral tumor bearing mice. In contrast, NV1042, NV1023 expressing interleukin 12, inhibited s.c. M6c tumor growth to a similar extent as G47Δ, but was less effective than NV1023 in intracerebral tumors. In the spontaneously arising mammary tumor model, when only the first arising tumor per mouse was treated, G47Δ inhibited the growth of a subset of tumors, and when all tumors were treated, G47Δ significantly delayed tumor progression. When the first mammary tumor was treated and the remaining mammary glands removed, NV1042 was more efficacious than G47Δ at inhibiting the growth and progression of injected tumors.

The molecular mechanisms which underlie the biological effects of exposure to ionizing radiation have particular relevance to radiation protection and risk assessment since most cancers are of epithelial origin. It is important to obtain a better understanding of radiation-induced oncogenic transformation in this cell type. Our previous studies have demonstrated that both X-rays and fission neutrons can transform immortalized human epidermal keratinocytes to a malignant phenotype. In both studies, exposure to ionizing radiation led to the development of morphologically altered cells and focus formation at confluence. These radiation-transformed cultures grew with an increased saturation density, exhibited anchorage-independent growth, and formed tumors in athymic mice. These radiation studies have been expanded to include a molecular analysis of the cellular ras genes and the tumor suppressor gene p53. Single-strand conformational polymorphism (SSCP) analysis and DNA sequencing demonstrated the absence of point mutations, deletions, and rearrangements in codons 12/13 and 61 in the Ha-ras, Ki-ras, or N-ras genes and exons 2–11 of the p53 tumor suppressor gene. Karyotypic analysis of these human keratinocytes indicated a loss of heterozygosity at the tumor suppressor loci RBI, DCC, APC, MTS1, and WT1 upon immortalization. Furthermore, karyotypic analysis of the X-ray-treated keratinocytes in relation to their tumorigenic potential indicated that the development of malignancy was associated with a duplication of material on chromosome 11 between regions q14 and q22. This additional material on chromosome 11 at q14/q22 was observed in all of the irradiated tumorigenic keratinocytes. A similar tandem duplication of the 11q13>q23 region has been observed in malignant lesions of oral squamous cell carcinoma and in head and neck tumors. Deletions in the same region were also observed in the irradiated non-tumorigenic cells. Together, these observations indicate that this region of chromosome 11 contains gene (s) with a role in tumorigenicity and that the duplication observed in chromosome 11 may be the primary event leading to radiation-induced transformation of these immortalized human keratinocytes. These studies demonstrate that mutations in either the cellular p53 or ras genes do not occur in these immortalized human epithelial cells (RHEK-1) when transformed to a malignant phenotype with ionizing radiation. Furthermore, specific regions on chromosome 11 may be a target for the transforming effects of ionizing radiation. © 1995 Wiley-Liss, Inc.

The immortal human prostate cell line, 267B1, has been used as a model system for investigating molecular events in carcinogenesis. Exposure of these cells to ionizing radiation results in progression to anchorage-independent growth. In addition, transfection of 267B1 cells with the activated Ki-ras oncogene results in tumorigenicity. We present here the further characterization of these cells, observing that both the immortalized 267B1 cell line and irradiated clones contain the wild type Ki-ras-2 gene and a Ki-ras pseudogene (Ki-ras-1 like). No mutations in p53 were observed. Cells expressing anchorage-independent growth following x-ray irradiation exhibited loss or rearrangements in numerous chromosomes, including chromosomes 10 and 18. These data are important for investigators using this model cell system for studies of carcinogenesis, gene expression, and signal transduction. © 1995 Wiley-Liss, Inc.

The biological effects of exposure to ionizing radiation have particular relevance to radiation protection and risk assessment. Since most cancers are of epithelial origin, it is important to obtain a better understanding of radiation-induced oncogenic transformation in this cell type. Our previous studies have demonstrated the potential of ionizing radiations to transform human epidermal keratinocytes (RHEK-1) [Thraves et al.: Proc Natl Acad Sci USA 87:1174–1177, 1990; Thraves et al.: Carcinogenesis (in press)] and have described the cellular characteristics, saturation density, anchorage-independent growth, and tumorigenicity of these transformed cells, together with a preliminary determination of the role of cellular ras genes. The present study has expanded these molecular studies to conclude a molecular analysis of all the ras genes and the tumor suppressor gene p53. Single-strand conformational polymorphism (SSCP) analysis and DNA sequencing demonstrated the absence of point mutations, deletions, and rearrangements in codons 12/13 and 61 in the Ha-ras or N-ras genes and exons 4–9 of the p53 tumor suppressor gene. These studies demonstrate that mutations in either the cellular p53 or ras genes do not occur in these immortalized human epithelial cells (RHEK-1) transformed to a malignant phenotype with ionizing radiation. © Wiley-Liss, Inc.

Aims Prolonged production of the free radical, nitric oxide (NO) by the enzyme inducible nitric oxide synthase (iNOS) plays an important role in tumour progression by promoting angiogenesis, invasion and inducing mutation in tumour suppressor gene. The purpose of this study is to evaluate the expression and intensity of iNOS in normal oral mucosa, precancer and oral squamous cell carcinoma (OSCC). Materials and methods An immunohistochemical study was performed using rabbit monoclonal antibody to iNOS on archival formalin fixed, paraffin embedded tissues of 5 normal oral mucosal samples, 10 Leukoplakia, 10 oral submucous fibrosis (OSMF) and 15 OSCC of different grades. Results A statistical significant difference was found between normal oral mucosa, precancer and OSCC in expression of iNOS (p value 0.0015). However, there was no statistically significant difference between the expressions of iNOS within premalignant groups (p value 0.5647) and histological sub-groups of OSCC (p value-0.5647). There was no statistically significant difference in the intensity of iNOS expression within the precancer group, OSCC sub-groups and between precancer and OSCC (p value-0.623). Conclusion The orderly increase in the expression of iNOS from normal, through precancer, to OSCC suggests the essential role played by iNOS in epithelial transformation and tumour formation.

Purpose: Our goal was to evaluate whether systemic administration of NV1042, an interleukin-12 (IL-12)–expressing oncolytic herpes simplex virus, and its noncytokine parental vector NV1023 are effective against preexisting metastatic prostate cancer in an immunocompetent mice model. Experimental Design: Metastatic TRAMP-C2 lung tumors established in C57Bl/6 or nude mice were treated on day 21 with four i.v. administrations of NV1042 or NV1023 and sacrificed on day 42 to assess virus efficacy and the potential mechanism of efficacy. Results: NV1042 or NV1023 treatment was similarly effective in eliminating extrapleural and hemorrhagic tumors present in mock-treated mice. However, NV1042 was further effective compared with NV1023 in controlling the growth of lung tumors (as determined by mean surface tumor nodule number, lung weights, and surface tumor burden) and in extending survival. NV1042-treated mice exhibited a transient increase of serum IL-12 1 day posttreatment, whereas IL-12 levels in tumor bearing lungs persisted a further 2 days at least. Only splenocytes from NV1042-treated mice secreted IFN-γ in response to TRAMP-C2 stimulation and displayed natural killer activity. The IL-12-mediated enhancement observed with NV1042 in the syngeneic model was abrogated in athymic mice treated in a similar manner, thus indicating a role for T cells in the augmented efficacy of NV1042 virus. Conclusions: Systemic administration of the IL-12-expressing NV1042 virus is more effective than its noncytokine parent, NV1023, against preestablished metastatic lung tumors. Given the clinical safety profile of NV1020, the parental vector of NV1023, and NV1042's enhanced efficacy and ability to activate the host immune system, NV1042 merits clinical consideration for treating metastatic prostate cancers.

Background: Cervical cancer is the most common gynecological cancer in Indian women. This study was initiated to assess whether the combination of paclitaxel and cisplatin with radiation was feasible in Indian women. Aims and Objectives: The aim of this study was to assess the immediate tumor response and toxicity of weekly cisplatin and paclitaxel along with radiotherapy in the treatment of cervical cancer. Materials and Methods: Women with primary untreated squamous cell carcinoma of the cervix with FIGO stages IB2 to IIIB were treated with weekly injections of cisplatin 30 mg/m2and paclitaxel 40 mg/m2 for 4 weeks along with radiotherapy. A total of 25 patients were enrolled in this study. Disease was assessed prior to treatment by pelvic examination and contrast enhanced computed tomography scan of the abdomen and pelvis. Response was assessed 6 weeks after completion of treatment using the same parameters. Clinical and radiological response was documented. The toxicity was assessed and was graded using the common toxicity criteria Version 3.0. Intention to treat analysis was used when reporting results. Results: A total of 23 patients completed the intended treatment. There was a complete response rate of 88%, 12% were not available for response assessment. The major toxicity was Grade 3 diarrhea (48%). The mean duration of treatment was 58 days. Conclusions: Combination chemotherapy with cisplatin and paclitaxel along with radiotherapy in patients with locally advanced squamous cell carcinoma of cervix had a high incidence of acute toxicity. There was no increase in immediate tumor response and progression free survival with this treatment regimen. Hence, this regimen offers no added benefit when compared to the chemo radiation with cisplatin alone.

We observed severe late effects in a patient treated with radiation therapy for breast cancer. Radiation survival studies of patient fibroblasts show an enhanced cellular radiation sensitivity (Do = 0.95 Gy). Genetic analysis reveals that the patient is heterozygous for a mutated ATM gene. Protein truncation test (PTT) and sequence analysis identified a truncation within the leucine zipper domain, corresponding to a fragment previously reported to exhibit dominant negative function. These findings demonstrate that ATM heterozygosity may be associated with enhanced clinical radiation sensitivity and suggest a clinical relevance to this truncation that results in a dominant negative-acting protein.

In a variety of human tumor tissues, including those of prostate and breast, CpG hypermethylation represents one of the mechanisms downregulating the expression of specific proteins, including tumor suppressor proteins. Using 267B1-XR cells generated by ionizing radiation-induced transformation of epithelial cells, derived from neonatal human prostate and immortalized by SV40 (267B1), we now report markedly low levels of expression of the cytoplasmic phosphoprotein stathmin, in addition to several proteins of the actin microfilaments and intermediate filaments that characterize the altered phenotype. Stathmin is emerging as a relay protein integrating signals from diverse pathways during differentiation and neoplastic progression. In this in vitro prostate carcinogenesis model system, where loss of specific-protein expression is a major feature of the transformed 267B1-XR cells, we employed 5-azacytidine treatment followed by 2D-PAGE to reveal if experimental genomic hypomethylation reinstated the levels of any of the differentially expressed proteins. Our data suggest that stathmin represents one such example.

It is generally accepted that the carcinogenic process in mammalian cell systems involves multiple steps that result in distinct phenotypic alterations associated with immortalization, progression, and conversion to malignancy. Prostate cancer, as the most common cancer in men, presents a particular challenge: not all histologic cancer progresses to clinical malignancies. The steps necessary for prostate carcinogenesis need to be defined and model systems expanded. We report the neoplastic transformation of an immortalized human prostate epithelial cell line (267Bl) by ionizing radiation.