Inhibiting Interleukin 36 Receptor Signaling Reduces Fibrosis in Mice With Chronic Intestinal Inflammation.

BACKGROUND & AIMS: Intestinal fibrosis is a long-term complication in inflammatory bowel diseases (IBD) that frequently results in functional damage, bowel obstruction, and surgery. Interleukin (IL) 36 is a group of cytokines in the IL1 family with inflammatory effects. We studied the expression of IL36 and its receptor, interleukin 1 receptor like 2 (IL1RL2 or IL36R) in the development of intestinal fibrosis in human tissues and mice. METHODS: We obtained intestinal tissues from 92 patients with Crohn's disease (CD), 48 patients with ulcerative colitis, and 26 patients without inflammatory bowel diseases (control individuals). Tissues were analyzed by histology to detect fibrosis and by immunohistochemistry to determine the distribution of fibroblasts and levels of IL36R ligands. Human and mouse fibroblasts were incubated with IL36 or control medium, and transcriptome-wide RNA sequences were analyzed. Mice were given neutralizing antibodies against IL36R, and we studied intestinal tissues from Il1rl2-/- mice; colitis and fibrosis were induced in mice by repetitive administration of DSS or TNBS. Bone marrow cells were transplanted from Il1rl2-/- to irradiated wild-type mice and intestinal tissues were analyzed. Antibodies against IL36R were applied to mice with established chronic colitis and fibrosis and intestinal tissues were studied. RESULTS: Mucosal and submucosal tissue from patients with CD or ulcerative colitis had higher levels of collagens, including type VI collagen, compared with tissue from control individuals. In tissues from patients with fibrostenotic CD, significantly higher levels of IL36A were noted, which correlated with high numbers of activated fibroblasts that expressed α-smooth muscle actin. IL36R activation of mouse and human fibroblasts resulted in expression of genes that regulate fibrosis and tissue remodeling, as well as expression of collagen type VI. Il1rl2-/- mice and mice given injections of an antibody against IL36R developed less severe colitis and fibrosis after administration of DSS or TNBS, but bone marrow cells from Il1rl2-/- mice did not prevent induction of colitis and fibrosis. Injection of antibodies against IL36R significantly reduced established fibrosis in mice with chronic intestinal inflammation. CONCLUSION: We found higher levels of IL36A in fibrotic intestinal tissues from patients with IBD compared with control individuals. IL36 induced expression of genes that regulate fibrogenesis in fibroblasts. Inhibition or knockout of the IL36R gene in mice reduces chronic colitis and intestinal fibrosis. Agents designed to block IL36R signaling could be developed for prevention and treatment of intestinal fibrosis in patients with IBD.

Expression of the DNA repair gene MLH1 correlates with survival in patients who have resected pancreatic cancer and have received adjuvant chemoradiation: NRG Oncology RTOG Study 9704.

BACKGROUND: The majority of patients with pancreatic cancer who undergo curative resection experience rapid disease recurrence. In previous small studies, high expression of the mismatch-repair protein mutL protein homolog 1 (MLH1) in pancreatic cancers was associated with better outcomes. The objective of this study was to validate the association between MLH1 expression and survival in patients who underwent resection of pancreatic cancer and received adjuvant chemoradiation. METHODS: Samples were obtained from the NRG Oncology Radiation Therapy Oncology Group 9704 prospective, randomized trial (clinicaltrials.gov identifier NCT00003216), which compared 2 adjuvant protocols in patients with pancreatic cancer who underwent resection. Tissue microarrays were prepared from formalin-fixed, paraffin-embedded, resected tumor tissues. MLH1 expression was quantified using fluorescence immunohistochemistry and automated quantitative analysis, and expression was dichotomized above and below the median value. RESULTS: Immunohistochemical staining was successfully performed on 117 patients for MLH1 (60 and 57 patients from the 2 arms). The characteristics of the participants who had tissue samples available were similar to those of the trial population as a whole. At the time of analysis, 84% of participants had died, with a median survival of 17 months. Elevated MLH1 expression levels in tumor nuclei were significantly correlated with longer disease-free and overall survival in each arm individually and in both arms combined. Two-year overall survival was 16% in patients who had low MLH1 expression levels and 53% in those who had high MLH1 expression levels (P < .0001 for both arms combined). This association remained true on a multivariate analysis that allowed for lymph node status (hazard ratio, 0.41; 95% confidence interval, 0.27-0.63; P < .0001). CONCLUSIONS: In the current sample, MLH1 expression was correlated with long-term survival. Further studies should assess whether MLH1 expression predicts which patients with localized pancreatic cancer may benefit most from aggressive, multimodality treatment. Cancer 2018;124:491-8. © 2017 American Cancer Society.

Evaluation of three commercial progesterone receptor assays in a single tamoxifen-treated breast cancer cohort.

Estrogen receptor and progesterone receptor status are routinely assessed using immunohistochemistry assays to assist in patient prognosis and clinical management. Three commonly utilized autostainer vendors-Dako, Leica and Ventana-provide ready-to-use progesterone receptor assays; however, they have never been directly compared in a single breast cancer cohort. We looked at three immunohistochemical progesterone receptor assays, in addition to original ligand-binding assay results, in a single retrospective, tamoxifen-treated breast cancer cohort to investigate inter- and intra-observer agreement, concordance, prognostic ability and measures of test performance. All immunohistochemical assays utilized the manufacturer's specified protocols. Five-year disease-free survival was the endpoint of interest, and multivariate models were adjusted for lymph node status, tumor grade, tumor size and human epidermal growth factor 2 status. All assays showed substantial to almost perfect agreement between the three observers (Dako κ=0.69-0.90; Leica κ=0.70-0.89; and Ventana κ=0.78-0.94) and concordance (Dako/Leica κ=0.81; Dako/Ventana κ=0.78; and Leica/Ventana κ=0.82). Univariate survival analyses showed that only the ligand-binding assay, Dako and Ventana assays achieved statistical significance. No statistically significant results were seen in multivariate models, although a strong trend was seen with the Ventana progesterone receptor assay. All assays performed similarly with regards to measures of test performance with ligand-binding assay set as the reference, and all immunohistochemical assays outperformed the ligand-binding assay in regards to 5-year disease-free survival. Despite similar agreement and concordance with the progesterone receptor assays, clear differences were noted with regards to 5-year disease-free survival. Additional survival analyses suggest that clinical utility of estrogen receptor assays vary when investigated in combination with progesterone receptor.

A systematic comparison of three commercial estrogen receptor assays in a single clinical outcome breast cancer cohort.

Breast cancers are routinely assessed for estrogen receptor status using immunohistochemical assays to assist in patient prognosis and clinical management. Specific assays vary between laboratories, and several antibodies have been validated and recommended for clinical use. As numerous factors can influence assay performance, many laboratories have opted for ready-to-use assays using automated stainers to improve reproducibility and consistency. Three commonly used autostainer vendors-Dako, Leica, and Ventana-all offer such estrogen receptor assays; however, they have never been directly compared. Here, we present a systematic comparison of three platform-specific estrogen receptor ready-to-use assays using a retrospective, tamoxifen-treated, breast cancer cohort from patients who were treated in Calgary, Alberta, Canada from 1985 to 2000. We found all assays showed good intra-observer agreement. Inter-observer pathological scoring showed some variability: Ventana had the strongest agreement followed closely by Dako, whereas Leica only showed substantial agreement. We also analyzed each estrogen receptor assay with respect to 5-year disease-free survival, and found that all performed similarly in univariate and multivariate models. Determination of measures of test performance found that the Leica assay had a lower negative predictive value than Dako or Ventana, compared with the original ligand-binding assay, while other measures-sensitivity, specificity, positive predictive value, and accuracy-were comparable between the three ready-to-use assays. When comparing against disease-free survival, the difference in negative predictive value between the vendor assays were not as extreme, but Dako and Ventana still performed slightly better than Leica. Despite some discordance, we found that all ready-to-use assays were comparable with or superior to the ligand-binding assay, endorsing their continued use. Our analysis also allowed for exploration of estrogen receptor-negative, progesterone receptor-positive cases, and we discovered that this phenotype was not consistent across the assays, suggesting this might be an artifact.

Fractal analysis of nuclear histology integrates tumor and stromal features into a single prognostic factor of the oral cancer microenvironment.

BACKGROUND: The lack of prognostic biomarkers in oral squamous cell carcinoma (OSCC) has hampered treatment decision making and survival in OSCC remains poor. Histopathological features are used for prognostication in OSCC and, although useful for predicting risk, manual assessment of histopathology is subjective and labour intensive. In this study, we propose a method that integrates multiple histopathological features of the tumor microenvironment into a single, digital pathology-based biomarker using nuclear fractal dimension (nFD) analysis. METHODS: One hundred and seven consecutive OSCC patients diagnosed between 1998 and 2006 in Calgary, Canada were included in the study. nFD scores were generated from DAPI-stained images of tissue microarray (TMA) cores. Ki67 protein expression was measured in the tumor using fluorescence immunohistochemistry (IHC) and automated quantitative analysis (AQUA®). Lymphocytic infiltration (LI) was measured in the stroma from haematoxylin-eosin (H&E)-stained TMA slides by a pathologist. RESULTS: Twenty-five (23.4%) and 82 (76.6%) patients were classified as high and low nFD, respectively. nFD was significantly associated with pathological tumor-stage (pT-stage; P = 0.01) and radiation treatment (RT; P = 0.01). High nFD of the total tumor microenvironment (stroma plus tumor) was significantly associated with improved disease-specific survival (DSS; P = 0.002). No association with DSS was observed when nFD of either the tumor or the stroma was measured separately. pT-stage (P = 0.01), pathological node status (pN-status; P = 0.02) and RT (P = 0.03) were also significantly associated with DSS. In multivariate analysis, nFD remained significantly associated with DSS [HR 0.12 (95% CI 0.02-0.89, P = 0.04)] in a model adjusted for pT-stage, pN-status and RT. We also found that high nFD was significantly associated with high tumor proliferation (P < 0.0001) and high LI (P < 0.0001), factors that we and others have shown to be associated with improved survival in OSCC. CONCLUSIONS: We provide evidence that nFD analysis integrates known prognostic factors from the tumor microenvironment, such as proliferation and immune infiltration, into a single digital pathology-based biomarker. Prospective validation of our results could establish nFD as a valuable tool for clinical decision making in OSCC.

Evaluation of E-cadherin, ß-catenin and vimentin protein expression using quantitative immunohistochemistry in nasopharyngeal carcinoma patients.

PURPOSE: Aberrant expression of proteins involved in epithelial-to-mesenchymal transition have been described in various cancers. In this retrospective study, we sought to evaluate E-cadherin, ß-catenin and vimentin protein expression in non-metastatic nasopharyngeal (NPC) patients treated with curative intent, examine their relationship with each other, and with clinical outcome measures. METHODS: Pre-treatment formalin-fixed paraffin-embedded biopsies of 140 patients treated between January 2000 and December 2007 were assembled into a tissue microarray (TMA). Automated quantitative immunohistochemistry (AQUA®) was performed on sequential TMA sections stained with fluorescent-labeled antibodies against E-cadherin, ß-catenin and vimentin. Cox proportional hazards regression was used to estimate the effect of cytoplasmic vimentin, cytoplasmic E-cadherin, ß-catenin nuclear/cytoplasmic ratio expression on overall survival and disease-free survival. RESULTS: The average age of the patients was 51.7 years (SD=12.1; range 18-85), 66% were male, 71% had a KPS ≥ 90% at the start of treatment and 65% had stage III/IV disease. After adjusting for performance status, WHO and stage, high E-cadherin levels over the 75th percentile were found to produce a significantly increased risk for both a worse overall survival (HR = 2.53, 95% CI 1.21, 5.27) and disease free survival (DFS; HR = 2.14, 95%CI 1.28, 3.59). Vimentin levels over the first quartile produced an increased risk for a worse DFS (HR = 2.21, 95% CI 1.11, 4.38). No association was seen between ß-catenin and survival. CONCLUSION: In this cohort of NPC patients, higher levels of E-cadherin and higher levels of vimentin were associated with worse outcomes. Further work is needed to understand the role of these epithelial mesenchymal transition proteins in NPC.

A subclass of acylated anti-inflammatory mediators usurp Toll-like receptor 2 to inhibit neutrophil recruitment through peroxisome proliferator-activated receptor gamma.

Toll-like receptors are host sentinel receptors that signal the presence of infectious nonself and initiate protective immunity. One of the primary immune defense mechanisms is the recruitment of neutrophils from the bloodstream into the infected tissue. Although neutrophils are important in host defense, they can also be responsible for damaging pathologies associated with excessive inflammation. Here, we report that the di-acylated TLR2 ligand lipoteichoic acid can directly inhibit neutrophil recruitment in vivo. This discovery allowed us to test the concept that conventional proinflammatory TLR2 ligands can be made to act as inhibitors through specific structural modifications. Indeed, lipopeptide TLR2 ligands, when modified at their acyl chains to contain linoleate, lose their capacity to induce inflammation and yield ligands that can directly inhibit the in vivo neutrophil recruitment initiated by a wide range of proinflammatory stimuli. The inhibitory capacity of LTA and these modified ligands requires the expression of TLR2, but is independent of the TLR2 signaling adaptor, MyD88. Instead, this inhibitory effect requires functional activity of the fatty acid and nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ). Therefore, these data support a model in TLR2 biology where structural modifications of these ligands can profoundly influence host-microbial interactions. These inhibitory TLR2 ligands also have broader implications with respect to their potential use in various inflammatory disease settings.

Bladder preservation therapy for muscle-invading bladder cancers on Radiation Therapy Oncology Group trials 8802, 8903, 9506, and 9706: vascular endothelial growth factor B overexpression predicts for increased distant metastasis and shorter survival.

BACKGROUND: From 1988 to 1999, the Radiation Therapy Oncology Group (RTOG) conducted four prospective studies (8802, 8903, 9506, 9706) of patients with clinical stage T2-4a muscle-invasive bladder cancer. Treatment was selective bladder preservation using transurethral surgery (TURBT) plus cisplatin-based induction and consolidation chemoradiation regimens, reserving radical cystectomy for invasive tumor recurrence. We investigated vascular endothelial growth factor (VEGF) pathway biomarkers in this unique clinical dataset (median follow-up of 3.1 years). METHODS: A total of 43 patients with tissue available from the entry TURBT were included in this analysis. Expression of VEGF ligands and receptors were quantified and scored by the AQUA platform (HistoRX, now Genoptix, Carlsbad, CA) and analyzed after median split. RESULTS: VEGF expression levels were not associated with increased rates of complete response to induction chemoradiation. Higher levels of cytoplasmic VEGF-B, VEGF-C, and VEGF-R2 were associated with decreased overall survival rates. The 3-year overall survival estimates for high and low expressers were 43.7% and 75% for VEGF-B cytoplasm (p = .01), 40.2% and 86.7% for VEGF-C cytoplasm (p = .01), and 49.7% and 66.7% for VEGF-R2 cytoplasm (p = .02). Higher expression levels of cytoplasm VEGF-B were associated with higher rates of distant failure (p = .01). CONCLUSIONS: Although VEGF ligands and receptors do not appear to be associated with complete response to induction chemoradiation for muscle-invasive bladder cancer, we report significant associations with overall survival and distant failure for certain VEGF family members.

Integrin ß7-mediated regulation of multiple myeloma cell adhesion, migration, and invasion.

Integrin-ß7 (ITGB7) mRNA is detected in multiple myeloma (MM) cells and its presence is correlated with MAF gene activation. Although the involvement of several integrin family members in MM-stoma cell interaction is well documented, the specific biologic functions regulated by integrin-ß7 in MM are largely unknown. Clinically, we have correlated integrin-ß7 expression in MM with poor survival outcomes post autologous stem cell transplantation and postsalvage therapy with bortezomib. Functionally, we have found that shRNA-mediated silencing of ITGB7 reduces MM-cell adhesion to extra-cellular matrix elements (fibronectin, E-cadherin) and reverses cell-adhesion-mediated drug resistance (CAM-DR) sensitizing them to bortezomib and melphalan. In addition, ITGB7 silencing abrogated MM-cell transwell migration in response to SDF1α gradients, reduced vessel density in xenografted tumors, and altered MM cells in vivo homing into the BM. Mechanistically, ITGB7 knockdown inhibited focal adhesion kinase (FAK) and Src phosphorylation, Rac1 activation, and SUMOylation, reduced VEGF production in MM-BM stem cell cocultures and attenuated p65-NF-κB activity. Our findings support a role for integrin-ß7 in MM-cell adhesion, migration, and BM homing, and pave the way for a novel therapeutic approach targeting this molecule.

Quantification of ER/PR expression in ovarian low-grade serous carcinoma.

OBJECTIVE: Case reports suggest that hormonal therapy may be a useful treatment option for low-grade serous carcinomas (LGSC) but the clinical value remains uncertain. We hypothesized that LGSCs show a constitutive high hormone receptor expression and that type diagnosis may be sufficient to initiate hormonal therapy. METHODS: We assessed ER and PR expression on 27 LGSC, 69 high-grade serous carcinomas (HGSC), 36 serous borderline tumors (SBOT), and five normal fallopian tubes using three different platforms/antibodies on tissue microarrays. Staining from the Leica Bond Max and DAKO PharmDx platforms was evaluated using the Allred score. Quantitative fluorescence immunohistochemistry was performed using the HistoRx AQUAnalysis platform. A second cohort of 12 LGSC and 183 HGSC was assessed using the HistoRx AQUAnalysis platform. Welch ANOVA or Fisher's Exact Test was used to compare differences in the histological types for each platform. Nonparametric bivariate density plots were used to graphically demonstrate the relationship between ER and PR for the various histological types. RESULTS: LGSC have higher ER and PR expression compared to HGSC but significantly less than FT and SBOT. Nonparametric bivariate density revealed two populations of LGSC: one fifth of LGSC are ER high/PR high expressers similar to SBOT but the majority show low ER/PR expression more like HGSC. CONCLUSIONS: Quantitative assessment of ER/PR expression using the HistoRx AQUAnalysis platform may be useful as a predictive diagnostic for hormonal therapy in LGSC, assuming that only the fraction of double high expressers benefit from hormonal treatment.

Low levels of Stat5a protein in breast cancer are associated with tumor progression and unfavorable clinical outcomes.

INTRODUCTION: Signal transducer and activator of transcripton-5a (Stat5a) and its close homologue, Stat5b, mediate key physiological effects of prolactin and growth hormone in mammary glands. In breast cancer, loss of nuclear localized and tyrosine phosphorylated Stat5a/b is associated with poor prognosis and increased risk of antiestrogen therapy failure. Here we quantify for the first time levels of Stat5a and Stat5b over breast cancer progression, and explore their potential association with clinical outcome. METHODS: Stat5a and Stat5b protein levels were quantified in situ in breast-cancer progression material. Stat5a and Stat5b transcript levels in breast cancer were correlated with clinical outcome in 936 patients. Stat5a protein was further quantified in four archival cohorts totaling 686 patients with clinical outcome data by using multivariate models. RESULTS: Protein levels of Stat5a but not Stat5b were reduced in primary breast cancer and lymph node metastases compared with normal epithelia. Low tumor levels of Stat5a but not Stat5b mRNA were associated with poor prognosis. Experimentally, only limited overlap between Stat5a- and Stat5b-modulated genes was found. In two cohorts of therapy-naïve, node-negative breast cancer patients, low nuclear Stat5a protein levels were an independent marker of poor prognosis. Multivariate analysis of two cohorts treated with antiestrogen monotherapy revealed that low nuclear Stat5a levels were associated with a more than fourfold risk of unfavorable outcome. CONCLUSIONS: Loss of Stat5a represents a new independent marker of poor prognosis in node-negative breast cancer and may be a predictor of response to antiestrogen therapy if validated in randomized clinical trials.

Bax expression measured by AQUAnalysis is an independent prognostic marker in oral squamous cell carcinoma.

BACKGROUND: Resistance to apoptosis is a hallmark of cancer and proteins regulating apoptosis have been proposed as prognostic markers in several malignancies. However, the prognostic impact of apoptotic markers has not been consistently demonstrated in oral squamous cell carcinoma (OSCC). This inconsistency in reported associations between apoptotic proteins and prognosis can be partly attributed to the intrinsic low resolution and misclassification associated with manual, semi-quantitative methods of biomarker expression measurement. The aim of this study was to examine the association between apoptosis-regulating proteins and clinical outcomes in oral squamous cell carcinoma (OSCC) using the quantitative fluorescence immunohistochemistry (IHC) based AQUAnalysis technique. METHODS: Sixty-nine OSCC patients diagnosed between 1998-2005 in Calgary, Alberta, Canada were included in the study. Clinical data were obtained from the Alberta Cancer Registry and chart review. Tissue microarrays (TMAs) were assembled from triplicate cores of formalin-fixed paraffin embedded pre-treatment tumour tissue. Bax, Bcl-2 and Bcl-XL protein expression was quantified using fluorescent IHC and AQUA technology in normal oral cavity squamous epithelium (OCSE) and OSCC tumour samples. Survival was analyzed using Kaplan-Meier plots and the Cox proportional hazard model. RESULTS: Bax expression was predominantly nuclear in OCSE and almost exclusively cytoplasmic in OSCC. No similar differences in localization were observed for Bcl-2 or Bcl-XL. Only Bax expression associated with disease-specific survival (DSS), with 5-year survival estimates of 85.7% for high Bax versus 50.3% for low Bax (p = 0.006), in univariate analysis. High Bax expression was also significantly associated with elevated Ki67 expression, indicating that increased proliferation might lead to an improved response to radiotherapy in patients with elevated Bax expression. In multivariate analyses, Bax protein expression remained an independent predictor of DSS in OSCC [HR 0.241 (0.078-0.745), p = 0.013]. CONCLUSIONS: The AQUA technique used in our study eliminates observer bias and provides reliable and reproducible estimates for biomarker expression. AQUA also provides essential measures of quality control that cannot be achieved with manual biomarker scoring techniques. Our results support the use of Bax protein expression as a prognostic marker in conjunction with other clinico-pathological variables when designing personalized treatment strategies for OSCC patients.

Epicutaneous Staphylococcus aureus induces IL-36 to enhance IgE production and ensuing allergic disease.

IgE induced by type 2 immune responses in atopic dermatitis is implicated in the progression of atopic dermatitis to other allergic diseases, including food allergies, allergic rhinitis, and asthma. However, the keratinocyte-derived signals that promote IgE and ensuing allergic diseases remain unclear. Herein, in a mouse model of atopic dermatitis-like skin inflammation induced by epicutaneous Staphylococcus aureus exposure, keratinocyte release of IL­36α along with IL-4 triggered B cell IgE class-switching, plasma cell differentiation, and increased serum IgE levels-all of which were abrogated in IL-36R-deficient mice or anti-IL­36R-blocking antibody-treated mice. Moreover, skin allergen sensitization during S. aureus epicutaneous exposure-induced IL-36 responses was required for the development of allergen-specific lung inflammation. In translating these findings, elevated IL­36 cytokines in human atopic dermatitis skin and in IL­36 receptor antagonist-deficiency patients coincided with increased serum IgE levels. Collectively, keratinocyte-initiated IL­36 responses represent a key mechanism and potential therapeutic target against allergic diseases.

The phytochemical piceatannol induces the loss of CBL and CBL-associated proteins.

Piceatannol is a naturally occurring bioactive stilbene with documented antileukemic properties. It has been extensively used as a Syk-selective protein tyrosine kinase inhibitor for the study of various signaling pathways. Herein, we show that the hydroxystilbene, piceatannol, and related catechol ring-containing compounds are able to induce the loss of the Cbl family of proteins. Normal cellular Cbl-regulatory mechanisms were not involved in this process. Screening of a small library of piceatannol-like compounds indicated that aromaticity and a catechol ring were required for the induction of Cbl loss. Further examination of these two chemical properties showed that the oxidative conversion of the catechol ring of piceatannol into a highly reactive O-benzoquinone was the cause of piceatannol-induced Cbl loss. Characterization of the Cbl selectivity of piceatannol-induced protein loss revealed that this compound was also able to induce the functional loss of specific Cbl-associated proteins involved in signaling pathways commonly associated with cancer. This work uncovers a new, piceatannol-dependent effect and shows a novel way in which this phenomenon can be exploited to inhibit disease-associated signaling pathways.

Loss of tumour-specific ATM protein expression is an independent prognostic factor in early resected NSCLC.

Ataxia-telangiectasia mutated (ATM) is critical in maintaining genomic integrity. In response to DNA double-strand breaks, ATM phosphorylates downstream proteins involved in cell-cycle checkpoint arrest, DNA repair, and apoptosis. Here we investigate the frequency, and influence of ATM deficiency on outcome, in early-resected non-small cell lung cancer (NSCLC). Tissue microarrays, containing 165 formalin-fixed, paraffin-embedded resected NSCLC tumours from patients diagnosed at the Tom Baker Cancer Centre, Calgary, Canada, between 2003 and 2006, were analyzed for ATM expression using quantitative fluorescence immunohistochemistry. Both malignant cell-specific ATM expression and the ratio of ATM expression within malignant tumour cells compared to that in the surrounding tumour stroma, defined as the ATM expression index (ATM-EI), were measured and correlated with clinical outcome. ATM loss was identified in 21.8% of patients, and was unaffected by clinical pathological variables. Patients with low ATM-EI tumours had worse survival outcomes compared to those with high ATM-EI (p < 0.01). This effect was pronounced in stage II/III patients, even after adjusting for other clinical co-variates (p < 0.001). Additionally, we provide evidence that ATM-deficient patients may derive greater benefit from guideline-recommended adjuvant chemotherapy following surgical resection. Taken together, these results indicate that ATM loss seems to be an early event in NSCLC carcinogenesis and is an independent prognostic factor associated with worse survival in stage II/III patients.

Molecular-Based Recursive Partitioning Analysis Model for Glioblastoma in the Temozolomide Era: A Correlative Analysis Based on NRG Oncology RTOG 0525.

IMPORTANCE: There is a need for a more refined, molecularly based classification model for glioblastoma (GBM) in the temozolomide era. OBJECTIVE: To refine the existing clinically based recursive partitioning analysis (RPA) model by incorporating molecular variables. DESIGN, SETTING, AND PARTICIPANTS: NRG Oncology RTOG 0525 specimens (n = 452) were analyzed for protein biomarkers representing key pathways in GBM by a quantitative molecular microscopy-based approach with semiquantitative immunohistochemical validation. Prognostic significance of each protein was examined by single-marker and multimarker Cox regression analyses. To reclassify the prognostic risk groups, significant protein biomarkers on single-marker analysis were incorporated into an RPA model consisting of the same clinical variables (age, Karnofsky Performance Status, extent of resection, and neurologic function) as the existing RTOG RPA. The new RPA model (NRG-GBM-RPA) was confirmed using traditional immunohistochemistry in an independent data set (n = 176). MAIN OUTCOMES AND MEASURES: Overall survival (OS). RESULTS: In 452 specimens, MGMT (hazard ratio [HR], 1.81; 95% CI, 1.37-2.39; P < .001), survivin (HR, 1.36; 95% CI, 1.04-1.76; P = .02), c-Met (HR, 1.53; 95% CI, 1.06-2.23; P = .02), pmTOR (HR, 0.76; 95% CI, 0.60-0.97; P = .03), and Ki-67 (HR, 1.40; 95% CI, 1.10-1.78; P = .007) protein levels were found to be significant on single-marker multivariate analysis of OS. To refine the existing RPA, significant protein biomarkers together with clinical variables (age, Karnofsky Performance Status, extent of resection, and neurological function) were incorporated into a new model. Of 166 patients used for the new NRG-GBM-RPA model, 97 (58.4%) were male (mean [SD] age, 55.7 [12.0] years). Higher MGMT protein level was significantly associated with decreased MGMT promoter methylation and vice versa (1425.1 for methylated vs 1828.0 for unmethylated; P < .001). Furthermore, MGMT protein expression (HR, 1.84; 95% CI, 1.38-2.43; P < .001) had greater prognostic value for OS compared with MGMT promoter methylation (HR, 1.77; 95% CI, 1.28-2.44; P < .001). The refined NRG-GBM-RPA consisting of MGMT protein, c-Met protein, and age revealed greater separation of OS prognostic classes compared with the existing clinically based RPA model and MGMT promoter methylation in NRG Oncology RTOG 0525. The prognostic significance of the NRG-GBM-RPA was subsequently confirmed in an independent data set (n = 176). CONCLUSIONS AND RELEVANCE: This new NRG-GBM-RPA model improves outcome stratification over both the current RTOG RPA model and MGMT promoter methylation, respectively, for patients with GBM treated with radiation and temozolomide and was biologically validated in an independent data set. The revised RPA has the potential to contribute to improving the accurate assessment of prognostic groups in patients with GBM treated with radiation and temozolomide and to influence clinical decision making. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00304031.

ATM, THMS, and RRM1 protein expression in nasopharyngeal carcinomas treated with curative intent.

BACKGROUND: In advanced nasopharyngeal carcinoma (NPC), biomarkers may help predict survival. METHODS: Tumoral expression of ataxia-telangiectasia mutated (ATM), thymidylate synthetase (THMS), and ribonucleotide reductase subunit M1 (RRM1), was correlated with survival in patients with nonmetastatic NPC using quantitative fluorescence immunohistochemistry with automated quantitative digital image analysis. RESULTS: Of the 146 patients included, 58 patients (40%) received concurrent chemoradiation therapy; the remainder was treated with radiation. Overall survival (OS) at 5 years was 71% (95% confidence interval [CI], 62% to 78%); disease-free survival (DFS) was 48% (95% CI, 39% to 57%). OS worsened for increasing values of ATM (hazard ratio [HR], 2.83; 95% CI, 1.01-7.94; p = .049) for values greater than the 75th percentile compared to less than the 25th percentile, but improved for tumors with higher THMS levels (HR, 0.44; 95% CI, 0.20-0.94; p = .033) for values greater than the 25th percentile compared to less than or equal to the 25th percentile. RRM1 was not associated with OS (p = .748). No biomarkers were associated with DFS. CONCLUSION: In our cohort, relative overexpression of ATM and low THMS levels were associated with worse OS. © 2015 Wiley Periodicals, Inc. Head Neck 38: E384-E391, 2016.

Association of keratinization with 5-year disease-specific survival in oropharyngeal squamous cell carcinoma.

IMPORTANCE: Keratinization is a histologic feature on hematoxylin-eosin staining associated with adverse outcomes in head and neck cancer, particularly oral cavity squamous cell carcinoma. However, the prognostic value of keratinization has not been demonstrated in oropharyngeal squamous cell carcinoma (OPSCC) in a large cohort of patients. OBJECTIVE: To quantify the prognostic value of keratinization in a large cohort of patients with OPSCC with subgroup analysis based on p16 status, basaloid differentiation, and smoking status. DESIGN, SETTING, AND PARTICIPANTS: Retrospective cross-sectional study using a prospectively collected database that identified 208 patients with OPSCC diagnosed and treated at a single tertiary cancer center from 2002 to 2009. Tissue microarrays (TMAs) were generated from 208 patient specimens stained with hematoxylin-eosin and immunohistochemical markers. Digital images from stained TMAs were scored for the presence of keratinization and/or basaloid differentiation and for p16 status. INTERVENTIONS: Patients were treated with curative intent with surgery, radiation, and/or chemotherapy. MAIN OUTCOMES AND MEASURES: The primary outcome measure was 5-year disease-specific survival (DSS) in OPSCC according to keratinization. Univariate and multivariate survival analyses were performed to estimate survival according to histopathologic profile and smoking status. RESULTS: In the 208 samples, 96 were keratinizing and 112 were nonkeratinizing. Patients with keratinizing tumors were more likely to have advanced-stage disease and be p16 negative. Keratinization was independently associated with adverse outcomes. The 5-year DSS was significantly higher for nonkeratinizing tumors (63.3%) compared with keratinizing tumors (44.8%; P = .007). In subgroup analysis, nonkeratinization was associated with improved DSS in those with nonbasaloid and p16-negative tumors and in patients who were smokers. When stratifying patients based on keratinization, p16-status, and smoking status, patients with p16-negative keratinizing tumors who were smokers had the lowest 5-year DSS (26.7%). CONCLUSIONS AND RELEVANCE: Patients with nonkeratinized OPSCC have improved survival compared with those with keratinizing tumors. Information on keratinization is most useful prognostically in those who have p16-negative and nonbasaloid tumors and in patients who are smokers. Survival can be stratified using keratinization, p16 status, and smoking status.

The prognostic value of HPV status and p16 expression in patients with carcinoma of the anal canal.

BACKGROUND: In anal cancer studies, the detection frequency of high-risk HPV (human papillomavirus) is variable, depending on the method used. There are limited data reporting results of different HPV detection techniques in the same clinical series, and very few correlating results with clinical outcome. OBJECTIVES: To evaluate tumor expression of p16/HPV16 using three different methods, and to determine their association with clinical outcome in patients with anal canal squamous cell carcinomas (SCC). DESIGN: This retrospective study included patients with anal canal SCC treated with definitive radiotherapy or chemoradiotherapy at a single institution between 1992 and 2005. Formalin-fixed paraffin-embedded tumor samples from 53 of the 89 (60%) patient pre-treatment biopsies were adequate for tissue microarray construction. HPV status was determined using: p16 expression by conventional immunohistochemistry (IHC) and quantitative IHC (AQUA), HPV genotype analysis by chromogenic in situ hybridization (CISH) and HPV linear array sub-typing. Expression status was correlated with clinical outcome. RESULTS: 80% (28/35) of patient tumors had high p16 expression using conventional IHC. HPV16 CISH was positive in 81% (34/42) of tumors, and 78% (28/36) of tumors were HPV subtype 16. HPV16 CISH correlated with p16 evaluated by conventional IHC (correlation coefficient 0.46; p = 0.01) and by p16 AQUA score (correlation coefficient 0.49; p = 0.001). A subset of cases (15%) had very high p16 quantitative IHC scores (>244) and were associated with a higher incidence of local or distant recurrence (p = 0.04). CONCLUSIONS: The vast majority (80%) of anal canal SCC in our series were positive for HPV16/p16, regardless of the testing method used. The exploratory analysis of automated quantitative IHC scoring was the only technique to define a subset of patients with a worse prognosis by p16 expression status on univariate analysis. Further exploration of the molecular mechanisms of treatment resistance in association with very high p16 expression is warranted.

Tumor cell apoptosis mediated by cytoplasmic ING1 is associated with improved survival in oral squamous cell carcinoma patients.

The ING1 epigenetic regulator and tumor suppressor plays a central role in apoptosis. The Ing1 gene is functionally inactivated in many cancer types but is rarely mutated. Although most studies have implicated the major ING1 isoform, p33ING1b, in nuclear apoptotic signalling, we recently discovered a novel and potent apoptosis-inducing effect of p33ING1b translocation to the mitochondria in response to DNA damage. In the present study, we examined the impact of cytoplasmic/mitochondrial localization of p33ING1b in oral squamous cell carcinoma (OSCC) patient samples and explored the therapeutic potential of adenovirally-overexpressed p33ING1b in OSCC cell lines in combination with ionizing radiation (IR) treatment. In contrast with previous reports, we found that p33ING1b protein and mRNA levels are higher in OSCC compared to normal epithelial cells. In OSCC patient samples, higher levels of intra-tumoral cytoplasmic p33ING1b correlated with increased apoptotic markers and significantly better patient survival. This association was strongest in patients who received post-operative radiotherapy. IR treatment induced p33ING1b translocation to the mitochondria and adenoviral-p33ING1b synergized with IR to kill OSCC cells. Our results identify a novel functional relationship between cytoplasmic p33ING1b and patient survival and highlight the potential for the use of p33ING1b as a therapeutic agent in combination with adjuvant radiotherapy in OSCC.