Debulking Hepatectomy for Colorectal Liver Metastasis Conveys Survival Benefit.

(1) Background: Despite advances in surgical technique and systemic chemotherapy, some patients with multifocal, bilobar colorectal liver metastases (CRLM) remain unresectable. These patients may benefit from surgical debulking of liver tumors in combination with chemotherapy compared to chemotherapy alone. (2) Methods: A retrospective study including patients evaluated for curative intent resection of CRLM was performed. Patients were divided into three groups: those who underwent liver resection with recurrence within 6 months (subtotal debulked, SD), those who had the first stage only of a two-stage hepatectomy (partially debulked, PD), and those never debulked (ND). Kaplan-Meier survival curves and log-rank test were performed to assess the median survival of each group. (3) Results: 174 patients underwent liver resection, and 34 patients recurred within 6 months. Of the patients planned for two-stage hepatectomy, 35 underwent the first stage only. Thirty-two patients were never resected. Median survival of the SD, PD, and ND groups was 31 months, 31 months, and 19.5 months, respectively (p = 0.012); (4) Conclusions: Patients who underwent a debulking of CRLM demonstrated a survival benefit compared to patients who did not undergo any surgical resection. This study provides support for the evaluation of intentional debulking versus palliative chemotherapy alone in a randomized trial.

Histopathological growth patterns of liver metastasis: updated consensus guidelines for pattern scoring, perspectives and recent mechanistic insights.

The first consensus guidelines for scoring the histopathological growth patterns (HGPs) of liver metastases were established in 2017. Since then, numerous studies have applied these guidelines, have further substantiated the potential clinical value of the HGPs in patients with liver metastases from various tumour types and are starting to shed light on the biology of the distinct HGPs. In the present guidelines, we give an overview of these studies, discuss novel strategies for predicting the HGPs of liver metastases, such as deep-learning algorithms for whole-slide histopathology images and medical imaging, and highlight liver metastasis animal models that exhibit features of the different HGPs. Based on a pooled analysis of large cohorts of patients with liver-metastatic colorectal cancer, we propose a new cut-off to categorise patients according to the HGPs. An up-to-date standard method for HGP assessment within liver metastases is also presented with the aim of incorporating HGPs into the decision-making processes surrounding the treatment of patients with liver-metastatic cancer. Finally, we propose hypotheses on the cellular and molecular mechanisms that drive the biology of the different HGPs, opening some exciting preclinical and clinical research perspectives.

Loss of mitochondrial ATPase ATAD3A contributes to nonalcoholic fatty liver disease through accumulation of lipids and damaged mitochondria.

Mitochondrial ATPase ATAD3A is essential for cholesterol transport, mitochondrial structure, and cell survival. However, the relationship between ATAD3A and nonalcoholic fatty liver disease (NAFLD) is largely unknown. In this study, we found that ATAD3A was upregulated in the progression of NAFLD in livers from rats with diet-induced nonalcoholic steatohepatitis and in human livers from patients diagnosed with NAFLD. We used CRISPR-Cas9 to delete ATAD3A in Huh7 human hepatocellular carcinoma cells and used RNAi to silence ATAD3A expression in human hepatocytes isolated from humanized liver-chimeric mice to assess the influence of ATAD3A deletion on liver cells with free cholesterol (FC) overload induced by treatment with cholesterol plus 58035, an inhibitor of acetyl-CoA acetyltransferase. Our results showed that ATAD3A KO exacerbated FC accumulation under FC overload in Huh7 cells and also that triglyceride levels were significantly increased in ATAD3A KO Huh7 cells following inhibition of lipolysis mediated by upregulation of lipid droplet-binding protein perilipin-2. Moreover, loss of ATAD3A upregulated autophagosome-associated light chain 3-II protein and p62 in Huh7 cells and fresh human hepatocytes through blockage of autophagosome degradation. Finally, we show the mitophagy mediator, PTEN-induced kinase 1, was downregulated in ATAD3A KO Huh7 cells, suggesting that ATAD3A KO inhibits mitophagy. These results also showed that loss of ATAD3A impaired mitochondrial basal respiration and ATP production in Huh7 cells under FC overload, accompanied by downregulation of mitochondrial ATP synthase. Taken together, we conclude that loss of ATAD3A promotes the progression of NAFLD through the accumulation of FC, triglyceride, and damaged mitochondria in hepatocytes.

Claudin-2 promotes colorectal cancer liver metastasis and is a biomarker of the replacement type growth pattern.

Claudin-2 promotes breast cancer liver metastasis by enabling seeding and early cancer cell survival. We now demonstrate that Claudin-2 is functionally required for colorectal cancer liver metastasis and that Claudin-2 expression in primary colorectal cancers is associated with poor overall and liver metastasis-free survival. We have examined the role of Claudin-2, and other claudin family members, as potential prognostic biomarkers of the desmoplastic and replacement histopathological growth pattern associated with colorectal cancer liver metastases. Immunohistochemical analysis revealed higher Claudin-2 levels in replacement type metastases when compared to those with desmoplastic features. In contrast, Claudin-8 was highly expressed in desmoplastic colorectal cancer liver metastases. Similar observations were made following immunohistochemical staining of patient-derived xenografts (PDXs) that we have established, which faithfully retain the histopathology of desmoplastic or replacement type colorectal cancer liver metastases. We provide evidence that Claudin-2 status in patient-derived extracellular vesicles may serve as a relevant prognostic biomarker to predict whether colorectal cancer patients have developed replacement type liver metastases. Such a biomarker will be a valuable tool in designing optimal treatment strategies to better manage patients with colorectal cancer liver metastases.

Cholesterol-binding translocator protein TSPO regulates steatosis and bile acid synthesis in nonalcoholic fatty liver disease.

Translocator protein (TSPO, 18 kDa) levels increase in parallel with the evolution of simple steatosis (SS) to nonalcoholic steatohepatitis (NASH) in nonalcoholic fatty liver disease (NAFLD). However, TSPO function in SS and NASH is unknown. Loss of TSPO in hepatocytes in vitro downregulated acetyl-CoA acetyltransferase 2 and increased free cholesterol (FC). FC accumulation induced endoplasmic reticulum stress via IRE1A and protein kinase RNA-like ER kinase/ATF4/CCAAT-enhancer-binding protein homologous protein pathways and autophagy. TSPO deficiency activated cellular adaptive antioxidant protection; this adaptation was lost upon excessive FC accumulation. A TSPO ligand 19-Atriol blocked cholesterol binding and recapitulated many of the alterations seen in TSPO-deficient cells. These data suggest that TSPO deficiency accelerated the progression of SS. In NASH, however, loss of TSPO ameliorated liver fibrosis through downregulation of bile acid synthesis by reducing CYP7A1 and CYP27A1 levels and increasing farnesoid X receptor expression. These studies indicate a dynamic and complex role for TSPO in the evolution of NAFLD.

Neutrophils expressing lysyl oxidase-like 4 protein are present in colorectal cancer liver metastases resistant to anti-angiogenic therapy.

Colorectal cancer liver metastases (CRCLM) that present with a replacement histopathological growth pattern (HGP) are resistant to neoadjuvant anti-angiogenic therapy. Surrogate biomarkers are not available to preoperatively identify patients with these tumors. Here we identify differentially expressed genes between CRCLM with a replacement HGP and those with a desmoplastic HGP using RNA sequencing. We demonstrate that LOXL4 is transcriptionally upregulated in replacement HGP CRCLM compared with desmoplastic HGP CRCLM and the adjacent normal liver. Interestingly, lysyl oxidase-like 4 (LOXL4) protein was expressed by neutrophils present in the tumor microenvironment in replacement HGP CRCLM. We further demonstrate that LOXL4 expression is higher in circulating neutrophils of cancer patients compared with healthy control patients and its expression can be induced by stimulation with lipopolysaccharide and TNF-α. Our study is the first to show the expression of LOXL4 in neutrophils and reveals the potential for LOXL4-expressing neutrophils to support the replacement HGP phenotype and to serve as a surrogate biomarker for this subtype of CRCLM. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Angiopoietin1 Deficiency in Hepatocytes Affects the Growth of Colorectal Cancer Liver Metastases (CRCLM).

Colorectal cancer liver metastases (CRCLM) that receive their blood supply via vessel co-option are associated with a poor response to anti-angiogenic therapy. Angiopoietins (Ang1 and Ang2) with their Tyrosine-protein kinase receptor (Tie2) have been shown to support vessel co-option. We demonstrate significantly higher expression of Ang1 in hepatocytes adjacent to the tumor region of human chemonaïve and treated co-opting (replacement histopathological growth patterns: RHGP) tumors. To investigate the role of the host Ang1 expression, Ang1 knockout (KO) mice were injected intra-splenically with metastatic MC-38 colon cancer cells that develop co-opting liver metastases. We observed a reduction in the number of liver metastases and interestingly, for the first time, the development of angiogenic driven desmoplastic (DHGP) liver metastases. In addition, in-vitro, knockout of Ang1 in primary hepatocytes inhibited viability, migration and invasion ability of MC-38 cells. We also demonstrate that Ang 1 alone promotes the migration and growth of both human and mouse colon cancer cell lines These results provide evidence that high expression of Ang1 in the host liver is important to support vessel co-option (RHGP lesions) and when inhibited, favours the formation of angiogenic driven liver metastases (DHGP lesions).

Vascularization of colorectal carcinoma liver metastasis: insight into stratification of patients for anti-angiogenic therapies.

Current treatment for metastatic disease targets angiogenesis. With the increasing data demonstrating that cancer cells do not entirely rely on angiogenesis but hijack the existing vasculature through mechanisms such as co-option of existing blood vessels, identification of targets has become of utmost importance. Our study looks at the vasculature of chemonaïve and treated colorectal carcinoma liver metastases (CRCLMs) to obtain a basic understanding of the microvessel density, type of vasculature (mature versus immature), and correlation with histopathological growth patterns that demonstrate unique patterns of angiogenesis. We performed immunohistochemistry on chemonaïve sections of desmoplastic histopathological growth pattern (DHGP) and replacement histopathological growth patterns (RHGP) lesions with CD31 [endothelial cell (EC) marker] and CD34/Ki67 double staining, which denotes proliferating ECs. The CD31 stains demonstrated a lower microvascular CD31 +ve capillary density in the DHGP versus RHGP lesions; and integrating both immunostains with CD34/Ki67 staining on serial sections revealed proliferating vessels in DHGP lesions and co-option of mature existing blood vessels in RHGP lesions. Interestingly, upon treatment with chemotherapy and bevacizumab, the RHGP lesions showed no necrosis whereas the DHGP lesions had almost 100% necrosis of the cancer cells and in most cases there was a single layer of viable cancer cells, just under or within the desmoplastic ring. The survival of these cells may be directly related to spatial location and possibly a different microenvironment, which may involve adhesion to different extracellular matrix components and/or different oxygen/nutrient availability. This remains to be elucidated. We provide evidence that DHGP CRCLMs obtain their blood supply via sprouting angiogenesis whereas RHGP lesions obtain their blood supply via co-option of existing vasculature. Furthermore current treatment regimens do not affect RHGP lesions and although they kill the majority of the cancer cells in DHGP lesions, there are cells surviving within or adjacent to the desmoplastic ring which could potentially give rise to a growing lesion.

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.

The Role of Prolactin in Bone Metastasis and Breast Cancer Cell-Mediated Osteoclast Differentiation.

BACKGROUND: Metastasis to the bone is a deleterious aspect of breast cancer and is a preferred site that results in bone loss. Hormones such as prolactin (PRL) have not yet been studied for their role in modulating the secondary tumor bone microenvironment. METHODS: We used quantitative immunohistochemistry with 134 samples of human primary breast cancer and 17 matched primary breast cancers and bone metastases. A Cox proportional hazards regression model was fitted to evaluate the associations between high prolactin receptor (PRLR) expression and time to bone metastasis, adjusting for estrogen receptor status, lymph node status, and chemotherapy status. We assessed osteoclast differentiation, osteoclast size, and measured pit formation in dentine slices. Statistical tests were two-sided. RESULTS: High PRLR expression in the primary breast tumor was associated with a shorter time to metastasis that includes bone (PRLRAQUA Max-per 100 unit hazard ratio = 1.04, 95% confidence interval = 1.00 to 1.07, P = .03). We observed the PRLR in rare samples of bone metastases and matched primary breast cancer. PRL treatment of breast cancer cells induced osteoclast differentiation and bone lysis via secreted factors and was abrogated by a PRLR antagonist (delta1-9-G129R-hPRL). We demonstrated that sonic hedgehog is a PRL-regulated cytokine in breast cancer cells and part of the mechanism that induces osteoclast differentiation. CONCLUSIONS: Our evidence indicates that PRL-PRLR can escalate the impact of breast cancer on bone metastasis and that the presence of the PRLR in the tumor microenvironment of breast cancer bone metastasis has the potential to modulate the microenvironment to induce lytic osteoclast formation.

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.

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.

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.

The significance of tumoral ERCC1 status in patients with locally advanced cervical cancer treated with chemoradiation therapy: a multicenter clinicopathologic analysis.

PURPOSE: ERCC1 (excision repair cross-complementation group 1) expression has been shown to be a molecular marker of cisplatin resistance in many tumor sites, but has not been well studied in cervical cancer patients. The purpose of this study was to measure tumoral ERCC1 in patients with locally advanced cervical cancer treated with chemoradiation therapy (CRT) in a large multicenter cohort, and to correlate expression with clinical outcome parameters. METHODS AND MATERIALS: A total of 264 patients with locally advanced cervical cancer, treated with curative-intent radical CRT from 3 major Canadian cancer centers were evaluated. Pretreatment formalin-fixed, paraffin-embedded tumor specimens were retrieved, and tissue microarrays were constructed. Tumoral ERCC1 (FL297 antibody) was measured using AQUA (R) technology. Statistical analysis was performed to determine the significance of clinical factors and ERCC1 status with progression-free survival (PFS) and overall survival (OS) at 5 years. RESULTS: The majority of patients had International Federation of Gynecology and Obstetrics (FIGO) stage II disease (n=119, 45%); median tumor size was 5 cm. OS was associated with tumor size (HR 1.16, P=.018), pretreatment hemoglobin status (HR 2.33, P=.00027), and FIGO stage. In addition, tumoral ERCC1 status (nuclear to cytoplasmic ratio) was associated with PFS (HR 2.33 [1.05-5.18], P=.038) and OS (HR 3.13 [1.27-7.71], P=.013). ERCC1 status was not significant on multivariate analysis when the model was adjusted for the clinical factors: for PFS (HR 1.49 [0.61-3.6], P=.38); for OS (HR 2.42 [0.94-6.24] P=.067). CONCLUSIONS: In this large multicenter cohort of locally advanced cervical cancer patients treated with radical CRT, stage, tumor size, and pretreatment hemoglobin status were significantly associated with PFS and OS. ERCC1 status appears to have prognostic impact on univariate analysis in these patients, but was not independently associated with outcome on multivariate analysis.

Assessment of ERCC1 and XPF protein expression using quantitative immunohistochemistry in nasopharyngeal carcinoma patients undergoing curative intent treatment.

PURPOSE: We sought to evaluate the prognostic/predictive value of ERCC1 and XPF in patients with nonmetastatic nasopharyngeal carcinoma (NPC) treated with curative intent. METHODS AND MATERIALS: ERCC1 and XPF protein expression was evaluated by immunofluorescence combined with automated quantitative analysis (AQUA) using the FL297 and 3F2 antibodies, respectively. ERCC1 and XPF protein expression levels were correlated with clinical outcomes. RESULTS: Patient characteristics were as follows: mean age 52 years (range, 18-85 years), 67% male, 72% Karnofsky performance status (KPS) ≥ 90%, World Health Organization (WHO) type 1/2/3 = 12%/28%/60%, stage III/IV 65%. With a median follow-up time of 50 months (range, 2.9 to 120 months), the 5-year overall survival (OS) was 70.8%. Median standardized nuclear AQUA scores were used as cutpoints for ERCC1 (n=138) and XPF (n=130) protein expression. Agreement between dichotomized ERCC1 and XPF scores was high at 79.4% (kappa = 0.587, P<.001). Neither biomarker predicted locoregional recurrence, DFS, or OS after adjustment for age and KPS, irrespective of stratification by stage, WHO type, or treatment. CONCLUSIONS: Neither ERCC1 nor XPF, analyzed by quantitative immunohistochemistry using the FL297 and 3F2 antibodies, was prognostic or predictive in this cohort of NPC patients.

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.

High stromal carbonic anhydrase IX expression is associated with nodal metastasis and decreased survival in patients with surgically-treated oral cavity squamous cell carcinoma.

Every year, approximately 25,000 patients are diagnosed with oral cavity squamous cell carcinoma (OCSCC) in the USA. The 5-year survival rate for OCSCC is approximately 40%. Intratumoral hypoxia confers poor prognosis and treatment failure but direct tumor oxygen measurement is challenging. Carbonic anhydrase IX (CAIX) is a marker of tissue hypoxia and we have recently shown that stromal CAIX is associated with reduced survival in patients with HPV-negative head and neck cancer. We examined the importance of this observation in OCSCC patients. We identified patients diagnosed and treated with OCSCC in Calgary (Alberta, Canada) between 1998 and 2005. Clinical and pathologic data were obtained from the Alberta Cancer Registry and chart review. Tissue microarrays (TMAs) were assembled from triplicate cores of archived tumor tissue. Stromal CAIX expression was assessed by quantitative immunohistochemistry (AQUA-HistoRx). The primary endpoint was disease-specific survival. We identified 102 patients with OCSCC; 87 patients had surgery as their primary treatment and adequate tumor tissue for TMA construction was available for all patients. CAIX expression was evaluable for 61 patients. High (top quartile) stromal CAIX expression was associated with significantly reduced 5-year disease-specific survival compared to low stromal CAIX expression (p<0.006). This study confirms our previously reported association between high stromal CAIX expression and significantly reduced overall survival in an independent, predominantly p16-negative, cohort of surgically treated OCSCC. Assessment of stromal CAIX expression could identify patients with the least favorable prognosis and inform therapeutic strategies in OCSCC.

CXCR4 overexpression is associated with poor outcome in females diagnosed with stage IV non-small cell lung cancer.

BACKGROUND: It has been proposed that the chemokine receptor, CXCR4, and its ligand, stromal cell-derived factor-1 (SDF-1), play a critical role in organ-specific tumor metastasis. High CXCR4 expression in resected non-small cell lung cancer (NSCLC) tumors is associated with poorer outcome; however, its effect on patient outcome in advanced NSCLC has not been explored. METHODS: After institutional ethical approval was obtained, demographic details, clinical variables, and outcome data were collected on consecutive NSCLC patients diagnosed at the Tom Baker Cancer Centre from 2003 to 2006 (Glans-Look Lung Cancer Database). Formalin-fixed paraffin-embedded diagnostic biopsies from stage IV patients were obtained and tissue microarrays generated. CXCR4 expression within NSCLC cells was analyzed by quantitative fluorescent immunohistochemistry using the HistoRx PM-2000 platform and then correlated with clinical outcome. RESULTS: Of 832 patients, 170 had samples suitable for tissue microarray generation and analysis. Automated immunohistochemistry for CXCR4 was successfully completed on all 170 patients. High expressors had a significantly poorer median overall survival of 2.7 months versus 5.6 months for the low expressors (p = 0.0468). This difference is driven by high-expressing females who have a median overall survival of 1.6 months versus 6.4 months for the low expressors (p = 0.006). CONCLUSIONS: CXCR4 is expressed in the majority of NSCLC tumors, and overexpression is associated with significantly poorer survival in stage IV NSCLC patients. Interestingly, this poor outcome is disproportionately represented in the female population. Our results suggest a gender-dependent difference in clinical outcome based on CXCR4 overexpression in stage IV NSCLC.

Glioma-associated oncogene family zinc finger 1 expression and metastasis in patients with head and neck squamous cell carcinoma treated with radiation therapy (RTOG 9003).

PURPOSE: Glioma-associated oncogene family zinc finger 1 (GLI1) expression was assessed to determine a potential role of hedgehog (Hh) signaling in head and neck squamous cell carcinoma (HNSCC). Additional proteins known to be modulated by Hh signaling, including beta-catenin (CTNNB1) and epidermal growth factor receptor (EGFR), were also assessed to determine the correlation among these distinct signaling pathways. PATIENTS AND METHODS: Nuclear GLI1 and CTNNB1 expression levels were determined in tumors from patients enrolled on Radiation Therapy Oncology Group (RTOG) 9003, a radiation fractionation trial. The results were also correlated with previously determined EGFR expression. The expression levels were evaluated in relation to three end points: time to metastasis (TTM), time to disease progression (TDP), and overall survival (OS). RESULTS: Among 1,068 eligible patients, data on GLI1, CTNNB1, and EGFR were available in 339, 164, and 300 patients, respectively. Although CTNNB1 expression did not differentiate prognosis, GLI1 was associated with poorer outcomes, adjusted for age, TNM stages, and Karnofsky performance score, and the significant influence persisted in a multivariable analysis (quartile 4 [Q4] v Q1 to Q3: TTM hazard ratio [HR], 2.7; 95% CI, 1.5 to 4.9; TDP HR, 1.6; 95% CI, 1.1 to 2.5; OS HR, 1.9; 95% CI, 1.4 to 2.7). The significance of GLI1 persisted in a multivariable analysis that included EGFR expression levels. CONCLUSION: These data suggest that Hh signaling may play an important role in metastasis and that GLI1 could serve as a marker in HNSCC, but the regulatory mechanisms and oncogenic significance need further investigation. Risk classification based on this analysis needs a validation in independent cohorts.

Detection of DNA damage in oocytes of small ovarian follicles following phosphoramide mustard exposures of cultured rodent ovaries in vitro.

Healthy oocytes are critical for producing healthy children, but little is known about whether or not oocytes have the capacity to identify and recover from injury. Using a model ovotoxic alkylating drug, cyclophosphamide (CPA), and its active metabolite, phosphoramide mustard (PM), we previously showed that PM (≥3µM) caused significant follicle loss in postnatal day 4 (PND4) mouse ovaries in vitro. We now investigate whether PM induces DNA damage in oocytes, examining histone H2AX phosphorylation (γH2AX), a marker of DNA double-strand breaks (DSBs). Exposure of cultured PND4 mouse ovaries to 3 and 0.1µM PM induced significant losses of primordial and small primary follicles, respectively. PM-induced γH2AX was observed predominantly in oocytes, in which foci of γH2AX staining increased in a concentration-dependent manner and peaked 18-24h after exposure to 3-10µMPM. Numbers of oocytes with ≥5 γH2AX foci were significantly increased both 1 and 8days after exposure to ≥1µMPM compared to controls. Inhibiting the kinases that phosphorylate H2AX significantly increased follicle loss relative to PM alone. In adult mice, CPA also induced follicle loss in vivo. PM also significantly decreased primordial follicle numbers (≥30µM) and increased γH2AX foci (≥3µM) in cultured PND4 Sprague-Dawley rat ovaries. Results suggest oocytes can detect PM-induced damage at or below concentrations which cause significant follicle loss, and there are quantitative species-specific differences in sensitivity. Surviving oocytes with DNA damage may represent an increased risk for fertility problems or unhealthy offspring.