Mie Konno

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.

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.

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.

Abstract A10: Stromal CAIX expression in oral squamous cell carcinoma and corresponding lymph node metastases

Background: Hypoxia is an almost ubiquitous feature of human solid tumours. However, the direct measurement of oxygen concentration in head and neck squamous cell carcinoma (HNSCC) has been conducted almost exclusively in the metastatic lymph nodes, not the primary tumor. Hypoxia is associated with resistance to both radiotherapy (RT) and chemotherapy in squamous cell cancers (SCCs). However, endogenous markers of hypoxia (EMH) have not reproducibly predicted clinical outcome. However, historically, the method used to assign hypoxic status according to directly measured oxygen concentration (polarographic electrode) and EMH (typically immunohistochemistry) have fundamentally differed. Methods: Demographic data and clinical outcomes of 61 patients with oral squamous cell carcinoma (OSCC) treated with primary surgery +/- adjuvant radiation were collected. Tissue microarray9s with representative samples of patients9 tumors were constructed from paraffin-embedded blocks. Automated quantitative immunohistochemistry (AQUA) was performed to measure expression and distribution of carbonic anhydrase IX (CAIX). Kaplan-Meier survival analysis and the log-rank test was used to assess the impact of vimentin-defined stromal CAIX (in both primary tumor and nodal metastases) on survival. Results: High stromal CAIX expression in the primary tumor was associated with significantly worse 5-year disease-specific survival. There was a statistically significant but relatively weak correlation between stromal CAIX expression in the primary tumor and the corresponding lymph nodes (r 2 :0.359; p=0.013). Stromal CAIX expression in the lymph node metastases was not significantly associated with 5-year disease-specific survival. Conclusions: Previous literature presenting the prognostic impact of directly measured hypoxia, using a polarographic electrode, was based predominantly on measurements from lymph nodes, not the primary tumor. Furthermore, comparisons with EMH were based solely on percentage of tumor cells that were positively stained, not the intensity of staining. We present the results of a tumor comparment-specific, quantitative fluorescent IHC method and the survival associated with EMH in the tumor microenvironment in the primary tumor and lymph node metastases. Citation Format: Shamir Chandarana, Pinaki Bose, Alexander C. Klimowicz, Stephanie K. Petrillo, Mie Konno, Luke Rudmik, Steven C. Nakoneshny, Wayne T. Matthews, Anthony M. Magliocco, Joseph C. Dort, Nigel T. Brockton. Stromal CAIX expression in oral squamous cell carcinoma and corresponding lymph node metastases. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A10.

Abstract P6-07-17: Proteomic screening of FFPE tissue identifies FKBP4 as an independent prognostic factor in hormone receptor positive breast cancers

Background: Adjuvant endocrine therapy reduces the risk of recurrence and death in hormone receptor positive breast cancer patients; however, 40–50% of estrogen receptor (ER) positive tumors are resistant to endocrine therapy. Identifying prognostic and predictive biomarkers to detect these non-responsive breast cancers at time of diagnosis may allow for improved clinical outcome for these patients. FKBP4 (FKBP52) is a co-chaperone protein that has been shown to regulate progesterone but not estrogen receptor activity, and was recently found to be highly expressed in early stage breast cancer compared to benign breast tissue. This evidence suggests that FKBP4 may play an important role in endocrine-responsive breast cancers. Methods: Global proteomic screening for biomarkers of aggressive breast cancer was performed on tissue samples from 24 patients with lymph node negative (LN−) disease and 24 patients with lymph node positive (LN+) disease randomly selected from the Calgary Tamoxifen Cohort, a retrospective cohort of breast cancer patients treated with adjuvant tamoxifen [n=511] from 1985–2000 at the Tom Baker Cancer Centre (Calgary, Canada). Liquid tissue lysates from FFPE tissue were analyzed by mass spectrometry and differentially expressed proteins were identified by the semi-quantitative spectral count method. FKBP4 was identified as an upregulated target in LN+ patients. The mRNA expression database of Kao et al. (BMC Cancer, 2011) was used to assess the prognostic potential of targets identified in the proteomic screen. FKBP4 protein expression was evaluated in tissue microarrays built from FFPE samples from the Calgary Tamoxifen Cohort using quantitative fluorescence immunohistochemistry and HistoRx AQUA analysis. Ten-year overall survival (OS) or five-year disease free survival (DFS) were the primary outcomes. Continuous variable FKBP4 data was dichotomized at the top quartile for both mRNA and protein expression analysis. Results: Univariate analysis demonstrated a significant association between high levels of FKBP4 mRNA and worse OS in ER+HER2− patients [n=182, HR=2.118 (1.070–4.192), p = 0.031] within the Kao et al database. Similarly, univariate analysis demonstrated that high levels of FKBP4 protein expression was associated with significantly worse DFS in ER+ HER2− patients [n=358, HR=1.632 (1.001–2.659), p = 0.049] in the Calgary Tamoxifen Cohort. FKBP4 was found to be an independent prognostic factor in both mRNA and protein expression cohorts using multivariate analysis adjusted for age, T stage, and lymph node status [OS: HR=2.786 (1.394–5.570), p = 0.004], or age, tumor size, tumor grade, and lymph node status [DFS: HR=1.875 (1.021–3.442), p = 0.043], respectively. Conclusions: Proteomic screening from FFPE tissue can identify new breast cancer biomarker candidates. Using this technique we have identified FKBP4 as an independent prognostic factor in ER+HER2− breast cancers, measured either by mRNA expression analysis or by quantitative protein expression analysis. Further studies are required to determine if FKBP4 may also be a predictive biomarker for tamoxifen response in ER+HER2− patients. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-07-17.

P5-12-03: An Innovative Quantification Method for Tamoxifen and Three Metabolites in Formalin-Fixed Paraffin-Embedded Tissues by Liquid Chromatography and Tandem Mass Spectrometry.

Background Tamoxifen (TAM) has been commonly used as a selective estrogen receptor modulator for the treatment and prevention of breast cancer. Although this drug is effective in many patients, some develop resistant and eventually relapse. Recent studies suggest that the cancer recurrence could possibly be due to changes in drug metabolism. TAM is metabolized by the cytochrome P450 enzyme pathway into several metabolites including 4-hydroxy-tamoxifen (4-OH), N-desmethyl-tamoxifen (DMT) and N-desmethyl-4-hydroxy-tamoxifen (endoxifen). These metabolites have variable potencies in suppressing estrogen-dependent breast cancer. Differences in metabolism may contribute to the clinical inter-individual variability in TAM response. The aim of our study was to provide a comprehensive evaluation of TAM and its metabolites through quantitative measurement in breast cancer patients to help better understand the pharmacological effects of TAM therapy. In the past, most methods used to measure the levels of TAM and its metabolites were in plasma or fresh/frozen tissue samples. These samples are typically not available retrospectively, and their long-term storage is expensive and laborious. Methods : We therefore explored the possibility to utilize formalin-fixed and paraffin-embedded (FFPE) tissues archived post breast surgery for quantification of TAM and its metabolites. Our laboratory has developed a rapid, sensitive and specific analytical method using liquid chromatography and tandem mass spectrometry (LC-MS/MS) for the measurement of TAM and its metabolites in FFPE tissues. The FFPE tissues were thin sectioned and deparaffinized by incubating twice with xylene for 10 min at room temperature. Sample clean-up was carried out subsequently using C2 solid-phase extraction, and detection was performed in the multiple-reaction monitoring mode with a triple quadrupole mass spectrometer. This method allows simultaneous quantification of TAM and three metabolites in FFPE tissues with a run time of 12 min. Results : The assay had good inter- and intra-assay precisions (2-6 %CV), and was linear over the range of 0.01-5 ng/g for 4-OH and endoxifen, and 0.1-50 ng/g for TAM and DMT. The extraction recoveries were between 83–88%. The validated method was successfully applied to analyze the FFPE tissues obtained from two groups of breast cancer patients. Patients were categorized into those with tumor recurrence (R) and those without recurrence (NR) after at least 2 months of 20 mg/d TAM treatment. Levels of TAM, 4-OH, DMT and endoxifen in FFPE tissues were compared between the two groups. Our preliminary data show that the ratio of DMT/TAM was significantly higher in the R (6.7, n = 13) than the NR patients (14.2, n = 9) (p Discussion : The assay described here not only allows accurate quantification of TAM and metabolites in FFPE tissues, but also opens up an incredible opportunity and new challenges for researchers to excavate precious information from FFPE tissues, especially when these archival samples represent the only source of biomaterial available. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-12-03.

P5-11-11: Automated Quantification Methods Improve the Accuracy of PR as an Independent Prognostic Factor in Tamoxifen Treated Breast Cancer Patients.

Background Adjuvant endocrine therapy reduces the risk of recurrence and death in hormone receptor positive breast cancer patients. However, 40–50% of estrogen receptor (ER) positive tumors are resistant to endocrine therapy. We have previously shown that quantitative measurement of ER protein expression has limited value as a prognostic marker in tamoxifen treated patients. The presence of progesterone receptor (PR) expression has shown promise as prognostic and/or predictive marker for endocrine therapy, including adjuvant tamoxifen, but reports are contradictory. Automation of scoring methods will improve the accuracy of PR scoring and its value as a prognostic factor. Methods : This retrospective study evaluated breast cancer patients treated with adjuvant tamoxifen (n=458) from 1985–2000 at the Tom Baker Cancer Centre, Calgary, Canada. Tissue microarrays were assembled from formalin fixed paraffin embedded tumor tissue. Clinico-pathologic data was obtained from chart review. Five-year disease-free survival (DFS) was the primary outcome. DAB-based PR staining was used to generate Allred PR scores, as well as to generate scores for PR percent area expression and PR integrated optical density (PR IOD) using the DAKO ACIS III scanner and image analysis software. PR tumor nuclear pixel intensity density scores were obtained using fluorescence-based PR staining, scanned and quantified with a HistoRx PM2000 scanner andAQUA image analysis software. Continuous variables were dichotomized using Xtile. Results : In our tamoxifen cohort, 5-year DFS was associated with tumor grade [HR 4.9(3.5−6.9), p Conclusions : We conclude that: 1) PR is an independent prognostic marker in our cohort, 2) using less subjective, automated quantitative scoring methods improves the value of PR as a prognostic biomarker, 3) AQUA appears superior to other automated methods and that 4) the incorporation of digital image analysis into practice would improve the prognostic value of PR expression in the clinical setting. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-11-11.