Microtubule associated proteins (MAPs) endogenously regulate microtubule stabilization and have been reported as prognostic and predictive markers for taxane response. The microtubule stabilizer, ...MAP-tau, has shown conflicting results. We quantitatively assessed MAP-tau expression in two independent breast cancer cohorts to determine prognostic and predictive value of this biomarker.
MAP-tau expression was evaluated in the retrospective Yale University breast cancer cohort (n = 651) using tissue microarrays and also in the TAX 307 cohort, a clinical trial randomized for TAC versus FAC chemotherapy (n = 140), using conventional whole tissue sections. Expression was measured using the AQUA method for quantitative immunofluorescence. Scores were correlated with clinicopathologic variables, survival, and response to therapy.
Assessment of the Yale cohort using Cox univariate analysis indicated an improved overall survival (OS) in tumors with a positive correlation between high MAP-tau expression and overall survival (OS) (HR = 0.691, 95% CI = 0.489-0.974; P = 0.004). Kaplan Meier analysis showed 10-year survival for 65% of patients with high MAP-tau expression compared to 52% with low expression (P = .006). In TAX 307, high expression was associated with significantly longer median time to tumor progression (TTP) regardless of treatment arm (33.0 versus 23.4 months, P = 0.010) with mean TTP of 31.2 months. Response rates did not differ by MAP-tau expression (P = 0.518) or by treatment arm (P = 0.584).
Quantitative measurement of MAP-tau expression has prognostic value in both cohorts, with high expression associated with longer TTP and OS. Differences by treatment arm or response rate in low versus high MAP-tau groups were not observed, indicating that MAP-tau is not associated with response to taxanes and is not a useful predictive marker for taxane-based chemotherapy.
There is critical need for standardization of HER2 immunohistochemistry testing in the clinical laboratory setting. Recently, the American Society of Clinical Oncology and the College of American ...Pathologists have submitted guidelines recommending that laboratories achieve 95% concordance between assays and observers for HER2 testing.
As a potential aid to pathologists for achieving these new guidelines, we have conducted an examination using automated quantitative analysis (AQUA analysis) to provide a standardized HER2 immunohistochemistry expression score across instruments (sites), operators, and staining runs.
We analyzed HER2 expression by immunohistochemistry in a cohort (n = 669) of invasive breast cancers in tissue microarray format across different instruments (n = 3), operators (n = 3), and staining runs (n = 3). Using light source, instrument calibration techniques, and a new generation of image analysis software, we produced normalized AQUA scores for each parameter and examined their reproducibility.
The average percent coefficients of variation across instruments, operators, and staining runs were 1.8%, 2.0%, and 5.1%, respectively. For positive/negative classification between parameters, concordance rates ranged from 94.5% to 99.3% for all cases. Differentially classified cases only occurred around the determined cut point, not over the entire distribution.
These data demonstrate that AQUA analysis can provide a standardized HER2 immunohistochemistry test that can meet current guidelines by the American Society of Clinical Oncology/College of American Pathologists. The use of AQUA analysis could allow for standardized and objective HER2 testing in clinical laboratories.
Tumor survival is influenced by interactions between tumor cells and the stromal microenvironment. One example is Endosialin (Tumor Endothelial Marker-1 (TEM-1) or CD248), which is expressed ...primarily by cells of mesenchymal origin and some tumor cells. The expression, as a function of architectural masking, of TEM-1 and its pathway-associated proteins was quantified and examined for association with five-year disease-specific survival on a colorectal cancer (CRC) cohort divided into training (n=330) and validation (n=164) sets. Although stromal expression of TEM-1 had prognostic value, a more significant prognostic signature was obtained through linear combination of five compartment-specific expression scores (TEM-1 Stroma, TEM-1 Tumor Vessel, HIF2α Stromal Vessel, Collagen IV Tumor, and Fibronectin Stroma). This resulted in a single continuous risk score (TAPPS: TEM-1 Associated Pathway Prognostic Signature) which was significantly associated with decreased survival on both the training set HR=1.76 (95%CI: 1.44-2.15); p<0.001 and validation set HR=1.38 (95%CI: 1.02-1.88); p=0.04. Importantly, since prognosis is a critical clinical question in Stage II patients, the TAPPS score also significantly predicted survival in the Stage II patient (n=126) cohort HR=1.75 (95%CI: 1.22-2.52); p=0.002 suggesting the potential of using the TAPPS score to assess overall risk in CRC patients, and specifically in Stage II patients.
Inherent to most tissue image analysis routines are user-defined steps whereby specific pixel intensity thresholds must be set manually to differentiate background from signal-specific pixels within ...multiple images. To reduce operator time, remove operator-to-operator variability, and to obtain objective and optimal pixel separation for each image, we have developed an unsupervised pixel-based clustering algorithm allowing for the objective and unsupervised differentiation of signal from background, and differentiation of compartment-specific pixels on an image-by-image basis. We used the Automated QUantitative Analysis (AQUA) platform, a well-established automated fluorescence-based immunohistochemistry image analysis platform used for quantification of protein expression in specific cellular compartments to demonstrate utility of this methodology. As a metric for cellular compartmentalization, we examined correlation of percentage nuclear volume with histologic grade in 3 serial sections of a large cohort (n=669) of invasive breast cancer samples. We observed a significant (P=0.002, 0.006, and 0.08) difference in mean percentage nuclear volume between low and high-grade tumors. Reproducibility of percentage nuclear volume was also significant (P<0.001) across 3 serial sections. We then quantified compartment-specific expression of 5 biomarkers in 3 cancer types for association with outcome: estrogen receptor (nuclear), progesterone receptor (nuclear), HER2 (membrane/cytoplasm), ERCC1 (nuclear), and PTEN (cytoplasm). All 5 markers showed an expected and significant (P<0.05) association with survival. This new clustering algorithm thus produces accurate and precise compartmentalization for assessment of target gene expression, and will enhance the efficiency and objectivity of the current Automated QUantitative Analysis and other image analysis platform.
To determine the safety and efficacy of gefitinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, in combination with radiation for newly diagnosed glioblastoma (GBM) patients.
...Between March 21, 2002, and May 3, 2004, Radiation Therapy Oncology Group (RTOG) 0211 enrolled 31 and 147 GBM patients in the phase 1 and 2 arms, respectively. Treatment consisted of daily oral gefinitnib started at the time of conventional cranial radiation therapy (RT) and continued post RT for 18 months or until progression. Tissue microarrays from 68 cases were analyzed for EGFR expression.
The maximum tolerated dose (MTD) of gefitinib was determined to be 500 mg in patients on non-enzyme-inducing anticonvulsant drugs (non-EIAEDs). All patients in the phase 2 component were treated at a gefitinib dose of 500 mg; patients receiving EIADSs could be escalated to 750 mg. The most common side effects of gefitinib in combination with radiation were dermatologic and gastrointestinal. Median survival was 11.5 months for patients treated per protocol. There was no overall survival benefit for patients treated with gefitinib + RT when compared with a historical cohort of patients treated with RT alone, matched by RTOG recursive partitioning analysis (RPA) class distribution. Younger age was significantly associated with better outcome. Per protocol stratification, EGFR expression was not found to be of prognostic value for gefitinib + RT-treated patients.
The addition of gefitinib to RT is well tolerated. Median survival of RTOG 0211 patients treated with RT with concurrent and adjuvant gefitinib was similar to that in a historical control cohort treated with radiation alone.
CONTEXTIncreased thymidylate synthase expression is a marker for decreased survival in colorectal cancer. OBJECTIVEThymidylate synthase localizes to both the nucleus and cytoplasm, but how the ...relationship of these expression levels affects colon cancer outcome has yet to be determined. DESIGNUsing AQUA, we assessed prognosis of thymidylate synthase expression as a function of subcellular localization in 2 retrospective cohorts of colorectal carcinoma. We used the first cohort (n = 599) as a training set, subsequently validating optimal expression cut points in the second cohort (n = 447). RESULTSA significant association between decreased 5-year disease-specific survival and increased nuclear expression (16% decreased survival 72% to 56% for the top 60% of nuclear-expressing tumors P < .001) and cytoplasmic expression (12% decreased survival 70% to 58% for the top 54% of cytoplasmic-expressing tumors P = .02) was observed for the training set. A higher nuclear to cytoplasmic ratio also correlated significantly with decreased survival (15% decreased survival 66% to 51% for the top 25% of tumors P < .001). Applying these findings to the validation set, as a function of time to recurrence, only the ratio (P = .03 expression ratio; P = .18 nuclear; P = .71 cytoplasmic) showed a significant association with decreased time to recurrence. Additionally, the expression ratio significantly added to the prognostic value given by the primary tumor pathologic classification and nodal status. CONCLUSIONSThese data suggest the relationship of nuclear to cytoplasmic thymidylate synthase expression, given as a ratio of continuous AQUA scores, to be a strong predictor of colon cancer survival.
Increased thymidylate synthase expression is a marker for decreased survival in colorectal cancer.
Thymidylate synthase localizes to both the nucleus and cytoplasm, but how the relationship of these ...expression levels affects colon cancer outcome has yet to be determined.
Using AQUA, we assessed prognosis of thymidylate synthase expression as a function of subcellular localization in 2 retrospective cohorts of colorectal carcinoma. We used the first cohort (n = 599) as a training set, subsequently validating optimal expression cut points in the second cohort (n = 447).
A significant association between decreased 5-year disease-specific survival and increased nuclear expression (16% decreased survival 72% to 56% for the top 60% of nuclear-expressing tumors P < .001) and cytoplasmic expression (12% decreased survival 70% to 58% for the top 54% of cytoplasmic-expressing tumors P = .02) was observed for the training set. A higher nuclear to cytoplasmic ratio also correlated significantly with decreased survival (15% decreased survival 66% to 51% for the top 25% of tumors P < .001). Applying these findings to the validation set, as a function of time to recurrence, only the ratio (P = .03 expression ratio; P = .18 nuclear; P = .71 cytoplasmic) showed a significant association with decreased time to recurrence. Additionally, the expression ratio significantly added to the prognostic value given by the primary tumor pathologic classification and nodal status.
These data suggest the relationship of nuclear to cytoplasmic thymidylate synthase expression, given as a ratio of continuous AQUA scores, to be a strong predictor of colon cancer survival.
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