Huan Cheng

Quantitative assessment shows loss of antigenic epitopes as a function of pre-analytic variables.

Pre-analytic variables, specifically cold ischemic time, have been implicated as key variables in the measurement of proteins by immunohistochemistry. To determine the significance and magnitude of antigenic loss due to pre-analytic variables, we have compared protein antigenicity in core needle biopsies, with essentially no cold ischemic time, with that in routinely processed tumor resection specimens. Two cohorts of matched core needle biopsies and tumor resections were collected with 20 matched pairs and 14 matched pairs, respectively. Both series were analyzed by quantitative immunofluorescence using the AQUA® method. Epitopes phospho-ERK, total ERK, phospho-AKT, total AKT, phospho-S6K1, total S6K1, estrogen receptor (ER), Ki67, cytokeratin and GAPDH were assessed. Detection levels for all phospho-epitopes were significantly decreased in tumor resections compared with biopsies while no significant change was seen in the corresponding total proteins. Of the other four proteins examined, ER and cytokeratin showed significant loss of antigenicity. This data suggest that measurement of phospho-protein antigenicity in formalin-fixed tissue by immunological methods is dramatically affected by pre-analytic variables. This study suggests that core needle biopsies are more accurate for assessment of tissue biomarkers.

Quantitative In Situ Measurement of Estrogen Receptor mRNA Predicts Response to Tamoxifen

Purpose Quantification of mRNA has historically been done by reverse transcription polymerase chain reaction (RT-PCR). Recently, a robust method of detection of mRNA utilizing in situ hybridization has been described that is linear and shows high specificity with low background. Here we describe the use of the AQUA method of quantitative immunofluorescence (QIF) for measuring mRNA in situ using ESR1 (the estrogen receptor alpha gene) in breast cancer to determine its predictive value compared to Estrogen Receptor α (ER) protein. Methods Messenger RNA for ER (ESR1) and Ubiquitin C (UbC) were visualized using RNAscope probes and levels were quantified by quantitative in situ hybridization (qISH) on two Yale breast cancer cohorts on tissue microarrays. ESR1 levels were compared to ER protein levels measured by QIF using the SP1 antibody. Results ESR1 mRNA is reproducibly and specifically measurable by qISH on tissue collected from 1993 or later. ESR1 levels were correlated to ER protein levels in a non-linear manner on two Yale cohorts. High levels of ESR1 were found to be predictive of response to tamoxifin. Conclusion Quantification of mRNA using qISH may allow assessment of large cohorts with minimal formalin fixed, paraffin embedded tissue. Exploratory data using this method suggests that measurement of ESR1 mRNA levels may be predictive of response to endocrine therapy in a manner that is different from the predictive value of ER.

Comparison of quantitative immunofluorescence with conventional methods for HER2/neu testing with respect to response to trastuzumab therapy in metastatic breast cancer.

CONTEXT Selection for trastuzumab therapy depends on a companion diagnostic assessment of HER2 by either immunohistochemistry (IHC) for protein overexpression or fluorescence in situ hybridization (FISH) to detect gene amplification. Although many studies have compared IHC to FISH, few have compared the tests to the true gold standard, tumor response. OBJECTIVE To compare HER2 testing by FISH and IHC along with a third immunofluorescence-based assay (automated quantitative analysis-tissue microarray [AQUA-TMA]) and to assess the value of each test for prediction of response to trastuzumab. DESIGN Immunohistochemistry and FISH assays were done on both whole slides (IHC-WS and FISH-WS) and on TMAs (IHC-TMA and FISH-TMA). AQUA was only done on TMAs (AQUA-TMA). Response was assessed according to modified Response Evaluation Criteria in Solid Tumors. RESULTS AQUA-TMA scores showed a significant linear relationship to both the FISH signal ratio and IHC scores on whole sections and TMAs. Assay assessment by outcome showed no association between response and FISH-WS ratio (P = .96), FISH-TMA (P = .55), IHC-WS (P = .75), or IHC-TMA (P = .06), but a significant relationship between AQUA score and categoric response was observed (P = .01). Assessed as a function of outcome using models of logistic regression, both AQUA-TMA and IHC-TMA were equally significant (P = .01). FISH-WS was the most sensitive assay, with a significantly higher true-positive fraction than all other tests except AQUA-TMA, although it was the least specific. IHC-TMA was the most specific assay. The lowest misclassification rate was achieved using AQUA-TMA (0.30). CONCLUSIONS Both AQUA-TMA and IHC-TMA were substantially more predictive than the FISH or IHC-WS tests. Although these results are derived from a small retrospective series, they suggest that accurate measurement of protein expression and unbiased selection of tissue for measurement may be key factors in prediction of response.

High SOX2 Levels Predict Better Outcome in Non-Small Cell Lung Carcinomas

Background SOX2 is an embryonic developmental transcription factor, which is important in the development of the respiratory tract. SOX2 overexpression is associated with aggressive disease in several tumor types. However, SOX2 overexpression and gene amplification associates with favorable outcome in lung squamous cell carcinomas (SCC) and dissimilar results have been reported in lung adenocarcinomas (ADC). The aim of the present study was to evaluate SOX2 expression in NSCLC and determine the relationship with clinico-pathological variables and outcome. Methods SOX2 protein levels were measured in tissue microarrays (TMAs) containing FFPE samples from two independent lung cancer cohorts (n = 340 & 307) using automated quantitative immunofluorescence (QIF). Assay validation was performed using FFPE preparations of cell lines with known SOX2 expression. Associations of SOX2 levels with main clinico-pathological characteristics and with overall survival were studied using uni-and multivariate analysis. Results SOX2 levels were higher in patients with SCC than in ADC in both cohorts (p value<0.0001). In the training cohort, NSCLC patients whose tumors showed high SOX2 (n = 245) had longer survival than those with low SOX2 levels (log rank p = 0.0002). Comparable results were observed in the second independent validation cohort, log rank p = 0.0113. SOX2 positive cases showed a 58% reduction in risk of death in Cox univariate analysis (hazards ratio-HR = 0.42 confidence interval-CI (0.36,0.73), p = 0.0002). SOX2 was associated with significantly longer survival independent of histology in multivariate analysis (hazards ratio-HR = 0.429 confidence interval-CI (0.295, 0.663), p = <0.001). Conclusions SOX2 is an independent positive prognostic marker in NSCLC. Increased SOX2 levels are more frequent in SCC than in ADC, but the association with better survival is independent from the histological subtype.

Stathmin expression and its relationship to microtubule-associated protein tau and outcome in breast cancer

Microtubule‐associated proteins (MAPs) endogenously regulate microtubule stability. Here, the prognostic value of stathmin, a destabilizing protein, was assessed in combination with MAP‐tau, a stabilizing protein, in order to evaluate microtubule stabilization as a potential biomarker.

EGFR expression is associated with decreased benefit from trastuzumab in the NCCTG N9831 (Alliance) trial

Background:Epidermal growth factor receptor (EGFR) has been hypothesised to modulate the effectiveness of anti-HER2 therapy. We used a standardised, quantitative immunofluorescence assay and a novel EGFR antibody to evaluate the correlation between EGFR expression and clinical outcome in the North Central Cancer Treatment Group (NCCTG) N9831 trial.Methods:Tissue microarrays were constructed that allowed analysis of 1365 patients randomly assigned to receive chemotherapy alone (Arm A), sequential trastuzumab after chemotherapy (Arm B) and chemotherapy with concurrent trastuzumab (Arm C). Measurement of EGFR was performed using the EGFR antibody, D38B1, on the fluorescence-based AQUA platform. The result was validated using an independent retrospective metastatic breast cancer cohort (n=130).Results:Epidermal growth factor receptor assessed as a continuous (logarithmic transformed) variable shows an association with disease-free survival in Arm C (P=0.009) but not in Arm A or B. High EGFR expression was associated with worse outcome (Hazard ratio (HR)=2.15; 95% CI 1.28–3.60, P=0.004). Validation in a Greek metastatic breast cancer cohort showed an HR associated with high EGFR expression of 1.92 (P=0.0073).Conclusions:High expression of EGFR appears to be associated with decreased benefit from adjuvant concurrent trastuzumab. Since other treatment options exist for HER2-driven tumours, further validation of these data may select patients for alternative or additive therapy.

High Frequency of Putative Ovarian Cancer Stem Cells With CD44/CK19 Coexpression Is Associated With Decreased Progression-Free Intervals In Patients With Recurrent Epithelial Ovarian Cancer

Objective: Epithelial ovarian cancer (EOC) cells with CD44 and CK19 coexpression may represent a subset of ovarian cancer stem cells (OCSCs). This study was conducted to evaluate the correlation of the frequency of putative OCSCs (CD44 + CK19 + OCSCs) with the clinicopathologic features and the prognostic value in patients with recurrent advanced stage EOC. Methods: A retrospective study was carried out on 33 patients with EOC and a uniformly treated tissue microarray was constructed. A multiplexed, immunofluorescence-based method of automated in situ quantitative measurement of protein analysis was used for evaluation of the frequency or density of CD44 + CK19 + OCSCs in EOC. Results: The mean follow-up time was 42.8 ± 27.1 months. High frequency of EOC cells with CD44+ or CD44+/CK19+ was associated with chemoresistance (P = .033 and P = .02, respectively). Using K-M analysis with log-rank test, a high frequency of putative OCSCs was associated with short disease-free interval (7.9 months vs 20.9 months, P = .019). In univariable analysis, the frequency of OCSCs, International Federation of Gynecology and Obstetrics stage and residual tumor volume were significant predictor variables and were entered into multivariable analysis (P = .019, .037, and .005, respectively). Although no independent significant predictor was found, the frequency of putative OCSCs was the most promising predictor variable compared with the other 2 variables (hazard ratio = 2.344, P = .052). Conclusion: Our findings suggest that high frequency of OCSCs (CD44+ and CK19+) in epithelial ovarian tumors correlates with short progression-free intervals.

In Situ Quantitative Measurement of HER2mRNA Predicts Benefit from Trastuzumab-Containing Chemotherapy in a Cohort of Metastatic Breast Cancer Patients

Background We sought to determine the predictive value of in situ mRNA measurement compared to traditional methods on a cohort of trastuzumab-treated metastatic breast cancer patients. Methods A tissue microarray composed of 149, classified as HER2-positive, metastatic breast cancers treated with various trastuzumab-containing chemotherapy regimens was constructed. HER2 intracellular domain(ICD), HER2 extracellular domain(ECD) and HER2 mRNA were assessed using AQUA. For HER2 protein evaluation, CB11 was used to measure ICD and SP3 to measure ECD of the HER2 receptor. In addition, HER2 mRNA status was assessed using RNAscope assay ERRB2 probe. Kaplan – Meier estimates were used for depicting time-to-event endpoints. Multivariate Cox regression models with backward elimination were used to assess the performance of markers as predictors of TTP and OS, after adjusting for important covariates. Results HER2 mRNA was correlated with ICD HER2, as measured by CB11 HER2, with ECD HER2 as measured by SP3 (Pearson’s Correlation Coefficient, r = 0.66 and 0.51 respectively) and with FISH HER2 (Spearman’s Correlation Coefficient, r = 0.75). All markers, HER2 mRNA, ICD HER2 and ECD HER2, along with FISH HER2, were found prognostic for OS (Log-rank p = 0.007, 0.005, 0.009 and 0.043 respectively), and except for FISH HER2, they were also prognostic for TTP Log-rank p = 0.036, 0.068 and 0.066 respectively) in this trastuzumab- treated cohort. Multivariate analysis showed that in the presence of pre-specified set of prognostic factors, among all biomarkers only ECD HER2, as measured by SP3, is strong prognostic factor for both TTP (HR = 0.54, 95% CI: 0.31–0.93, p = 0.027) and OS (HR = 0.39, 95%CI: 0.22–0.70, p = 0.002). Conclusions The expression of HER2 ICD and ECD as well as HER2 mRNA levels was significantly associated with TTP and OS in this trastuzumab-treated metastatic cohort. In situ assessment of HER2 mRNA has the potential to identify breast cancer patients who derive benefit from Trastuzumab treatment.

Comparison of HER2 and Phospho-HER2 Expression between Biopsy and Resected Breast Cancer Specimens Using a Quantitative Assessment Method

Background HER2/Neu (ErbB-2) overexpression, which occurs in 15–20% of breast cancer cases, is associated with better response to treatment with the drug trastuzumab. PhosphoHER2 (pHER2) has been evaluated for prediction of response to trastuzumab. Both markers are heterogeneously detected and are potentially subject to loss as a consequence of delayed time to fixation. Here, we quantitatively assess both markers in core needle biopsies (CNBs) and matched tumor resections to assess concordance between the core and the resection and between HER2 and pHER2. Methods A selected retrospective collection of archival breast cancer cases yielded 67 cases with both core and resection specimens. Both HER2 and pTyr1248HER2 were analyzed by the AQUA® method of quantitative immunofluorescence on each specimen pair. Results Both HER2 immunoreactivity (P<0.0001) and pTyr1248HER2 immunoreactivity (P<0.0001) were lower in resections relative to CNB specimens. However, clinical implications of this change may not be evident since no case changed from 3+ (CNB) to negative (resection). Assessment of pTyr1248HER2 showed no direct correlation with HER2 in either CNB or resection specimens. Conclusions The data suggest that measurement of both HER2 and phospho- Tyr1248HER2, in formalin-fixed tissue by immunological methods is significantly affected by pre-analytic variables. The current study warrants the adequate handling of resected specimens for the reproducible evaluation of HER2 and pHER2. The level of pTyr1248HER2, was not correlated to total HER2 protein. Further studies are required to determine the significance of these observations with respect to response to HER2 directed therapies.

Abstract 705: HER2 assessment to define outcomes for breast cancer patients treated with neoadjuvant therapy

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: HER2 expression or amplification is now an established companion diagnostic test. Now, as HER2-targeting neoadjuvant therapy for breast cancer increases, the accuracy of the companion diagnostic tests are revisited. Specifically, we seek to determine with the level of expression of the target (HER2 protein) is associated with pathologic response. In this study, we quantitatively assess HER2 expression both before and after the neoadjuvant therapies to breast cancer in the BrUOG BR-211B ([NCT00617942][1]) trial. Methods: We evaluated core biopsy samples from breast cancer patients enrolled in a preoperative clinical trial using trastuzumab, nab-paclitaxel and carboplatin combination therapy. Tumor core biopsies were taken before and after 10 days of either trastuzumab or nab-paclitaxel treatment. Combination chemotherapy was given for 6 cycles. Automated quantitative analysis (AQUA), a fluorescent-based method for analysis of in situ protein expression, was used to assess HER2 and phospho-HER2 expression in the BruOG Cohort, a study where patients who serially received Herceptin and Abraxane were biopsied both before and after treatment. This study assesses the HER2 positive patient subset (n=27) Results: Of 18 evaluable patients, the 8 with a PathCR showed a mean HER2 levels of 10251 compared with 4766 in the patients without PathCR (p=0.0047). This is notable in that a HER2 score of 2000 is equivalent to HER2 2+ in previous studies. Measurement of Phospho-HER2 showed no difference in PathCR vs Non-PathCR groups. A small subset (9) cases were measured for HER2 immediately after treatment, but the study has accrued an insufficient number of cases to statistically determine if there is a reduction in HER2 immediately following treatment. Conclusions: High levels of HER2 are associated with PathCR in the neoadjuvant setting. Levels of Phospho-HER2 are not associated with response. Further studies are underway to determine changes in levels of HER2 immediately after treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 705. doi:1538-7445.AM2012-705 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00617942&atom=%2Fcanres%2F72%2F8_Supplement%2F705.atom