OncoPlex Diagnostics (United States)

Abstract Current cancer treatment regimens rely on the use of chemotherapy agents that inhibit DNA replication and repair machinery. Several proteins are involved in this mechanism and these proteins have been identified as predictive biomarkers of response for many chemotherapy agents. While it has been demonstrated that expression levels of these biomarkers can predict chemotherapy response, chemotherapy in both the adjuvant and metastatic settings for NSCLC are not based on biomarker analysis. We have developed a quantitative, multiplex, mass spectrometry based diagnostic assay that can identify multiple biomarkers associated with chemotherapy benefit. At the time of abstract submission, the expression levels of each biomarker were surveyed in 23 NSCLC patients, and the biomarker level for each chemotherapy agent (ERCC1 and XRCC1 for Platinum based chemotherapy; FR alpha and GART for pemetrexed; hENT1 and RRM1 for gemcitabine; and TUBB3 for taxane) was evaluated with clinical outcome. We are extending these analyses with a population of 100 NSCLC tumors with associated outcome. Our pilot study shows a wide range of expression for each biomarker in patient biopsies. Among these biomarkers, FR alpha in NSCLC shows over 4 orders of magnitude in expression range. Also as expected, each biomarker level was found to be correlated with clinical outcome. For example, patients with high level of FR alpha showed improved response to pemetrexed; patients with lower level of TUBB3 showed improved response to taxane and patients with lower level of hENT1 showed poorer response to gemcitabine. Our preliminary retrospective data analysis suggests that the ChemoPlex SRM assay can help predict response to specific chemotherapeutic agents. This test can be used to characterize individual tumor biology and identify actionable targets that can better inform patient care. Citation Format: Eunkyung An, Tae-Jung Kim, Manish Monga, Kathleen Bengali, Alexi Drilea, Joseph Reilly, Marlene Darfler, Jon Burrows, Todd Hembrough. ChemoPlex SRM assay predicts response to specific chemotherapeutic agents in NSCLC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 566. doi:10.1158/1538-7445.AM2015-566

AimsTo demonstrate clinical application of a mesodissection platform that was developed to combine advantages of laser-based instrumentation with the speed/ease of manual dissection for automated dissection of tissue off standard glass slides.MethodsGenomic analysis for KRAS gene mutation was performed on formalin fixed paraffin embedded (FFPE) cancer patient tissue that was dissected using the mesodissection platform. Selected reaction monitoring proteomic analysis for quantitative Her2 protein expression was performed on FFPE patient tumour tissue dissected by a laser-based instrument and the MilliSect instrument.ResultsGenomic analysis demonstrates highly confident detection of KRAS mutation specifically in lung cancer cells and not the surrounding benign, non-tumour tissue. Proteomic analysis demonstrates Her2 quantitative protein expression in breast cancer cells dissected manually, by laser-based instrumentation and by MilliSect instrumentation (mesodissection).ConclusionsSlide-mounted tissue dissection is commonly performed using laser-based instruments or manually scraping tissue by scalpel. Here we demonstrate that the mesodissection platform as performed by the MilliSect instrument for tissue dissection is cost-effective; it functions comparably to laser-based dissection and which can be adopted into a clinical diagnostic workflow.

e19057 Background: The diagnosis of adenocarcinoma (ADC) vs. squamous cell carcinoma (SCC) in NSCLC is critical since new therapies (e.g. pemetrexed) are restricted to non-squamous NSCLC. Most diagnoses are made based on histopathology, though IHC is often performed as well. Since IHC is not quantitative, interpretation of intensity is subjective, and reproducibility can be problematic. Using the Liquid Tissue-SRM mass spectrometry (MS) platform, we have developed a quantitative, multiplexed clinical diagnostic assay to measure the adeno/squamous markers TTF-1/CK7 (ADC) and K5 and p63/40 (SCC) in FFPE tissue. This proteomic assay is epitope independent, and highly reproducible. Methods: Recombinant K5, K7, TTF-1 and p63/p40, were subjected to trypsin digestion mapping to identify peptides for MS analysis. Heavy isotope labeled peptides were synthesized as positive control peptides for quantitation. The multiplexed assay was technically validated on cell lines, and human tumor tissues. Results: ADC/SCC proteomic analysis was performed on a training set of 39 NSCLC tissues from U. of Chicago (20 ADC and 19 SCC). Principal Component Analysis was used to define optimal quantitative cutoffs for each of the four markers for the two tumor subtypes. The ADC diagnosis was unequivocal for all 20 ADC samples, however within the 19 SCC’s, one sample appeared to be ADC and two samples had markers for both ADC and SCC. Proteomic analysis of a test set of 194 NSCLC tumors (98 ADC and 98 SCC based on pathology reports) from MD Anderson Cancer Center was consistent with the observations in the training set. 93/98 ADC cases were confirmed by proteomic analysis, four ADC cases showed mixed or SCC phenotypes. Proteomic analysis confirmed 58/98 SCC cases but identified 27 cases as mixed ADC SCC or ADC based on TTF1/K5 expression. Further studies are ongoing to determine the clinical utility of these findings. Conclusions: These data demonstrate that quantitative proteomic analysis can define cut offs for ADC and SCC Biomarkers profiles in NSCLC tumors. These profiles can determine ADC, SCC and mixed phenotypes and provide physicians with accurate information to help them stratify their patients to the right evidence based treatment therapy.

Abstract 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.

As the most commonly used fluoroquinolone in the United States since 1987, ciprofloxacin has exerted the greatest selective pressure on S. pneumoniae and provides a valuable marker to evaluate the actual and potential emergence of fluoroquinolone resistance in this species. Analysis of susceptibility results obtained with 5,640 strains collected from throughout the United States showed that only 16 (0.3%) of the isolates demonstrated MICs of > or =4 microg/ml. The prevalence of this phenotype was significantly higher (P < 0.05) among penicillin-resistant populations, among isolates from patients >64 years old, and among respiratory isolates. However, >99% of strains had MICs of <4 microg/ml regardless of the risk group examined, and the MIC population distributions were the same for each risk group. These findings demonstrate that the phenotype of a MIC of > or =4 microg/ml remains uncommon after 10 years of ciprofloxacin use; however, these findings are no reason to become complacent with regard to appropriate use of fluoroquinolones and the need to carefully track resistance trends. Equally important is careful analysis of data that result from surveillance in terms of risk factors and other associated trends so that resistance and susceptibility, and their consequences, are neither over- nor underestimated.