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Purpose: Elevated levels or increases in circulating tumor cells (CTC) portend poor prognosis in patients with epithelial cancers. Less is known about CTCs as surrogate endpoints or their use for predictive biomarker evaluation. This study investigated the utility of CTC enumeration and characterization using the CellSearch platform, as well as mutation detection in circulating tumor DNA (ctDNA), in patients with advanced non–small cell lung cancer (NSCLC). Experimental Design: Forty-one patients were enrolled in a single-arm phase II clinical trial of erlotinib and pertuzumab. Peripheral blood was analyzed for CTC enumeration, EGFR expression in CTCs, and detection of oncogenic mutations in CTCs and ctDNA. Changes in CTC levels were correlated with 2[18F]fluoro-2-deoxy-d-glucose–positron emission tomographic (FDG-PET) and computed tomographic (CT) imaging and survival endpoints. Results: CTCs were detected (≥1 CTC) at baseline in 78% of patients. Greater sensitivity for mutation detection was observed in ctDNA than in CTCs and detected mutations were strongly concordant with mutation status in matched tumor. Higher baseline CTC counts were associated with response to treatment by Response Evaluation Criteria in Solid Tumors (RECIST, P = 0.009) and decreased CTC counts upon treatment were associated with FDG-PET and RECIST response (P = 0.014 and P = 0.019) and longer progression-free survival (P = 0.050). Conclusion: These data provide evidence of a correlation between decreases in CTC counts and radiographic response by either FDG-PET or RECIST in patients with advanced NSCLC. These findings require prospective validation but suggest a potential role for using CTC decreases as an early indication of response to therapy and ctDNA for real-time assessment of mutation status from blood. Clin Cancer Res; 18(8); 2391–401. ©2012 AACR.

Background Evaluation of cancer biomarkers from blood could significantly enable biomarker assessment by providing a relatively non-invasive source of representative tumor material. Circulating Tumor Cells (CTCs) isolated from blood of metastatic cancer patients hold significant promise in this regard. Methodology/Principal Findings Using spiked tumor-cells we evaluated CTC capture on different CTC technology platforms, including CellSearch® and two biochip platforms, and used the isolated CTCs to develop and optimize assays for molecular characterization of CTCs. We report similar performance for the various platforms tested in capturing CTCs, and find that capture efficiency is dependent on the level of EpCAM expression. We demonstrate that captured CTCs are amenable to biomarker analyses such as HER2 status, qRT-PCR for breast cancer subtype markers, KRAS mutation detection, and EGFR staining by immunofluorescence (IF). We quantify cell surface expression of EGFR in metastatic lung cancer patient samples. In addition, we determined HER2 status by IF and FISH in CTCs from metastatic breast cancer patients. In the majority of patients (89%) we found concordance with HER2 status from patient tumor tissue, though in a subset of patients (11%), HER2 status in CTCs differed from that observed in the primary tumor. Surprisingly, we found CTC counts to be higher in ER+ patients in comparison to HER2+ and triple negative patients, which could be explained by low EpCAM expression and a more mesenchymal phenotype of tumors belonging to the basal-like molecular subtype of breast cancer. Conclusions/Significance Our data suggests that molecular characterization from captured CTCs is possible and can potentially provide real-time information on biomarker status. In this regard, CTCs hold significant promise as a source of tumor material to facilitate clinical biomarker evaluation. However, limitations exist from a purely EpCAM based capture system and addition of antibodies to mesenchymal markers could further improve CTC capture efficiency to enable routine biomarker analysis from CTCs.

Background: The PI3K/AKT pathway is frequently activated in cancer by multiple mechanisms including PI3K activating mutations, PTEN loss, RTK activation, and other means. GDC-0068 is a potent, highly selective, ATP-competitive pan-AKT inhibitor that is currently in Phase 1a and Phase 1b clinical development. Here, we describe the cell line screening efforts and xenograft studies that provide rationale for a predictive biomarker strategy both for single agent studies and combinations with chemotherapeutic agents. Methods: Cell line screening using GDC-0068 as a single agent and in combination with other agents provided a means of identifying predictive markers across various indications and drug combinations. Single agent screens were performed with GDC-0068 in 9 prostate, 54 breast, and 18 ovarian cell lines. We evaluated combination activity of GDC-0068 with docetaxel, doxorubicin, rapamycin and MEK inhibitor in a panel of 24 cell lines and combination activity of GDC-0068 with 5FU/Cisplatin (Folfox) in a panel of 11 gastric and 15 head and neck cancer cell lines. We also evaluated both single agent activity and combination activity in select xenograft models. Results: Single agent screens identified PTEN loss, PIK3CA activating mutations, and RTK activation (HER2 in breast) as key predictive markers in breast, ovarian and prostate cell lines. Similarly, in prostate cancer xenograft models, the highest tumor growth inhibition was seen in models with PI3K pathway activation, e.g. via PTEN loss. In the GDC-0068 combination studies, MEK inhibitors provided the most significant synergy across different targeted and chemotherapy agents tested (rapamycin, docetaxel, doxurubicin). In combination with 5FU/Cisplatin, we observed additive effects in gastric and head and neck cell lines and this response was best observed in cell lines with pathway activation: PTEN loss, PI3K mutations or amplifications, and/or high pAKT. The in vitro results were recapitulated in vivo with the combination of GDC-0068 with either docetaxel or carboplatin enhancing the antitumor efficacy compared to either single agent alone in multiple tumor xenograft models. All combinations were well tolerated as assessed by animal body weights and mortalities. Conclusions: Across various indications, PTEN loss or PI3K pathway activation via PIK3CA activating mutations are strong predictive biomarkers of GDC-0068 activity either as a single agent or in combination studies and provide a strong diagnostic hypothesis for evaluation in the clinic. Based on these results and others, GDC-0068 is currently being tested for single agent activity in a diagnostically selected Phase 1a expansion cohort in breast and prostate cancer, and in multiple combination Phase Ib trials. 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 966. doi:1538-7445.AM2012-966

The PI3K/Akt pathway is frequently activated in human cancers. A number of the components of this pathway are activated, mutated, amplified and deleted in a wide variety of human cancers highlighting the key role of this pathway. PTEN is a negative regulator of this pathway that is frequently inactivated in a variety of tumors. PTEN loss could be useful as a predictive biomarker to identify the tumors that are dependent on the PI3K/Akt pathway and therefore likely to respond to PI3K/Akt targeted agents. PTEN is frequently deleted in prostate cancer with incidence as high as 75% reported in advanced disease. Both genetic models of PTEN loss in prostate cancer as well as associations of PTEN deletions with poor clinical outcome support a role for PTEN in prostate cancer progression. Determining PTEN status is challenging in metastatic prostate cancer since biopsies are infrequently performed in this setting. Circulating tumor cells (CTCs) could provide an alternate non-invasive source of tumor cells and relatively higher counts of CTCs have been reported in prostate cancer compared to other indications. Here we evaluate the utility of CTCs in determining PTEN status of patients with castration resistant prostate cancer (CRPC). We evaluate whether we can detect PTEN loss in CRPC patient CTCs and in their respective tissue. We developed a PTEN FISH assay to score PTEN in spiked CTCs in blood and compared PTEN loss in CTCs (captured on the Cellsearch platform) with archival tissue available from CRPC patients. Our data suggests that predictive biomarker analysis in CTCs is possible and CTCs could be potentially used to predict PTEN loss. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3153. doi:10.1158/1538-7445.AM2011-3153

Background: Evaluation of cancer biomarkers from blood and other accessible tissues could significantly enable biomarker assessment by providing a relatively noninvasive source of representative tumor material. Circulating tumor cells (CTCs) isolated from blood of metastatic cancer patients hold significant promise in this regard. Methodology/Principal Findings: Using a tumor-cell spike-in model system we evaluated CTC capture on different CTC technology platforms, including CellSearch® and two biochip platforms, and used the isolated CTCs to develop and optimize assays for molecular characterization of CTCs. We report similar performance for the various platforms tested in capturing CTCs, and find that capture efficiency is dependent on the level of EpCAM expression. We demonstrate that captured CTCs are amenable to biomarker analyses such as HER2 status, qRT-PCR for breast cancer subtype markers, KRAS mutation detection, and EGFR staining by immunofluorescence (IF). We demonstrate that cell surface expression of EGFR can be quantitated in CTCs from metastatic lung cancer patient samples. In addition, we determined HER2 status by IF and FISH in CTCs from metastatic breast cancer patients and in the majority of patients (89%) found concordance with HER2 status from patient tumor tissue, while in a subset of patients (11%), HER2 status had changed from the primary tumor at diagnosis. Surprisingly, we found CTC counts to be higher in ER+ patients in comparison to HER2+ and triple negative patients despite their more aggressive phenotype. This may be explained by our findings that the basal-like molecular subtype of breast cancer has low EpCAM expression and a more mesenchymal phenotype and CTCs will likely not be efficiently captured using EpCAM alone in tumors that arise from this subtype. Conclusions/Significance: Our data suggests that molecular characterization from captured CTCs is possible and can inform us of the patients’ current biomarker status. In this regard, CTCs hold significant promise as a source of tumor material to facilitate clinical biomarker evaluation. However, limitations exist from a purely EpCAM-based capture system and addition of antibodies to mesenchymal markers in addition to EpCAM could further improve CTC capture efficiency to enable routine biomarker analysis from CTCs.

Introduction: Tumor tissue collection is essential for biomarker assessment but can be difficult in tumor types such as non small-cell lung cancer (NSCLC) where often no surgery is performed and diagnosis is done with biopsies yielding only very limited tissue quantities. Also, in cases where primary tissue is available, the samples may not be representative of patient's current disease, i.e. in the case of hormone refractory prostate cancer (HRPC). Rationale: Evaluation of cancer biomarkers (both predictive and pharmacodynamic) from blood and other biological material could significantly enable our ability to generate biomarkers by providing an accessible and relevant source of samples that can be analyzed. Successful assessment of biomarkers that do not require access to tumor “tissue” could improve the inclusion of biomarker evaluations in clinical development and across our oncology practice. Results: We report on our efforts to evaluate Circulating Tumor Cells (CTC) for utility in tumor biomarker assessment. These efforts included evaluating multiple technologies for isolation and molecular analysis of CTCs from blood. By spiking tumor cells into blood as a model system for CTCs we have developed assays for biomarker evaluation in CTCs including: Fluorescence in situ hybridization (FISH), Immunofluoresecnce (IF), qRT-PCR and mutation detection. Validation of these assays in patient blood is ongoing and early data suggests that biomarker assessment is indeed possible from CTCs. Using blood samples from metastatic, HER2+ breast cancer patients; we were able to evaluate the HER2 status from CTCs using immunofluorescence and/or FISH. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5768.