Tam Pham

Detection of EpCAM-Negative and Cytokeratin-Negative Circulating Tumor Cells in Peripheral Blood

Enrichment of rare circulating tumor cells (CTCs) in blood is typically achieved using antibodies to epithelial cell adhesion molecule (EpCAM), with detection using cytokeratin (CK) antibodies. However, EpCAM and CK are not expressed in some tumors and can be downregulated during epithelial-to-mesenchymal transition. A micro-fluidic system, not limited to EpCAM or CK, was developed to use multiple antibodies for capture followed by detection using CEE-Enhanced (CE), a novel in situ staining method that fluorescently labels the capture antibodies bound to CTCs. Higher recovery of CTCs was demonstrated using antibody mixtures compared to anti-EpCAM. In addition, CK-positive breast cancer cells were found in 15 of 24 samples (63%; range 1–60 CTCs), while all samples contained additional CE-positive cells (range 1–41; median = 11; P = .02). Thus, antibody mixtures against a range of cell surface antigens enables capture of more CTCs than anti-EpCAM alone and CE staining enables the detection of CK-negative CTCs.

A Novel Platform for Detection of CK+ and CK− CTCs

UNLABELLED Metastasis is a complex, multistep process that begins with the epithelial-mesenchymal transition (EMT). Circulating tumor cells (CTC) are believed to have undergone EMT and thus lack or express low levels of epithelial markers commonly used for enrichment and/or detection of such cells. However, most current CTC detection methods target only EpCAM and/or cytokeratin (CK) to enrich epithelial CTCs, resulting in failure to recognize other, perhaps more important, CTC phenotypes that lack expression of these markers. Here, we describe a population of complex aneuploid CTCs that do not express CK or CD45 antigen in patients with breast, ovarian, or colorectal cancer. These cells were not observed in healthy subjects. We show that the primary epithelial tumors were characterized by similar complex aneuploidy, indicating conversion to an EMT phenotype in the captured cells. Collectively, our study provides a new method for highly efficient capture of previously unrecognized populations of CTCs. SIGNIFICANCE Current assays for CTC capture likely miss populations of cells that have undergone EMT. Capture and study of CTCs that have undergone EMT would allow a better understanding of the mechanisms driving metastasis.

FISH-based determination of HER2 status in circulating tumor cells isolated with the microfluidic CEE™ platform.

Determination of HER2 status in breast cancer patients is considered standard practice for therapy selection. However, tumor biopsy in patients with recurrent and/or metastatic disease is not always feasible. Thus, circulating tumor cells (CTCs) are an alternative source of tumor cells for analysis of HER2. An antibody cocktail for recovery of variable, high- and low-, EpCAM-expressing tumor cells was developed based on FACS evaluation and then verified by CTC enumeration (based on CK and CD45 staining) with comparison to EpCAM-only and with CellSearch® (n=19). HER2 fluorescence in situ hybridization (FISH) on all (CK+ and CK-) captured cells was compared to HER2 status on the primary tumors (n=54) of patients with late stage metastatic/recurrent breast cancer. Capture of low EpCAM-expressing tumor cells increased from 27% to 76% when using the cocktail versus EpCAM alone, respectively. Overall, CTC detection with the OncoCEE™ platform was better compared to CellSearch® (68% vs. 89%, respectively), and a 93% concordance in HER2 status was observed. HER2 FISH analysis of CK+ and CK- CTCs is feasible using the CEE™ platform. Although larger clinical studies are warranted, the results demonstrate adequate sensitivity and specificity as needed for incorporation into laboratory testing.

Discordance in HER2 gene amplification in circulating and disseminated tumor cells in patients with operable breast cancer

Human epidermal growth factor receptor 2 (HER2) gene amplification in circulating tumor cells (CTCs) and disseminated tumor cells (DTCs) might be useful for modifying Herceptin therapy in breast cancer. In the process of investigating the utility of a microfluidic platform for detecting HER2 gene amplification in these cells, we observed novel results on discordance of HER2 status. Peripheral blood (8.5 mL) and bone marrow (BM) (7.5–10 mL) were collected prospectively from patients with clinical stages I–IV breast cancer. Mononuclear cells were recovered, stained with cytokeratin (CK), CD45, and DAPI, and processed through microfluidic channels for fluorescence in situ hybridization (FISH). A ratio of HER2:CEP17 >2 in any CK+/CD45 or CK−/CD45 cell was regarded as positive for HER2 gene amplification. Peripheral blood from 95 patients and BM from 78 patients were studied. We found CK+/CD45−/DAPI+ CTCs in 27.3% of patients. We evaluated HER2 gene amplification by FISH in 88 blood and 78 BM specimens and found HER2+ CTCs in 1 of 9 (11.1%) and HER2+ DTCs (27.2%) in 3 of 11 patients with HER2+ primary tumor. Among patients with a HER2− primary tumor, 5 of 79 had HER2+ CTCs (6.3%) and 14 of 67 had HER2+ DTCs (20.8%). The overall rate of discordance in HER2 status was 15% between primary tumor and CTCs and 28.2% between primary tumor and DTCs. HER2 was amplified in CTCs and DTCs in a portion of both HER2+ and HER2− primary tumors. HER2 discordance was more frequent for DTCs. The clinical implications of evaluating HER2 status in CTCs and DTCs in breast cancer needs to be established in prospective clinical trials. The cell enrichment and extraction microfluidic technology provides a sensitive platform for evaluation of HER2 gene amplification in CTCs and DTCs.

Abstract 4568: Estrogen receptor and progesterone receptor immunocytochemistry staining in circulating tumor cells as compared to primary tumor or metastatic biopsy

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: Hormone receptor (estrogen receptor (ER) and progesterone receptor (PR)) status in all breast cancer patients is recommended by immunohistochemistry (IHC) and is considered standard practice for selection of treatment options. However, the analytical sensitivity of IHC in detecting low levels of ER/PR is often poor and likely due to methodological variation. Because biopsy is not often feasible in all patients presenting with recurrent and/or metastatic breast disease, circulating tumor cells (CTCs) offer an attractive alternative source of tumor tissue for determining ER/PR status and can be monitored more readily to enable a more effective course of treatment. Experimental Design: Twenty ml of peripheral blood was collected prospectively from 14 patients diagnosed with late stage metastatic/recurrent breast cancer. CTCs were isolated using the microfluidic OncoCEETM platform. A cocktail of antibodies was utilized for CTC capture and detection with an expanded anti-cytokeratin (CK) cocktail mixture and anti-CD45. ER/PR protein expression was assessed by immunocytochemistry (ICC) on the cells captured within the microchannels and compared to IHC performed on the primary tumor or metastatic biopsy. Results: CK+/CD45-/DAPI+ cells were located and assessed for ER/PR immunocytochemistry. Among the 14 cases a high concordance (12/14; 86%) in ER/PR status between primary tumor/metastatic biopsy and CTCs was observed. Two cases were found to be discordant where one was positive by IHC and negative on the CTCs and the other was negative on by IHC on the primary tumor/metastatic biopsy and positive on the CTCs. However, both cases that were discordant had low numbers of CTCs detected. Conclusions: There is significant heterogeneity between ER/PR protein expression in CTCs and primary tumor/metastatic biopsy and this status may change over time due to therapy. ER/PR ICC on CTCs from peripheral blood using the OncoCEETM platform is shown to be feasible with high concordance (86%) in ER/PR status between primary tumor/metastatic biopsy (by IHC) and CTCs (by ICC). The significance of heterogeneity at the ER/PR protein level in CTCs ascertaining to the prognosis and predictive response to anti-estrogen therapy needs further evaluation in larger prospective clinical 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 4568. doi:1538-7445.AM2012-4568

Abstract P2-01-10: Immunocytochemistry staining for estrogen and progesterone receptor in circulating tumor cells: Concordance between primary and metastatic tumors.

Introduction: Estrogen (ER) and progesterone receptor (PR)) status is recommended by immunohistochemistry (IHC) and is considered standard practice for selection of treatment options in all breast cancer patients. Because biopsy is not often feasible in every patient presenting with recurrent and/or metastatic disease, circulating tumor cells (CTCs) offer an attractive alternative source of tumor tissue for determining ER/PR status. In addition, CTCs enable monitoring for more effective course of treatment. Experimental Design: Twenty ml of peripheral blood was collected prospectively from 34 patients diagnosed with late stage metastatic/recurrent breast cancer. CTCs were isolated using the microfluidic OncoCEE platform. A cocktail of antibodies was utilized for CTC capture, and detection was accomplished with an expanded anti-cytokeratin (CK) cocktail mixture and anti-CD45. ER/PR protein expression was assessed by immunocytochemistry (ICC) on CK+/CD45- CTCs captured directly within the microchannel and then compared to IHC performed on the primary and/or metastatic tumor. Results: CK+/CD45−/DAPI+ cells were detected in 22 of 34 (65%) patients with late stage breast cancer and assessed for ER/PR immunocytochemistry. Among the 22 cases with one or more CTCs, a concordance of 75% (15/20) and 90% (9/10) in ER/PR status between primary and metastatic tumor was observed, respectively. An overall concordance of 86% (19/22) was achieved. Five cases were discordant based on primary tissue alone; however, two of these cases are concordant when compared to the metastatic biopsy. Thus, only three cases were found to be discordant: all three were positive by IHC on the primary and/or metastatic tumor but negative by CTCs and all three had relatively low numbers of CTCs detected. Conclusions: There is significant heterogeneity of ER/PR protein expression in CTCs and primary/metastatic tumor biopsy. In addition, hormonal status may change over time due to therapy. ER/PR ICC on CTCs using the OncoCEE platform is shown to be feasible, with high concordance (86%) as compared to primary and/or metastatic biopsy (by IHC). The significance of heterogeneity at the ER/PR protein level in CTCs related to the prognosis and predictive response to anti-estrogen therapy needs further evaluation in larger prospective clinical trials. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-01-10.

Abstract 1553: Redefining CTCs: Detection of cytokeratin-negative circulating tumor cells (CTCs)

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction: There is growing evidence that CTCs display an epithelial-mesenchymal transition phenotype. Current enrichment techniques rely upon epithelial markers for capture (anti-EpCAM antibodies and cytokeratin [CK]) and may miss important populations of circulating tumor cells. We sought to develop a new method for identification of CTCs. Methods: After IRB approval, blood from patients with ovarian or colorectal carcinoma was collected. Cell Enrichment and Extraction (CEE) technology, a microfluidic-based device using an antibody cocktail targeting epithelial and mesenchymal markers, was utilized for the capture and analysis of rare cells in blood. We enumerated cells that were CK+ and/or contained complex aneuploidy by fluorescent in situ hybridization (FISH). Blood samples from healthy volunteers served as a negative control. Fresh frozen tumor samples were analyzed on a subset of patients to search for concordance of molecular alterations with CTCs. An orthotopic ovarian cancer model was used to evaluate effects of chemotherapy on CTCs. Results: Enumeration of CK+ cells identified an average of 1 and 2 cells per 10 mL blood in ovarian and colorectal cancer patients, independent of stage or tumor grade. Enumeration did not correlate with serum tumor marker levels. The majority of CK+ cells had complex aneuploidy. Ovarian cancer patients had equal numbers of CK- and CK+ complex aneuploid cells (p=1.0). A three-fold increase in CK- over CK+ complex aneuploid cells was noted in patients with colorectal cancer, although not statistically significant (p<0.19). The total number of complex aneuploid cells did not correlate with serum tumor markers. Similar patterns of complex aneuploidy were identified in patients’ primary tumors as were seen in their CK- cells from blood, indicating that the cells identified in circulation were indeed CTCs. No CK+ or complex aneuploid cells were found in healthy volunteers. In the orthotopic mouse model, compared to tumor-bearing controls, chemotherapy treatment initially lead to a 17-fold increase in apoptotic appearing CTCs (p<0.02) followed by a 15-fold decrease in CTCs (p<0.03), which also reflected tumor response. Discussion: Here, we have developed and characterized a novel and robust method for detection of CTCs. The power of this approach lies in its ability to detect CTCs with either an epithelial or EMT-positive phenotype. Our findings suggest current enrichment techniques may be missing an important population of cells. Complex aneuploid CK- cells challenge the current definition of a CTC and merit further development in clinical settings. 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 1553. doi:10.1158/1538-7445.AM2011-1553

Abstract 319: Comparison of fluorescence in situ hybridization of estrogen receptor genetic locus with protein expression in invasive breast carcinoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL INTRODUCTION: Evaluation of estrogen receptor (ESR1) status is recommended in all breast cancer patients and is generally performed using immunohistochemistry (IHC). However, the analytical sensitivity of IHC in detecting low levels of ESR1 amplification is often poor and likely due to methodological variation. Though FISH (fluorescent in-situ hybridization) has been proposed as an alternative approach for detection of ESR1 gain, results have been controversial with few studies evaluating concordance between FISH and IHC. In this study, we describe the performance of FISH using an ESR1 probe and correlation of the results with IHC for ESR protein expression. METHODS: FISH and IHC for ESR1 was performed on adjacent sections of formalin fixed paraffin embedded tumor sections from 32 patients with invasive breast cancer enrolled for treatment at MD Anderson Cancer Center. For FISH, pretreatment of the slides with the paraffin pretreatment kit III (Abbott Laboratories) was performed followed by incubation using the ESR1/Cen6 probe set (Zytovision). The number of fluorescent signals for each the ESR1 and centromere 6 were counted in a minimum of 200 non-overlapping, intact nuclei. Nuclei were characterized as normal (two signals for each locus), monosomy (one signal for ER and two signals for the centromere) or trisomy (three signals for ER and two signals for the centromere). IHC on primary tumor tissue sections was carried out in a Bond-maX machine (Leica Microsystems) with primary ER antibody (clone 6F11, Novocastra) at dilution of 1:35 and antigen retrieval using citrate buffer. Nuclear positivity in the tumor cells was expressed as percentage and categorized as negative, low positive or positive based on nuclear staining of 0%, 1-10% and >10% respectively. RESULTS: Of the 32 samples that were successful for both FISH and IHC, a comparison was made to determine concordance of FISH signals to ER IHC results. We calculated the percentage of cells having ≫3 ESR1 signals and those that contained <2 ESR1 signals in all 32 cases. Based on a FISH percentage cutoff of 2.0, cases could be classified into three groups: negative (n=7), equivocal (n=8) and amplified (n=10) with seven cases being discordant when compared to IHC results. The p-value for the ratio of ER negative to ER positive cohort was found to be statistically significant (p=0.026). Based on these criteria we observe a concordance of 75% between the two technologies. CONCLUSION: 1. There is a significant heterogeneity between the gene amplification status and protein overexpression of ESR1. 2. The gene status of ESR1 ranges from negative, equivocal and amplified in both ER negative and ER immunopositive cases. 3. The significance of heterogeneity at the ESR1 gene locus in ascertaining the prognosis and predictive response to antiestrogen therapy needs further evaluation in larger prospective clinical trials. 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 319. doi:10.1158/1538-7445.AM2011-319

Abstract 5172: Fluorescence labeling of cytokeratin-negative circulating tumor cells in peripheral blood: A new paradigm for the study of cancer progression

Introduction: The measurement of circulating tumor cells (CTCs) in blood generally requires either anti-EpCAM for capture, anti-cytokeratin (CK) for detection, or both. However, EpCAM and CK are absent in some tumor cells, and both may be down-regulated during neoplastic progression or during epithelial-to-mesenchymal transition (EMT). Here we show capture on a micro-fluidic system using single or multiple antibodies, and CTC detection with CEE-Enhanced (CE), a novel in situ staining method that fluorescently labels the capture antibodies bound to the CTCs. Methods: Buffy coat cells isolated from 8 mL of blood were pre-incubated with either anti-EpCAM alone or with a mixture of antibodies to epithelial and mesenchymal cell surface antigens. CTCs were captured on a micro-fluidic channel. Identification of CTCs was determined with anti-CK and with CE to detect cells with the capture antibodies bound to the cell surface antigens. All cells scored as positive for CK or CE were, by definition, CD45-negative and DAPI-positive. Results: Using anti-EpCAM alone for capture, significantly more CTCs were detected by CE staining than with anti-CK in breast and prostate cancers. This indicated that CK-negative CTCs were captured but not detected and that some EpCAM-positive CTCs were CK-negative. Results using capture antibody mixtures varied from sample to sample but gave up to 2-fold higher CK-positive cells than anti-EpCAM only, and as much as 4-fold higher CE-positive cells. Control blood from healthy donors was CK and CE-negative. All CK-positive cells co-stained with CE, as determined with different fluorescent labels. In a clinical study of stage IV breast cancer, CTCs were isolated with an antibody mixture and sequentially stained with CK and CE. Fifteen of 24 samples (63%) contained CK-positive cells (range 1-60 CTCs) while 24 of 24 samples (100%) contained additional CE-positive cells (range 1-41; median=11; Wilcoxon test, p=0.02). The modest correlation coefficient (r = 0.57, p=0.004) for the number of CE-positive and CK-positive cells in each sample suggests that one or more different phenotypes of CTCs were being detected. Amplified Her2 was detected by FISH in isolated CK-positive CTCs, and also in CK-negative, CE-positive CTCs from Her2-positive patients, indicating these were tumor cells. Conclusions: The CEE-Enhanced staining technology enables the detection of CK-negative CTCs. This novel detection method, based on the in situ labeling of antibodies used for capture, greatly expands single and multi-antibody approaches to the study of rare circulating cells. Specifically, this allows the exploration and study of circulating cells not expressing CK such as highly de-differentiated tumor cells, stem cells or those tumor cells undergoing EMT. The clinical significance of these CK-negative CTCs is under investigation. 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 5172. doi:10.1158/1538-7445.AM2011-5172

P5-01-08: Immunocytochemistry Staining of Estrogen Receptor in Circulating Tumor Cells as Compared to Primary Tumor.

Introduction: Estrogen receptor (ER) status in all breast cancer patients is recommended by immunohistochemistry (IHC) and is considered standard practice for selection of treatment options. However, the analytical sensitivity of IHC in detecting low levels of ER is often poor and likely due to methodological variation. Because biopsy is not often feasible in all patients presenting with recurrent and/or metastatic breast disease, circulating tumor cells (CTCs) offer an attractive alternative source of tumor tissue for determining ER status and can be monitored more readily to enable a more effective course of treatment. Experimental Design: Twenty ml of peripheral blood was collected prospectively from # patients diagnosed with late stage metastatic/recurrent breast cancer. CTCs were isolated using the microfluidic OncoCEE™ platform. A cocktail of antibodies was utilized for CTC capture and detection with an expanded anti-cytokeratin (CK) cocktail mixture and anti-CD45. ER protein expression was assessed by immunocytochemistry (ICC) on the cells captured within the microchannels and compared to IHC performed on the primary tumor. Results: CK+/CD45- cells were detected in 23 of 27 cases (85%). Among the 23 cases in which CK+ cells were detected, only moderate concordance (16/23; 70%) in ER status between primary tumor and CTCs was observed. Conclusions: There is significant heterogeneity between ER protein expression in CTCs and primary tumor. ER ICC on CTCs from peripheral blood using the OncoCEE™ platform is shown to be feasible but the comparison likely compromised by lack of tumor staging at the time of blood draw. Given that IHC was in some cases performed 5–10 years pre-blood draw and that tumors have the potential to convert, ER status only moderately correlates to the IHC status in the primary tumor. The significance of heterogeneity at the ER protein level in CTCs ascertaining to the prognosis and predictive response to anti-estrogen therapy needs further evaluation in larger prospective clinical trials. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-01-08.