Circulating tumor cells in metastatic breast cancer patients treated with immune checkpoint inhibitors - a biomarker analysis of the ALICE and ICON trials.

Immune checkpoint inhibitors (ICIs) have been introduced in breast cancer (BC) treatment and better biomarkers are needed to predict benefit. Circulating tumor cells (CTCs) are prognostic in BC, but knowledge is limited on CTCs in the context of ICI therapy. In this study, serial sampling of CTCs (CellSearch system) was evaluated in 82 patients with metastatic BC enrolled in two randomized trials investigating ICI plus chemotherapy. Programmed death-ligand 1 (PD-L1) expression on CTCs was also measured. Patients with ≥ 2 CTCs per 7.5 mL at baseline had gene expression profiles in tumor suggestive of increased T-cell activity, including increased tumor inflammation signature (TIS) in both triple-negative (P = 0.010) and hormone receptor-positive (P = 0.024) disease. Patients with luminal A BC had higher CTC levels. The association between CTC status and outcome was most apparent 4 weeks into therapy. PD-L1 expression in CTCs was observed in 6/17 CTC-positive patients and was associated with inferior survival. In conclusion, our study indicates that CTC numbers may inform on tumor immune composition, as well as prognosis. These findings suggest a potential of using CTCs as an accessible biomarker source in BC patients treated with immunotherapy.

Preoperative CTC-Detection by CellSearch® Is Associated with Early Distant Metastasis and Impaired Survival in Resected Pancreatic Cancer.

In patients with presumed pancreatic ductal adenocarcinoma (PDAC), biomarkers that may open for personalised, risk-adapted treatment are lacking. The study analysed the impact of CTCs-presence on the patterns of recurrence and survival in 98 patients resected for PDAC with 5-10 years of follow-up. Preoperative samples were analysed by the CellSearch® system for EpCAM+/DAPI+/CK+/CD45-CTCs. CTCs were detected in 7 of the 98 patients. CTCs predicted a significantly shorter median disease-free survival (DFS) of 3.3 vs. 9.2 months and a median cancer specific survival (CSS)of 6.3 vs. 18.5 months. Relapse status was confirmed by imaging for 87 patients. Of these, 58 patients developed distant metastases (DM) and 29 developed isolated local recurrence (ILR) as the first sign of cancer relapse. All patients with CTCs experienced DM. pN-status and histological grade >2 were other independent risk factors for DM, but only CTCs predicted significantly shorter cancer-specific, disease-free and post-recurrence survival. Preoperative parameters did not affect clinical outcome. We conclude that CTC presence in resected PDAC patients predicted early distant metastasis and impaired survival. Preoperative CTCs alone or in combination with histopathological factors may guide initial treatment decisions in patients with resectable PDAC in the future.

Cytokeratin-positive cells in the bone marrow from patients with pancreatic, periampullary malignancy and benign pancreatic disease show no prognostic information.

BACKGROUND: Pancreatic and periampullary carcinoma are aggressive tumours where preoperative assessment is challenging. Disseminated tumour cells (DTC) in the bone marrow (BM) are associated with impaired prognosis in a variety of epithelial cancers. In a cohort of patients with presumed resectable pancreatic and periampullary carcinoma, we evaluated the frequency and the potential prognostic impact of the preoperative presence of DTC, defined as cytokeratin-positive cells detected by immunocytochemistry (ICC). METHODS: Preoperative BM samples from 242 patients selected for surgical resection of presumed resectable pancreatic and periampullary carcinoma from 09/2009 to 12/2014, were analysed for presence of CK-positive cells by ICC. The median observation time was 21.5 months. Overall survival (OS) and disease-free survival (DFS) were calculated by Kaplan-Meier and Cox regression analysis. RESULTS: Successful resections of malignant tumours were performed in 179 of the cases, 30 patients resected had benign pancreatic disease based on postoperative histology, and 33 were deemed inoperable intraoperatively due to advanced disease. Overall survival for patients with resected carcinoma was 21.1 months (95% CI: 18.0-24.1), for those with benign disease OS was 101 months (95% CI: 69.4-132) and for those with advanced disease OS was 8.8 months (95% CI: 4.3-13.3). The proportion of patients with detected CK-positive cells was 6/168 (3.6%) in resected malignant cases, 2/31 (6.5%) in advanced disease and 4/29 (13.8%) in benign disease. The presence of CK-positive cells was not correlated to OS or DFS, neither in the entire cohort nor in the subgroup negative for circulating tumour cells (CTC). CONCLUSIONS: The results indicate that CK-positive cells may be present in both patients with malignant and benign diseases of the pancreas. Detection of CK-positive cells was not associated with differences in prognosis for the entire cohort or any of the subgroups analysed. TRIAL REGISTRATION: clinicaltrials.gov ( NCT01919151 ).

Circulating Tumor Cells are an Independent Predictor of Shorter Survival in Patients Undergoing Resection for Pancreatic and Periampullary Adenocarcinoma.

OBJECTIVE: We evaluated the prognostic impact of circulating tumor cells (CTCs) for patients with presumed resectable pancreatic and periampullary cancers. SUMMARY OF BACKGROUND DATA: Initial treatment decisions for this group are currently taken without a reliable prognostic marker. The CellSearch system allows standardized CTC-testing and has shown excellent specificity and prognostic value in other applications. METHODS: Preoperative blood samples from 242 patients between September 2009 and December 2014 were analyzed. One hundred seventy-nine patients underwent tumor resection, of whom 30 with stage-I tumors and duodenal cancer were assigned to the low-risk group, and the others to the high-risk group. Further 33 had advanced disease, 30 benign histology. Observation ended in December 2016. Cancer-specific survival (CSS) and disease-free survival (DFS) were calculated by log-rank and Cox regression. RESULTS: CTCs (CTC-positive; ≥1 CTC/7.5 mL) were detected in 6.8% (10/147) of the high-risk patients and 6.2% (2/33) with advanced disease. No CTCs (CTC-negative) were detected in the low-risk patients or benign disease. In high-risk patients, median CSS for CTC-positive versus CTC-negative was 8.1 versus 20.0 months (P < 0.0001), and DFS 4.0 versus 10.5 months (P < 0.001). Median CSS in advanced disease was 7.7 months. Univariate hazard ratio (HR) of CTC-positivity was 3.4 (P < 0.001). In multivariable analysis, CTC-status remained independent (HR: 2.4, P = 0.009) when corrected for histological type (HR: 2.7, P = 0.030), nodal status (HR: 1.7, P = 0.016), and vascular infiltration (HR: 1.7, P = 0.001). CONCLUSION: Patients testing CTC-positive preoperatively showed a detrimental outcome despite successful tumor resections. Although the low CTC-rate seems a limiting factor, results indicate high specificity. Thus, preoperative analysis of CTCs by this test may guide treatment decisions and warrants further testing in clinical trials.

Detection of Circulating Tumor Cells at Surgery and at Follow-Up Assessment to Predict Survival After Two-Stage Liver Resection of Colorectal Liver Metastases.

BACKGROUND: The presence of circulating tumor cells (CTCs) is negatively associated with survival after resection of colorectal liver metastases (CLM). The current study aimed to determine the prognostic value of CTCs and disseminated tumor cells (DTCs) at the time of surgery and the prognostic value of CTCs at follow-up assessment, for patients scheduled to undergo two-stage hepatectomy with portal vein embolization (PVE) for CLM. METHODS: Samples were collected at surgery (blood and bone marrow) and at follow-up assessment (blood) for the period 2008 through 2011. In this study, CTCs were detected with the CellSearch system, and DTCs were detected using standard immunocytochemical analysis. RESULTS: Of 24 patients, 18 completed both stages, and no patients were lost to follow-up. The median overall survival (OS) was 37 months, and the median recurrence-free survival (RFS) was 7 months. At surgery, CTCs were found in nine patients (38 %), and their presence was associated with reduced OS (p < 0.001) and RFS (p = 0.006). Follow-up CTC status was available for 11 patients. All eight patients with positive CTC status experienced recurrence. Two of three patients with negative CTC status remained recurrence free. In seven patients (32 %), DTCs were detected but were not associated with OS or RFS. CONCLUSIONS: The presence of CTCs at surgery is associated with worse OS and RFS for patients undergoing two-stage hepatectomy with PVE for CLM. Analysis of CTCs should be explored further for their potential to assist in treatment decisions and monitoring for CLM patients.

Clinical outcome with correlation to disseminated tumor cell (DTC) status after DTC-guided secondary adjuvant treatment with docetaxel in early breast cancer.

PURPOSE: The presence of disseminated tumor cells (DTCs) in bone marrow (BM) predicts survival in early breast cancer. This study explores the use of DTCs for identification of patients insufficiently treated with adjuvant therapy so they can be offered secondary adjuvant treatment and the subsequent surrogate marker potential of DTCs for outcome determination. PATIENTS AND METHODS: Patients with early breast cancer who had completed six cycles of adjuvant fluorouracil, epirubicin, and cyclophosphamide (FEC) chemotherapy underwent BM aspiration 2 to 3 months (BM1) and 8 to 9 months (BM2) after FEC. Presence of DTCs in BM was determined by immunocytochemistry using pan-cytokeratin monoclonal antibodies. If one or more DTCs were present at BM2, six cycles of docetaxel (100 mg/m(2), once every 3 weeks) were administered, followed by DTC analysis 1 and 13 months after the last docetaxel infusion (after treatment). Cox regression analysis was used to evaluate disease-free interval (DFI). RESULTS: Of 1,066 patients with a DTC result at BM2 and available follow-up information (median follow-up, 71.9 months from the time of BM2), 7.2% were DTC positive. Of 72 docetaxel-treated patients analyzed for DTCs after treatment, 15 (20.8%) had persistent DTCs. Patients with remaining DTCs had markedly reduced DFI (46.7% experienced relapse) compared with patients with no DTCs after treatment (adjusted hazard ratio, 7.58; 95% CI, 2.3 to 24.7). The docetaxel-treated patients with no DTCs after treatment had comparable DFI (8.8% experienced relapse) compared with those with no DTCs both at BM1 and BM2 (12.7% experienced relapse; P = .377, log-rank test). CONCLUSION: DTC status identifies high-risk patients after FEC chemotherapy, and DTC monitoring status after secondary treatment with docetaxel correlated strongly with survival. This emphasizes the potential for DTC analysis as a surrogate marker for adjuvant treatment effect in breast cancer.

Disseminated tumor cells as selection marker and monitoring tool for secondary adjuvant treatment in early breast cancer. Descriptive results from an intervention study.

BACKGROUND: Presence of disseminated tumor cells (DTCs) in bone marrow (BM) after completion of systemic adjuvant treatment predicts reduced survival in breast cancer. The present study explores the use of DTCs to identify adjuvant insufficiently treated patients to be offered secondary adjuvant treatment intervention, and as a surrogate marker for therapy response. METHODS: A total of 1121 patients with pN1-3 or pT1c/T2G2-3pN0-status were enrolled. All had completed primary surgery and received 6 cycles of anthracycline-containing chemotherapy. BM-aspiration was performed 8-12 weeks after chemotherapy (BM1), followed by a second BM-aspiration 6 months later (BM2). DTC-status was determined by morphological evaluation of immunocytochemically detected cytokeratin-positive cells. If DTCs were present at BM2, docetaxel (100 mg/m², 3qw, 6 courses) was administered, followed by DTC-analysis 1 month (BM3) and 13 months (BM4) after the last docetaxel infusion. RESULTS: Clinical follow-up (FU) is still ongoing. Here, the descriptive data from the study are presented. Of 1085 patients with a reported DTC result at both BM1 and BM2, 94 patients (8.7%) were BM1 positive and 83 (7.6%) were BM2 positive. The concordance between BM1 and BM2 was 86.5%. Both at BM1 and BM2 DTC-status was significantly associated with lobular carcinomas (p = 0.02 and p = 0.03, respectively; chi-square). In addition, DTC-status at BM2 was also associated with pN-status (p = 0.009) and pT-status (p = 0.03). At BM1 28.8% and 12.8% of the DTC-positive patients had ≥2 DTCs and ≥3 DTCs, respectively. At BM2, the corresponding frequencies were 47.0% and 25.3%. Of 72 docetaxel-treated patients analyzed at BM3 and/or BM4, only 15 (20.8%) had persistent DTCs. Of 17 patients with ≥3 DTCs before docetaxel treatment, 12 patients turned negative after treatment (70.6%). The change to DTC-negativity was associated with the presence of ductal carcinoma (p = 0.009). CONCLUSIONS: After docetaxel treatment, the majority of patients experienced disappearance of DTCs. As this is not a randomized trial, the results can be due to effects of adjuvant (docetaxel/endocrine/trastuzumab) treatment and/or limitations of the methodology. The clinical significance of these results awaits mature FU data, but indicates a possibility for clinical use of DTC-status as a residual disease-monitoring tool and as a surrogate marker of treatment response. TRIAL REGISTRATION: Clin Trials Gov NCT00248703.

Disseminated tumor cells in bone marrow following definitive radiotherapy for intermediate or high-risk prostate cancer.

BACKGROUND: The purpose of this study was to explore the prevalence of disseminated tumor cells (DTCs) in bone marrow (BM) of clinically progression-free prostate cancer (PC) patients at least 2 years after curatively intended radiotherapy (RT) with or without adjuvant hormone treatment. METHODS: All patients were T(1-3)N(0)M(0) with intermediate or high risk of progression. Median time from RT to BM sampling was 5 years (2-8). A standardized immunocytochemical method applying the anticytokeratin antibodies AE1/AE3 was used for DTCs detection in 130 patients. Morphological characterization of immunostained cells was performed to exclude false positive cells. The post-treatment BM was explored in relation to pre-treatment risk factors, treatment strategy and serum levels of Testosterone and PSA at the time of BM sampling. Longitudinal changes in BM status were studied in a sub-group of 109 patients who also had donated BM prior to treatment. RESULTS: Post-treatment BM-aspirates were positive for DTCs in 17% of cases without correlation to any of the tested variables. Out of 14 patients who had DTCs in BM prior to treatment, all but one had become post-treatment negative. Out of 95 patients with pre-treatment negative BM status, 18 (19%) had become post-treatment positive. CONCLUSIONS: DTCs in BM were found in 17% of clinically progression-free PC patients following RT. The detection of these cells may provide PSA-independent prognostic information remaining to be explored by prolonged follow-up.

Comparison of the clinical significance of occult tumor cells in blood and bone marrow in breast cancer.

Immunocytochemical (ICC) detection of disseminated tumor cells (DTC) in bone marrow (BM) in early breast cancer is an independent prognostic factor. The significance of circulating tumor cells (CTC) in peripheral blood (PB) needs further exploration and comparison to DTC detection. PB and BM were prospectively collected from 341 breast cancer patients median 40 months after operation. PB samples were analyzed for tumor cells by a negative immunomagnetic technique (10x10(6) cells/test). BM aspirates were analyzed by standard ICC (2x10(6) cells/test). CTC were present in 10% of the patients and DTC in 14%. Thirty-seven relapses and 14 breast cancer deaths have occurred at median 66 months after diagnosis. Both CTC-status and DTC-status were significantly associated with disease free survival (DFS) (event rate: CTC-positive 26.5% vs. CTC-negative 9.1%; DTC-positive 29.2% vs. DTC-negative 7.8%) (p<0.001/p<0.001, log rank) and breast cancer specific survival (event rate: CTC-positive 17.6% vs. CTC-negative 2.6%; DTC-positive 12.5% vs. DTC-negative 2.7%) (p<0.001/p<0.001). The presence of both CTC and DTC (n=8) resulted in an especially poor prognosis (p<0.001). In node negative patients, DTC-status, but not CTC-status, predicted differences in DFS (p=0.006 vs. p=0.503). Excluding 23 patients with breast cancer-related events prior to the sample collections, CTC detection was not significantly associated with DFS/distant-DFS (p=0.158/0.193), in contrast to DTC detection (p<0.001/<0.001). Presence of CTC and absence of DTC did not affect DFS (p=0.516). Applied to early stage disease, CTC analysis of increased volumes of PB appears less sensitive and prognostic than standard DTC analysis. Currently, this does not support an exchange of BM with PB for analysis of occult tumor cells.