Presence Of CK-positive Cells Research Articles

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

BackgroundPancreatic 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).MethodsPreoperative 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.ResultsSuccessful 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).ConclusionsThe 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 registrationclinicaltrials.gov (NCT01919151).

Abstract 1587: Detection of PD-L1 and PD-1 positive circulating tumor cells (CTCs) in non-small cell lung cancer (NSCLC) patients treated with nivolumab

Abstract Introduction: Circulating tumor cells (CTCs) are considered as a “liquid biopsy” that allows the assessment of tumor changes over time. Tumor cells may escape from the immune system through the activation of PD-1/PD-L1 axis. Targeting these molecules with monoclonal antibodies has shown encouraging results at many types of cancers, including NSCLC. In the current study we investigated the expression of PD-1/PD-L1 molecules on the CTCs isolated from NSCLC patients treated with Nivolumab. Methods: CTCs were isolated based on their size using the ISET platform from 27 patients before treatment, after 1 cycle and 3 cycles. CTCs were detected with Giemsa staining and immunofluorescence (IF) experiments, using either pancytokeratin (A45-B/B3) (CK7)/PD-1/CD45 or (A45-B/B3)(CK7)/PD-L1/CD45 combination of antibodies and analysis with the ARIOL system. Spiking experiments using the NSCLC cell lines: H460, H1299, HCC827 and SKMES in normal blood were used to evaluate the detection method. Results: Giemsa evaluation in Nivolumab-treated patients at baseline (25 evaluable samples), after the 1st (9 evaluable samples) and the 3rd (8 evaluable patients) cycle of treatment showed that CTCs could be detected in 48% (12/25), 33.3% (3/9) and 50% (4/8) of patients, respectively. IF could also reveal the presence of CK-positive cells in 44.4% (12/27), 22% (2/9) and 75% (6/8) patients, respectively. PD-1 (+) CTCs were detected in 33.3% (4/12) of patients at baseline, in, 0% after the 1 and 16.7% (1/6) of patients after the 3rd cycle. The same percentages were identified for PD-L1 expression in the same cohort of patients. The expression of PD-1 at baseline was associated with poorer OS (p=0.022) and PFS (p=0.011), while the expression of PD-L1 was associated with shorter PFS (p=0.011). Multivariate analysis revealed that the presence of CK-positive cells is an independent prognostic factor for OS (p=0.028) Conclusion: Nivolumab reduced the number of PD-1- and PD-L1-expressing CTCs in advanced NSCLC patients. Furthermore the expression of both markers at baseline is associated with the clinical outcome. Citation Format: Galatea Kallergi, Anastasios Koutsopoulos, Eleni Lagoudaki, Despoina Agouraki, Eleni-Kiriaki Vetsika, Anastasia Voumvouraki, Stuart S. Martin, Filippos Koinis, Christos Stournaras, Vassilis Georgoulias, Athanasios Kotsakis. Detection of PD-L1 and PD-1 positive circulating tumor cells (CTCs) in non-small cell lung cancer (NSCLC) patients treated with nivolumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1587.

Can the condition of the cell microenvironment of mediastinal lymph nodes help predict the risk of metastases in non-small cell lung cancer?

Background: The aim of this study was to analyze the properties of the immune cell microenvironment of regional lymph nodes (LNs) positive for lung cancer. Methods: Twenty-four patients operated on for stages T1 and T2 of the NSCLC, were enrolled in the study. Peripheral blood and LN tissue were obtained from different lymph node sites and levels. As a control, LN tissue was taken from patients diagnosed with emphysema or pneumothorax. The cells from randomly chosen LN were tested by multi-color flow cytometry. Separate portions of LN were snap-frozen and examined for the presence of cytokeratin positive cells (CK). Propensity for apoptosis, level of TCR zeta chain expression of T cells and the number and maturation status of dendritic cells were confronted with the presence of CK-positive cells. Results: The presence of metastases correlated with the downregulation of TCR zeta, especially CD8(+) T cells. The most striking feature was the reduction in the number of myeloid CD11c(+) dendritic cells in the LN of patients with LN metastases. This could be a reflection of the immunodeficient state observed in lung cancer patients. Even in the absence of metastases in the regional LN, the same type of changes in the LN microenvironment were observed in those LN located nearer the primary tumor. Conclusions: The preliminary results of this study suggest that this approach may be helpful as an independent tumor staging factor. It is also worth noting that part of the staging process could also be based on features describing the immune cells in the peripheral blood.

Multicenter study on the detection of disseminated tumor cells in primary breast cancer.

Abstract Abstract #5021 Background: Detection of disseminated tumor cells (DTC) in bone marrow (BM) has prognostic value in primary breast cancer (BC) and is currently under investigation for therapy stratification and monitoring. Anti-cytokeratin (CK) antibodies (Abs) have been established as markers for detection of such DTC in patients with BC. Here we present a direct comparison of the two Abs most frequently used for DTC detection over the past 10 years.
 Material and Methods: We screened BM samples from 391 BC patients of which 64 were node-positive (N+). All patients were free of any signs of overt metastases (stage M0, pT1-3, pN0-3). Tumor cells in BM were enriched by Ficoll-Hypaque gradient and the mononuclear cell (MNC) fraction was spun on slides. For each patient 2*106 MNC were analyzed per Ab, using A45-B/B3 (A45) and AE1/AE3 (AE) Abs in parallel. A45 staining was, however, performed a couple of years later than AE staining, on cytospins stored at -80°C. All samples were screened automatically for the presence of CK-positive cells. By morphological evaluation only TC-compatible cells were scored positive. Median follow-up times ranged from 5 to 118 months.
 Results: For both Abs, the number of DTC revealed was 5 or less. Of the 391 patients, 24 patients (6.1%) were positive for A45, and 41 patients (10.5%) for AE, respectively. Concordance obtained by cross-analysis for the two Abs was 84.4%, whereas the overlap in positive cases was only 3.2%. Regarding immunohistopathological characteristics AE- and A45-positive BM were found significantly more frequent in tumors of higher nodal status (p=0.003 and p=0.026), and for AE also in hormone receptor negative (HR-) compared to HR+ patients (p=0.006). Positivity for AE and/or A45 staining in BM correlated with high nodal status and large tumor size (p=0.001 and p= 0.016, respectively). Survival analyses revealed shorter relapse-free survival for A45-positive patients (p=0.026). In node-positive patients, a significantly worse prognosis regarding overall and BC specific survival was observed for AE-positive (p=0.0245 and p=0.0125) and for AE- and/or A45-positive patients (p=0.035 and p=0.044, respectively). In node-negative patients, A45-positivity was correlated to a significantly shorter relapse-free survival (p=0.032). Immunocytochemical double staining of BM samples from 26 patients (M+ and M0) revealed subpopulations of DTC labeled with both Abs (in 8/26 cases) and other DTC exclusively stained for either A45 or AE (4/26).
 Discussion: Our results indicate that the two anti-cytokeratin Abs most frequently used for DTC detection in BM do not give identical results. They appear to identify at least partially different subpopulations of DTC in BC patients. This implies a possible advantage of the combined use of these Abs in future trials using DTC diagnostics. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5021.

Micrometastases in bone marrow: prognostic indicators for pancreatic cancer.

Minimal residual disease in patients with operable pancreatic carcinoma is frequently missed by current noninvasive tumour staging. We applied an immunocytochemical cytokeratin assay that allows identification of individual pancreatic carcinoma cells disseminated to bone marrow. Prior to therapy, bone marrow was aspirated from the upper iliac crest of 48 patients with ductal adenocarcinoma of the pancreas at various disease stages and an age-matched control group of 33 noncarcinoma patients. Tumor cells in cytologic bone marrow preparations were detected with monoclonal antibodies (mAbs) CK2, KL1, and A45-B/B3 to epithelial cytokeratins (CK) using the alkaline phosphatase antialkaline phosphatase method. CK-positive cells were found in 14 (48.4%) of 31 cancer patients treated with curative intent and in 10 (58.8%) of 18 patients with extended disease. The overall frequency of these cells was 1 to 83 per 5x10(5) mononuclear cells with no significant differences between patients at different tumor stages and lymph node involvement. After a median follow-up of 22.8 months (range 3-48 months), 6 (40.0%) of 15 patients who underwent complete surgical resection but had tumor cells in bone marrow presented with distant metastasis and 7 (46.7%) had local relapse compared to none of 12 corresponding patients without such cells (p<0.05). Univariate survival analyses revealed that the presence of CK-positive cells was predictive of reduced overall survival. In conclusion, anticytokeratin mAbs are reliable probes for the immunocytochemical detection of single pancreatic cancer cells disseminated to bone marrow. Thus the described technique may help identify patients with pancreatic cancer and at potentially high risk of early metastatic relapse. The results promise to be of important assistance for determining prognosis and the consequences in therapy of early stage pancreatic cancer.