2019 ASCO | Research progress of circulating tumor cells CTCs in tumor immunotherapy


From May 31 to June 4, 2019, the 55th ASCO Annual Meeting will continue to gather in Chicago. The theme of this year's meeting is:

2019 ASCO | 循环肿瘤细胞CTCs在肿瘤免疫治疗中的研究进展

ASCO conference content has many research progress on liquid biopsy. Today we will look at the relevant reports of circulating tumor cells CTCs in immunotherapy and tumor immunology through ASCO conference.


1 PD-L1 expression on circulating tumor cells and prognosis of breast cancer patients.

PD-L1 expression and prognosis in circulating tumor cells of breast cancer patients]

● Research area: Peking Union Medical College, China

● Summary link: http://abstracts.asco.org/239/AbstView_239_247627.html

research backgroundAt present, some patients can benefit from PD1/PD-L1 inhibitor treatment, and there are clinical trials for specific breast cancer patients. Although we know that the prognosis of breast cancer is related to CTCs and is critical for the prediction and surveillance of molecular targeted therapy, the expression of PD-L1 in CTCs has been poorly studied, and the predictive biomarker response to PD-L1 checkpoint inhibitors is lacking. Therefore, this study attempted to investigate the relationship between the expression of PD-L1 in CTCs and the prognosis and clinicopathological features of breast cancer.

research method: The expression of CTCs and PD-L1 genes on CTCs in 20 patients with primary breast cancer was analyzed by gene probe hybridization. The relationship between clinicopathological features and PD-L1 expression in CTCs was analyzed by chi-square test. Kaplan-Meier and univariate Cox proportional hazards models were used to compare the survival of patients with high pd-l1 expression and low pd-l1 expression.

Research Results:

(1) Of the 20 patients, 15 patients had more than 1 CTC detected in their 7.5ml peripheral blood, and in these 15 patients, at least 1 CTC per patient showed PD-L1;

(2) The expression of PD-L1 on CTC is divided into low expression, medium expression and high expression, and then low expression 1 point, medium expression 2 points and high expression 3 points are given, PD-L1 the total score of high expression is ≥ 3, while the total score of PD-L1 low expression is <3; We found that PD-L1 expression on CTC is related to tumor size (P = 0.012) lymph node status (P = 0.001) and PR status (P = 0.037), there was a significant difference between T2 and T3 in the large tumor group (P = 0.003); while there was a statistical difference between N1 vs N3 (P = 0.000) and N2 vs N3 (P = 0.015) in the lymph node status group;

(3) The study data showed that the overall survival rate of patients with high PD-L1 expression on CTC was worse than that of low PD-L1 expression on CTC (P = 0.004) (table); univariate Cox proportional hazards model analysis showed that high PD-L1 expression on CTC was an independent prognostic factor.

2019 ASCO | 循环肿瘤细胞CTCs在肿瘤免疫治疗中的研究进展

Conclusion: PD-L1 on CTCs is indeed associated with some adverse clinicopathological features; high expression of PD-L1 on CTCs is an independent prognostic factor for shorter survival and is associated with shorter survival.


2 Role of the expression of PD-L1 and CD47 on circulating tumor cells (CTCs) in the prediction of outcome in metastatic breast cancer (mBC) patients.

The role of PD-L1 and CD47 expression on circulating tumor cells (CTCs) in the prognosis of patients with metastatic breast cancer (mBC)]

Study area: Greece

Summary Link:http://abstracts.asco.org/239/AbsView_239_267255.html

research backgroundDetection of the innate immune checkpoint (CD47) and adaptive immune checkpoint (PD-L1) in CTCs may assist in monitoring immune escape. In this study, the correlation between CD47 and/or PD-L1 expression and clinical characteristics in CTCs of patients with metastatic breast (MBC) was evaluated.

research method: Blood collection was performed on 98 patients with MBC before the start of first-line therapy. Triple immunofluorescence staining of peripheral blood mononuclear cells (PBMC) for cytokeratin (CK), CD47 and PD-L1 was performed and 1*106 PBMCs per patient were analyzed using an Ariol microscope system. Using MDA.MB. 231 breast cancer cells were used as control, and the expression levels of CD47 and PD-L1 in CK CTCs were standardized.

Research Results:

(1) CK + CTCs were detected in 22.4%(22/98) of patients; high expression of CD47 and PD-L1 was identified in 41.9 and 11.6% of CTCs, respectively, while high expression of both markers was observed in 9.1% of CTCs;

(2) CTCs with high expression of at least one marker (CD47-high and/or PD-L1-high) were identified in 11.2% of patients, although there was a differential distribution among patients with triple-negative, hormone receptor-positive, and HER2-positive primary tumors (27.3, 12.5, 0%, respectively, p = 0.040);CD47-high and/or PD-L1-high CTCs were associated with disease progression (27.8 vs 5.6, p = 0.005) and shorter PFS [median 5.8(4.1 to 7.5)vs 13.3(11.4 to 15.2) months, p = 0.010]], the detection of only PD-L1-high CTCs was associated with reduced OS [median 23.8(5.8-41.8)vs 35.7(29.9-41.4) months, p = 0.043]];

(3) Multivariate Cox regression analysis showed that CD47-high and/or PD-L1-high CTCs predicted an increased risk of recurrence [HR:2.7(95% CI:1.3~5.7); P = 0.009], while PD-L1-high CTCs was associated with a high risk of death [HR:4.8(95% CI:1.4~16.6); P = 0.011]].

ConclusionImmune checkpoint CD47 and PD-L1 are co-expressed in a subset of CTCs of mBC patients; detection of high CD47 and/or PD-L1 expression on CTCs is associated with triple negative tumors, disease progression, and poor patient outcomes, and can be used to improve prognosis in mBC; patients carrying this CTC population can benefit from anti-CD47 and anti-PD-L1 immunotherapy.


3 Circulating tumor cell (CTC) PD-L1 and interferon regulatory factor-1 (IRF-1) expression as biomarkers of anti-PD-(L)1 response.

Circulating tumor cell (CTC) PD-L1 and interferon regulatory factor -1 (IRF-1) expression as biomarkers of anti-PD- (L) 1 response]

Study area: United States

Summary Link:http://abstracts.asco.org/239/AbstView_239_269835.html

research backgroundAnti-PD-L1 checkpoint inhibitors (CPI) have significantly improved the survival of patients with a variety of solid tumors due to their durable response and tolerability. Unfortunately, only a small percentage of patients with solid tumors (PTs) have objective responses. It was hypothesized that PD-L1 and IRF-1 expression in CTCs might be correlated with CPI response and a preliminary assessment of their expression was made.

research method: Anti-PD-L1 monotherapy or combination therapy in patients with metastatic solid tumors. 7.5 cc of blood was drawn for CTC assessment at 3 time points: baseline, after 1 CPI dose, 3-6 months after starting CPI, or when CPI was stopped. Peripheral blood CTCs were isolated with Accuyte kits and subjected to CK/EPCAM, anti-CD45, nuclear staining, PD-L1 and IRF-1 staining. CTCs are then identified with Cytefinder software.

Research Results:

(1) 16 patients have been registered, with a median age of 71 years (28-83) and a median ECOG = 1(0-3);50% are women; 50% of them are NSCLC,13% are head and neck cancer, 13% are breast cancer, and 24% are other cancer types;

(2) During the baseline period, CTCs were identified in the blood of 13 patients (#CTCs range from 0 to 101), of which 12 patients received imaging scans since the beginning of CPI treatment. According to RECIST 1.1 (Efficacy Evaluation Criteria for Solid Tumors 1.1 Edition), 3 patients had partial response (PR) and 1 patient had stable disease (SD),8 had disease progression (PD); patients with PR had more frequent IRF-1 staining on CTCs in the baseline period (table), and 1/3 of CTCs PD-L1 positive;

(3) The results of 6 cases with tumor tissue PD-L1 staining can be used for correlation analysis, of which 3 cases are PD-L1 + (positive is defined as tumor tissue PD-L1 ≥ 1%); 1 patient with tumor tissue PD-L1 + has PD-L1 + CTCs; Among the 3 patients with tumor tissue PD-L1-, 1 patient has a mixed PD-L1 +/PD-L1-CTC group, no PD-L1 staining was detected for the other 2 CTCs.

2019 ASCO | 循环肿瘤细胞CTCs在肿瘤免疫治疗中的研究进展

Conclusion: Isolation and enumeration of CTCs are feasible in different solid tumors. PD-L1-resistant PR patients may have higher IRF-1 CTCs; however, this result is preliminary and additional pts and samples are being evaluated.


4 Quantification of activated and exhausted immune cell populations and simultaneous characterization of CTCs in peripheral blood of cancer patients receiving checkpoint inhibitor therapy

Quantification of activated and depleted immune cell populations in peripheral blood and characterization of CTCs in cancer patients treated with checkpoint inhibitors]

Study area: United States

Summary Link:http://abstracts.asco.org/239/AbstiView_239_269185.html

research background: Immune checkpoint inhibitors (ICIs) are becoming the standard treatment for many tumors, but a significant proportion of patients do not respond well to ICIs. Individual biomarkers such as PD-L1 are not sufficiently accurate in predicting patient benefit. The study sought to expand the Epic Sciences noninvasive liquid biopsy platform to use CTC molecular analysis and immune cell changes to identify predictive peripheral blood biomarkers for ICIs.

research methodBaseline and treatment-time blood samples were collected from prostate, kidney, and bladder cancer patients treated with ICIs and sent to EPIC Science. Nucleated cells were plated on glass slides and immunoplate stained for CTCs (pan-ck,CD45,pd-l1, and dapi) with activated cells (cd4,cd8,ki-67, and dapi), depleted immune cells (cd8,ki-67,pd-1,lag-3,tim-3, and dapi). Changes in immune cells and circulating tumor cell populations were assessed with high-throughput digital pathology.

Research ResultsCTC was detected in 73%(24/33) of the patients, of which PD-L1 + CTC was detected in 12%(4/33) of the patients. No PD-L1 + CTCs were detected in the 9 treated samples tested. Compared to baseline in 14 matched samples, 57%(8/14) of patients had an increase in activated CD4 + leukocytes and 36%(5/14) of patients had an increase in activated CD8 + leukocytes in the treated sample. The depletion analysis was performed on a subset of matched samples (6 out of 14 samples). In the baseline and treatment samples, one patient had higher CD8 + leukocyte consumption than the healthy donor control group, and two patients had lower CD8 + leukocyte consumption than the control group. No change in depleted CD8 + leukocytes was observed from baseline to the treatment period.

Conclusion: Developed a liquid biopsy-based platform that can simultaneously measure the biomarkers of CTCs and leukocytes in a single peripheral blood sample. To detect changes in activated and depleted immune cell populations after ICIs treatment, and to evaluate the expression of PD-L1 on CTCs, the next step is to stratify immune cells and rare cell populations.

5 Clinical implication of monitoring PD-L1 circulating tumor cells (CTCs) in patients with non-small cell lung cancer (NSCLC) receiving immunotherapy.

Clinical Significance of Monitoring PD-L1 Circulating Tumor Cells (CTCs) in Patients with Non-small Cell Lung Cancer (NSCLC) Receiving Immunotherapy]

Study area: United States

Summary Link:http://abstracts.asco.org/239/AbstView_239_269637.html

research backgroundCTCs are the cellular components of liquid biopsies, and detection of these cells can help identify patients with residual disease and poor prognosis. However, it is unclear whether different types of CTCs can be used to monitor cancer sternness (Stemness) and the patient's immunotherapy response. Currently, there is limited clinical data for monitoring cancer stem cell markers (such as CD44 or CD166) and immunotherapy targets (including PD-L1) in CTCs. This study hypothesized that CTC positivity with dynamic monitoring of these markers is associated with response to immune checkpoint inhibitor therapy in NSCLC patients.

research method: As part of a prospective analysis of peripheral blood cells, 30 NSCLC patients receiving immunotherapy (pembrolizumab or Atozolizumab) or combination chemotherapy were included. Blood samples were collected every 3-4 weeks up to 24 weeks. Peripheral blood mononuclear cells were isolated and analyzed for CD45, non-leukocyte CTCs were identified for CD166 and CD4, and PD-L1 and cancer stem cell markers in potential CTCs were identified. Patients were evaluated for response according to the Recist criteria. The samples at each time point were statistically analyzed using one-way ANOVA.

Research Results:

(1) The decrease in PD-L1 CD45-CTC detected at the second time point was most strongly associated with the initial response to treatment; conversely, an increase in the number of PD-L1 CD45-cells was found in patients with disease progression compared to patients with stable disease (SD) or partial response (PR) (p = 0.01);

(2)CD45-cells, CD44 CD45-cells, CD166 CD45-cells showed no significant correlation with clinical outcomes;

(3) Baseline PD-L1 levels on CTCs were not correlated with tissue PD-L1 levels.

ConclusionWe report the feasibility and potential clinical significance of monitoring CTC surface markers in blood before and after the start of immunotherapy, and find that the added value of PD-L1 CTCs in non-responders to immunotherapy needs to be further verified.

If there are any mistakes or objections, please leave a message to correct them.


2019 ASCO | 循环肿瘤细胞CTCs在肿瘤免疫治疗中的研究进展

China CTC Industry Alliance is an industry alliance jointly initiated by 10 domestic CTC R & D enterprises and formally established on September 30, 2016. The purpose of the alliance is to "promote the academic exchange of CTC, promote the clinical application of CTC, and establish the industry standard of CTC", so as to contribute to the prevention and treatment of chronic diseases in cancer patients and cancer high-risk groups in China. The companies forming the alliance are Beijing Boao Jingdian Biotechnology Co., Ltd., Beijing Zhongkonatai Biotechnology Co., Ltd., Hangzhou Huadesen Biotechnology Co., Ltd., Yishan Biotechnology Co., Ltd., Zhuhai Lizhu Shengmei Medical Diagnostic Technology Co., Ltd., Shanghai Baihuikang Biotechnology Co., Ltd., Yichang Meiguang Silicon Valley Life Technology Co., Ltd., and Guangzhou Anfang Biotechnology Co., Ltd.

China CTC Industry Alliance relies on domestic academic organizations such as China Society of Clinical Oncology, Laboratory Medicine Branch of Chinese Medical Association, Pathology Branch of Chinese Medical Association, and connects relevant medical institutions, universities, scientific research institutes and technical enterprises to better develop liquid biopsy technology, promote the liquid biopsy market more greatly, promote the status of liquid biopsy more highly, amplify the sound of liquid biopsy more strongly, and realize the clinical transformation of liquid biopsy more quickly, more accurate for the Chinese people to carry out cancer prevention and treatment services.