Baseline Circulating Tumor Cells Research Articles

Clinical significance of circulating tumour cells and tumour marker detection in the chemotherapeutic evaluation of advanced colorectal cancer.

AimSystemic chemotherapy combining biological targeted therapies is the standard therapy for patients with metastatic colorectal cancer (mCRC), but effective markers are needed to identify clinical responders. Circulating tumour cells (CTCs) have been associated with prognosis in patients with mCRC. This study aimed to explore the relationship between CTC number and the clinical response of patients with advanced CRC.MethodEpithelial cell adhesion molecule‐independent enrichment and CD45− fluorescence in situ hybridization immunofluorescence were used to detect peripheral blood CTCs in 79 patients with advanced CRC. Fisher's exact test and Spearman's rank correlation coefficient were used to analyse the correlation between CTC number and efficacy of chemotherapy. Kaplan–Meier and Cox regression analyses were used to evaluate progression‐free survival (PFS).ResultsAmong the evaluable patients, CTCs were significantly correlated with clinical response (r =4.891, p = 0.031). High CTC numbers were associated with a poor treatment response (r = −0.250, p = 0.027). Dynamic decrease in CTC number was associated with clinical response (p = 0.046). High baseline CTC number and carcinoembryonic antigen levels were prognostic factors for unfavourable PFS in multivariable analysis [hazard ratio (HR) = 3.30, p = 0.011 and HR = 2.04, p = 0.044, respectively]. Compared with the CTC‐positive group, the CTC‐negative group showed superior PFS (median PFS 15.53 vs. 9.43 months, p = 0.041) among CRC patients receiving first‐line treatment.ConclusionCTC number is a feasible biomarker for predicting outcomes in mCRC patients receiving systemic chemotherapy.

OAB-008: Identification of high-risk Multiple Myeloma with a plasma cell Leukemia-like transcriptomic profile

<h3>Background</h3> Primary plasma cell leukemia (pPCL) is an aggressive subtype of multiple myeloma that is characterized by ≥20% circulating tumor cells (CTCs), whereas CTC levels in newly diagnosed multiple myeloma (NDMM) are <20%. Currently, there is no molecular marker for pPCL. It therefore remains to be elucidated if certain NDMM tumors molecularly resemble pPCL and if this has any prognostic significance in the context of conventional high-risk (HR) markers in NDMM. Aims: (1) To construct and validate a transcriptomic classifier for PCL-like disease. (2) To test its value as independent prognostic marker in NDMM. <h3>Methods</h3> Transcriptomic data were generated of CD138-enriched bone marrow (BM) plasma cells from both NDMM patients enrolled in the Cassiopeia (NCT02541383) and HO143 (EudraCT 2016-002600-90) trials and pPCL patients from the EMN12/HO129 (EudraCT 2013-005157-75) trial. NDMM CTC levels were determined with the EuroFlow NGF protocol. HR FISH was defined as the presence of either t(4;14), t(14;16) or del17p.154 NDMM and 29 pPCL patients were divided into a discovery and validation cohort to construct and test a classifier for PCL-like disease. Subsequently, data from 8 additional NDMM cohorts were used to assess the association of PCL-like status with progression-free survival (PFS) and overall survival (OS) in Cox proportional hazards (PH) models including conventional HR markers. <h3>Results</h3> Baseline CTC levels were determined in 297 NDMM and 51 pPCL patients. CTCs could be detected in 87% of NDMM patients, with a limit of detection <10-5. CTC levels were positively associated with tumor burden (BM plasmacytosis). In the discovery cohort, 1700 genes correlated with high CTC levels (FDR <0.05), independently of tumor burden. These included genes involved in cell adhesion, cell migration and proliferation. After optimization by leave-one-out cross-validation, a classifier for PCL-like disease was built with a selection of 54 genes. This showed a sensitivity of 93% to identify pPCL in the validation cohort, but also classified 10% of NDMM tumors as PCL-like. In a cohort of 2139 NDMM patients, Cox PH regression analyses confirmed a significant and independent association of PCL-like status with PFS and OS, in the context of either R-ISS stage, ISS stage, HR FISH, SKY92 HR or UAMS70 HR status. PCL-like status in combination with R-ISS, age and treatment status showed a hazard ratio of 1.64 (95% CI, 1.30-2.07, p<0.0001) for PFS and 1.89 (95% CI, 1.42-2.50, p<0.0001) for OS. PCL-like status conferred a median OS of only 13.2 months among NDMM patients with R-ISS III. <h3>Conclusions</h3> (1) pPCL cannot only be identified clinically, but also molecularly. (2) PCL-like status is a novel marker for HR disease in NDMM that identifies patients with a tumor transcriptome similar to pPCL and has independent prognostic value in the context of conventional HR markers. (3) PCL-like status could help detect NDMM patients with early stage or borderline pPCL.

Prognostic impact and potential predictive role of baseline circulating tumor cells in locally advanced head and neck squamous cell carcinoma

ObjectivesThe prognostic impact of circulating tumor cells (CTCs) or circulating tumor microemboli (CTM) in locally advanced head and neck squamous cell carcinoma (LA-HNSCC) is yet to be determined, with conflicting results in previous trials. The role of induction chemotherapy (ICT) in the management of LA-HNSCC is controversial with no predictive biomarkers to guide treatment strategy in this scenario. The aim of this trial is to determine the prognostic impact of CTCs and CTM, their biomarkers expression by immunocytochemistry (ICC), and its potential role as predictors of ICT benefit in LA-HNSCC. Materials and MethodsProspective study, with newly diagnosed stage III/IV non-metastatic LA-HNSCC patients treated with curative intent. Blood samples analyzed for CTCs and CTM before treatment using the ISET method. ResultsA total of 83 patients were included. CTCs counts were an independent prognostic factor for overall survival (OS; HR: 1.17; 95 %CI: 1.05–1.31; p = 0.005) and progression free survival (PFS; HR:1.14; 95 %CI: 1.03–1.26; p = 0.007). Using the Lausen and Schumacher technique, 2.8 CTCs/mL for OS and 3.8 CTCs/mL for PFS were defined as the best cut-offs. CTM were detected in 27.7% of patients, correlating with worse PFS (HR = 2.70; IC95%: 1.30–5.58; p = 0.007). MRP-7 expression in CTM correlated with worse OS (HR = 3.49; 95 %CI: 1.01–12.04; p = 0.047) and PFS (HR = 3.62; 95 %CI: 1.08–12.13; p = 0.037). CTCs counts were predictive of complete response to treatment (OR = 0.74; 95 %CI: 0.58–0.95; p = 0.022) and high counts (cut-off 3.8/mL) and CTM were potential predictors of ICT benefit. ConclusionCTCs/CTM had significant prognostic impact and potential role as predictors of ICT benefit in LA-HNSCC.

Abstract 602: Prognostic utility of circulating tumor cells/cell clusters in head and neck cancers

Abstract Background: Metastases remains the major cause of death in head and neck cancer (HNC) patients. Circulating tumour cells (CTCs) that are shed from primary and metastatic sites and circulate in patients' peripheral blood, represent an important window into disease status. Minimally invasive techniques are required to identify at time of diagnosis, which patients will have worse prognosis due to distant relapse. Method: We collected bloods from treatment naïve, locoregionally advanced HNC patients (n=72) at the time of diagnosis, and CTCs were isolated using a spiral microfluidic device. CTCs were then immobilised onto glass slides and identified by using immunofluorescence staining (Cytokeratin/CD45/DAPI). We then correlated the CTC numbers of each patient to their clinical outcome based on either PET/CT scan and/or clinical assessment after definitive treatment. Results: CTCs were detected in 39/72 (54%) of patients (ranging from 1-18 CTCs/10 mL blood) at diagnosis. CTC numbers were correlated to TNM staging (p&amp;lt;0.01), especially the Node staging (p&amp;lt;0.01). Patients with ≥2 CTCs at baseline showed significantly worse prognosis than those with no CTCs detected (P&amp;lt;0.001). We also found that baseline CTCs is an independent predictor of the adverse clinical outcome (residual disease, recurrence, and equivocal clinical presentation) even after adjusting to other prognostic factors (hazard ratio=4.613, p&amp;lt;0.05). Patients with CTC cluster had poor prognosis compared to patients with single CTCs. Conclusion: We were able to predict disease progression based on CTC numbers, thus providing potential additional prognostic information for locally advanced HNC patients. The ability to detect CTCs at diagnosis allows us to stratify the risk in patients as well as to understand the fundamental mechanisms by which CTCs spread. The clinical benefit of establishing the use of CTCs in HNC is likely to translate into better patient selection for treatment intensification and/or de-intensification strategies. Citation Format: Xi Zhang, Kai Tang, Brett Hughes, Sarj Vasani, Zhen Liu, Majid Warkiani, Gunter Hartel, Rahul Ladwa, Liz Kenny, Chamindie Punyadeera. Prognostic utility of circulating tumor cells/cell clusters in head and neck cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 602.

Abstract 597: Comprehensive analysis of blood-based biomarkers to immunotherapy using circulating tumor cells, peripheral blood cells and circulating tumor DNA in advanced non-small cell lung cancer

Abstract Background: This study aimed to investigate the feasibility of liquid biopsy using circulating tumor cells (CTCs), peripheral blood cells (PBCs) and circulating tumor DNA (ctDNA) as predictive blood-based biomarkers to immunotherapy in advanced non-small cell lung cancer (NSCLC). Methods: This study included patients with advanced NSCLC receiving pembrolizumab or atezolizumab from July 2019 to June 2020. Blood was collected in K2-EDTA tube before each administration from cycle 1 to 4 (C1-4), and PBC counts (absolute neutrophil count[ANC], neutrophil-to-lymphocyte ratio[NLR], derived NLR[dNLR], platelet-to-lymphocyte ratio[PLR]) were calculated at each cycle. CTCs were enriched by using CD-PRIMETM system, the antibody-independent size-based isolation method, and identified by single positive cells (EpCAM/CK+CD45-) in BioViewCCBSTM system. Cell-free DNA was purified and extracted from plasma at baseline and C4 or end-of-treatment. Results: Among 83 response-evaluable patients, objective response rate was 19% and durable clinical benefit (DCB) were 34%. Baseline (C1) CTC, PBC count and ctDNA amount did not show a significant difference according to best response and DCB. However, patients with lower NLR, dNLR, PLR and ctDNA level at C1 showed significant benefit in progression-free survival (PFS) and overall survival (OS) (p&amp;lt;0.05 for all). Patients with decreased CTC at C2 compared to C1 showed benefit in median PFS (6.2 vs 2.3, p=0.078) and OS (not reached [NR] vs 6.8, p=0.021) than those with increased CTC count. Low dNLR (≤2.0) at C1 and decreased CTC at C2 compared to C1 were the independently significant factors for survival. Conclusion: Comprehensive analysis of CTC, PBC count and ctDNA amount at baseline and their changes during treatment could be potential biomarkers to predict the survival benefit and may help risk-group stratification in patients with advanced NSCLC who received anti-PD-1/PD-L1 immunotherapy. Citation Format: Cheol-Kyu Park, Hyun-Ju Cho, In-Jae Oh, Young-Chul Kim. Comprehensive analysis of blood-based biomarkers to immunotherapy using circulating tumor cells, peripheral blood cells and circulating tumor DNA in advanced non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 597.

Prospective Evaluation of a Circulating Tumor Cell Sensitivity Profile to Predict Response to Cisplatin Chemotherapy in Metastatic Breast Cancer Patients.

BackgroundCisplatin (cDDP) has regained interest for metastatic breast cancer (MBC) patients, given the platinum sensitivity in subtypes and better manageable toxicity. Here, the primary aim was to determine whether molecular characteristics of circulating tumor cells (CTCs) could identify patients responding to cDDP and to describe the outcomes to cDDP monotherapy in a large group of MBC patients pretreated with anthracycline- and taxane-based treatments.MethodsBased on cell line data, a CTC-cDDP-sensitivity profile was generated. Applying an A’Herns single-stage phase II design, further investigation was considered worthwhile if 5/10 patients with a favorable profile responded to cDDP. Patients received 70mg/m2 cDDP every three weeks, CTCs were enumerated and the CTC-cDDP-sensitivity profile was determined. In total, 65 heavily pretreated MBC patients (77% received ≥2 lines of previous chemotherapy for MBC) were eligible for the per-protocol analysis. Primary endpoint was response rate, secondary endpoints included best observed response, progression-free survival (PFS) and overall survival (OS).ResultsThe best observed response during cDDP therapy was a partial response in 7% and stable disease in 56% of the patients. None of the patients with a favorable CTC-cDDP-sensitivity profile had a response. The median baseline CTC count was 8 (range 0-3254). Patients with <5 CTCs had a better PFS and OS than patients with ≥5 CTCs (median PFS 4.5 months (95%CI 2.38-6.62) vs. 2.1 months [(95%CI 1.34-2.80)(p=0.009)] and median OS 13.1 months (95%CI 9.89-16.33) vs. 5.6 months [(95%CI 3.60-7.64)(p=0.003)]. No other factors than CTC count were associated with outcome to cDDP therapy, including triple-negative breast cancer versus ER-positive tumors.ConclusionsThe CTC-cDDP-sensitivity profile was unable to select patients responding to cDDP monotherapy. In an unselected group of heavily pretreated MBC patients, cDDP yields outcomes comparable to other chemotherapeutic regimens for heavily pretreated MBC patients. CTC count was the only factor associated with outcome in these patients.Clinical Trial Registration(https://www.trialregister.nl/trial/3885, identifier NTR4046)

Prognostic Role of Circulating Tumor Cells in Metastatic Renal Cell Carcinoma: A Large, Multicenter, Prospective Trial.

Circulating tumor cells (CTCs) correlate with adverse prognosis in patients with breast, colorectal, lung, and prostate cancer. Little data are available for renal cell carcinoma (RCC). We designed a multicenter prospective observational study to assess the correlation between CTC counts and progression-free survival (PFS) in patients with metastatic RCC treated with an antiangiogenic tyrosine kinase inhibitor as a first-line regimen; overall survival (OS) and response were secondary objectives. CTC counts were enumerated by the CellSearch system at four time points: day 0 of treatment, day 28, day 56 and then at progression, or at 12 months in the absence of progression. One hundred ninety-five eligible patients with a median age of 69 years were treated with sunitinib (77.5%) or pazopanib (21%). At baseline, 46.7% of patients had one or more CTCs per milliliter (range, 1 to 263). Thirty patients had at least three CTCs, with a median PFS of 5.8 versus 15 months in the remaining patients (p=.002; hazard ratio [HR], 1.99), independently of the International Metastatic RCC Database Consortium score at multivariate analysis (HR, 1.91; 95% confidence interval [CI], 1.16-3.14). Patients with at least three CTCs had a shorter estimated OS of 13.8 months versus 52.8 months in those with fewer than three CTCs (p=.003; HR, 1.99; multivariate analysis HR, 1.67; 95% CI, 0.95-2.93). Baseline CTC counts did not correlate with response; neither did having CTC sequencing counts greater than or equal to one, two, three, four, or five. We provide prospective evidence that the presence of three or more CTCs at baseline is associated with a significantly shorter PFS and OS in patients with metastatic RCC. This prospective study evaluated whether the presence of circulating tumor cells (CTCs) in the peripheral blood correlates with activity of first-line tyrosine kinase inhibitors in metastatic renal cell carcinoma (RCC). This study demonstrated that almost half of patients with metastatic RCC have at least one CTC in their blood and that those patients with at least three CTCs are at increased risk of early progressive disease and early death due to RCC. Studies incorporating CTC counts in the prognostic algorithms of metastatic RCC are warranted.

Correlation of baseline circulating tumor cells (CTC) and associated genomic profile with survival outcomes in patients (pts) with metastatic castration-sensitive prostate cancer (mCSPC) in a real-world cohort.

5077 Background: We recently published, in the context of SWOG1216 trial in pts with mCSPC, that higher baseline CTC level were associated with inferior survival outcomes (Goldkorn. Agarwal, CCR, 2021). Here in, we validate these findings in a real world population of mCSPC and interrogate tumor genomic profile with respect to the CTC level. Methods: Eligibility criteria: new mCSPC receiving ADT without or with intensification (docetaxel or novel hormonal therapy) and enumeration of baseline CTCs by FDA cleared Cell Search CTC assay. Gene alterations were determined by comprehensive genomic profiling (CGP) of tumor tissue (Foundation Medicine). CTC counts were categorized as 0, 1-4 and ≥5/7.5 ml. Relationships between CTC counts and number (no.) of genes altered and individual gene alterations were assessed via Kruskal-Wallis and chi-squared tests, respectively. Relationships between progression-free survival (PFS), overall survival (OS) and individual mutations were assessed via log-rank tests. Relationships between CTC counts, PFS and OS were assessed by Cox proportional hazards models, both unadjusted and adjusted for multiple variables (Table). Results: Overall 103 pts were eligible. Median age: 67 yrs, Gleason score: 9, PSA at ADT initiation: 41 ng/mL. 67 (65%) pts had de-novo metastatic disease and 44 (43%) pts underwent ADT intensification therapy. Pts with greater CTC counts tended to have greater no. of altered genes (p=0.017), greater no. of total alterations (p=0.017) and higher rate of TP53 mutations (p=0.036). In univariate analyses (UVA) and multivariable analyses (MVA), both CTC counts and no. of genes altered were strongly associated with both PFS and OS (Table). CGP of tumors with respect to CTC counts will be presented in meeting. Conclusions: Herein, we validate our previous findings from SWOG1216 trial of association of higher CTC level with inferior survival outcomes in a real world mCSPC cohort. The CTC enriched population is associated with a distinct tumor genomic landscape, which may guide further drug development in this pt population at the highest risk of progression and/or death.[Table: see text]

A phase (Ph) 1b/2 study of ribociclib (R) in combination with docetaxel (D) plus prednisone (P) in metastatic castration-resistant prostate cancer (mCRPC).

5043 Background: The survival benefit of D in mCRPC is modest. CDK4/6 inhibitors such as R have shown synergistic activity with taxanes in pre-clinical cancer models. We sought to determine the safety and efficacy of R + D + P in mCRPC patients (pts). Methods: This was a Ph 1b/2 multicenter, open-label single arm trial of mCRPC pts with progression (PD) on ≥ 1 prior androgen receptor signaling inhibitor (ARSi) who had not previously received D for mCRPC (NCT02494921). Pts were treated with escalating doses of R in combination with D + P for 6-9 cycles, followed by single agent maintenance R until radiographic or clinical PD. The Ph 2 primary endpoint was 6-month (mo) radiographic progression-free survival (rPFS) rate by PCWG2 criteria, with a target rate of 55% and null hypothesis of 35%. Ph 2 pts underwent baseline circulating tumor cell (CTC) enumeration and genome sequencing (Epic Sciences). Cox proportional hazard model and log-rank test were used to test for associations between rPFS and CTC burden and copy number (CN) variants, respectively. Results: 43 pts were enrolled from 11/2015 to 6/2019. Median age was 68 (range 55-84). 20.9% of pts had visceral metastases. 33 (77%) had PD on prior abiraterone, 27 (63%) on enzalutamide, and 17 (40%) on both. In Ph 1b, 19 pts were enrolled. In the first cohort (D 75 mg/m2 day [d] 1, R 200 mg/d d2-14 of every 21d cycle), 2 pts experienced DLTs (febrile neutropenia [FN] and grade 4 neutropenia). With an alternative dosing schema of D 60 mg/m2 on d1, and R daily on d1-4 and 8-15 of cycle, with daily G-CSF support on d5-7, the MTD was not reached and D 60 mg/m2 + R 400 mg/d was chosen as the recommended Ph 2 dose (RP2D). In total, 30 pts were treated at RP2D; median number of D cycles was 8.5 and 60% went on to receive maintenance R. The Ph 2 primary endpoint was met with a 6-mo rPFS rate of 65% (95% CI 50-85%). Median rPFS was 8.0 mos (95% CI 4.1-10.0). PSA response rate (RR) defined as ≥50% reduction was 27.6% (95% CI 12.7-47.2%) and objective RR was 30.8% (95% CI 9.1-61.4%). Among pts treated at RP2D, the most common grade ≥3 treatment-related adverse events were neutropenia (n= 11, 36.7%), lymphocytopenia (n=3, 10%); no cases of FN were observed. Baseline CTC burden was associated with an increased risk of radiographic PD or death (HR 1.038, 95% CI 1.001-1.074, p = 0.038). Pts harboring CTCs without MYC (4/11 pts) or CDK6 CN gain (7/11 pts) had prolonged rPFS compared to those with gene amplification (median rPFS 10.76 vs 4.11 mos, p = 0.03, and 7.01 vs 1.92 mos, p = 0.053, respectively). Conclusions: The combination of R + D was well tolerated and showed promising activity in mCRPC pts who had progressed on an ARSi. The Ph 2 study met its primary endpoint, with an encouraging 6-mo rPFS rate of 65%. Lack of MYC or CDK6 amplification on CTC sequencing was associated with longer rPFS. Funding: Novartis Pharmaceuticals, PCF YIA. Managed by the PCCTC. Clinical trial information: NCT02494921.

Sensitive and dynamic CTCs measurement for prediction and monitoring of PD-1 therapy in GC/EGJC patients: Results from a pilot phase 2 study.

e16094 Background: Programmed death-ligand 1 (PD-L1) expression is currently being explored as a predictive biomarker for anti-PD-1antibody treatment in patients with advanced gastric or esophagogastric junction cancer (GC/EGJC) patients. Circulating tumor cells (CTCs) can be collected non-invasively and sequentially, and may allow real-time monitoring of immune activation in tumor. The aim of this study was to investigate whether PD-L1 expression on CTCs can accurately represent tumor PD-L1 expression and the potential of CTCs as a companion diagnosis (CDx) biomarker. Methods: We evaluated the CTCs counts and PD-L1 status on CTCs during treatment in a phase 2 clinical trial, where patients with previously treated gastric cancer received JS001 in combination with Apatinib. The multi-center trial was registered with ClinicalTrials.gov, number NCT04190745. Blood samples were collected from patients. Sixteen patients with GC were analyzed to investigate the association between clinical outcome and changes of CTC counts pre and post JS001 plus Apatinib. The correlation between the binary clinical outcome (SD/PR/CR vs. PD) and the change in CTCs counts pre and post treatment was analyzed using a logistic regression adjusting for baseline CTCs counts. PD-L1 expression on CTCs were analyzed to determine whether they were in accordance with tissue biopsy. Results: CTCs were detected in all the patients (16/16，100%) ranging from 1 to 33 CTCs/ml. PD-L1+ CTCs were detected in 12 of 16 patients (75%) ranging from 1 to 5 PD-L1 + CTCs/ml. PD-L1 expression in tumor tissue was positive in 11 of 13 patients (84.6%, positive means CPS &gt; = 1), the sensitivity of PD-L1+ CTCs is 90.9% (10/11) if using biopsy as gold standard. The only false negative patient though with CPS 40%, got no benefit from immunotherapy. The specificity of PD-L1+ CTCs detection is 100%, and the accuracy is 90.9%. Overall response rate of this regimen is 26.6% (4/15). Disease control rate is 53.3% (8/15). Progression free survival (PFS) was 2.43 months in all the evaluable patients. However, patients with PD-L1+ CTCs had a longer PFS compared to patients without PD-L1+ CTC (3.30months vs 1.37months, p = 0.001). Overall Survival is not reached. Change of CTCs counts after treatment was significantly correlated with clinical response (p = 0.024). The logistic model McFadden goodness of fit score was 0.435, which is a strong correlation value. Notably, one of the patients who had pseudo-progression shows remarked decreased CTCs count. Conclusions: Our results reveal the potential of CTCs as a noninvasive CDx biomarker in patients with advanced GC/EGJC.

Value of Early Circulating Tumor Cells Dynamics to Estimate Docetaxel Benefit in Metastatic Castration-Resistant Prostate Cancer (mCRPC) Patients.

Simple SummaryThe prognostic role of CTC enumeration in mCRPC patients has been established in several studies, demonstrating a higher prognostic performance than post-treatment changes in PSA levels in patients treated with AR signaling inhibitors, but not taxanes. We carried out a pooled analysis of two prospective studies in mCRPC patients treated with docetaxel. The results of this study showed a greater ability of early changes in circulating tumor cells (CTCs) compared to PSA response endpoints to predict overall survival in metastatic castration-resistant prostate cancer (mCRPC) patients treated with docetaxel. These results encourage the clinical usefulness of CTC enumeration to determine the outcome of mCRPC patients.Circulating tumor cell (CTC) enumeration and changes following treatment have been demonstrated to be superior to PSA response in determining mCRPC outcome in patients receiving AR signaling inhibitors but not taxanes. We carried out a pooled analysis of two prospective studies in mCRPC patients treated with docetaxel. CTCs were measured at baseline and 3–6 weeks post treatment initiation. Cox regression models were constructed to compare 6-month radiographical progression-free survival (rPFS), CTCs and PSA changes predicting outcome. Among the subjects, 80 and 52 patients had evaluable baseline and post-treatment CTC counts, respectively. A significant association of higher baseline CTC count with worse overall survival (OS), PFS and time to PSA progression (TTPP) was observed. While CTC response at 3–6 weeks (CTC conversion (from ≥5 to <5 CTCs), CTC30 (≥30% decline in CTC) or CTC0 (decline to 0 CTC)) and 6-month rPFS were significantly associated with OS (all p < 0.005), the association was not significant for PSA30 or PSA50 response. CTC and PSA response were discordant in over 50% of cases, with outcome driven by CTC response in these patients. The c-index values for OS were superior for early CTC changes compared to PSA response endpoints, and similar to 6-month rPFS. Early CTC declines were good predictors of improved outcomes in mCRPC patients treated with docetaxel in this small study, offering a superior and/or earlier estimation of docetaxel benefit in comparison to PSA or rPFS that merits further confirmation in larger studies.

Circulating Tumor Cell Genomic Evolution and Hormone Therapy Outcomes in Men with Metastatic Castration-Resistant Prostate Cancer.

Men with circulating tumor cell (CTC) AR-V7-positive metastatic castration-resistant prostate cancer (mCRPC) have worse outcomes when treated with enzalutamide/abiraterone. However, most men lack CTC AR-V7 detection, and additional predictive biomarkers are needed. We conducted a retrospective secondary analysis of the prospective PROPHECY trial (NCT02269982) of men with mCRPC undergoing treatment with enzalutamide/abiraterone, analyzing pooled CTC and germline DNA for whole-genome copy-number alterations (CNA) in 73 samples from 48 men over time along with pooled CTC and germline whole-exome sequencing on 22 paired samples before and following progression on androgen receptor (AR) inhibitor therapy to identify somatic genomic alterations associated with acquired resistance. We observed broad interpatient and longitudinal CTC genomic heterogeneity from AR-V7-negative men with mCRPC, including common gains of KDM6A, MYCN, and AR, and loss of ZFHX3, BRCA1, and PTEN. Men who had progression-free survival of ≤3 months despite enzalutamide/abiraterone treatment were more likely to have baseline CTC genomic loss of CHD1, PTEN, PHLPP1, and ZFHX3 and gains of BRCA2, KDM5D, MYCN, and SPARC. After progression on abiraterone/enzalutamide, we observed clonal evolution of CTCs harboring TP53 mutations and gain of ATM, KDM6A, and MYC, and loss of NCOR1, PTEN, RB1, and RUNX2. CTC genomic findings were independently confirmed in a separate cohort of mCRPC men who progressed despite prior treatment with abiraterone/enzalutamide (NCT02204943). IMPLICATIONS: We identified common and reproducible genomic alterations in CTCs from AR-V7-negative mCRPC men associated with poor outcomes during enzalutamide/abiraterone treatment, including CNAs in genes linked to lineage plasticity and epigenetic signaling, DNA repair, AR, TP53/RB1, PTEN, and WNT pathways.

Influence of BRAF and PIK3CA mutations on the efficacy of FOLFIRI plus bevacizumab or cetuximab as first-line therapy in patients with RAS wild-type metastatic colorectal carcinoma and

BackgroundWe explored the influence of BRAF and PIK3CA mutational status on the efficacy of bevacizumab or cetuximab plus 5-fluorouracil/leucovorin and irinotecan (FOLFIRI) as first-line therapy in patients with RAS wild-type metastatic colorectal cancer (mCRC).Patients and methodsVISNÚ-2 was a multicentre, randomised, phase II study. Patients with RAS wild-type mCRC and <3 circulating tumour cells/7.5 ml blood were stratified by BRAF/PIK3CA status (wild-type versus mutated) and number of affected organs (1 versus >1), and allocated to bevacizumab (5 mg/kg every 2 weeks) or cetuximab (400 mg/m2 then 250 mg/m2 weekly) plus FOLFIRI [irinotecan 180 mg/m2, leucovorin 400 mg/m2, 5-fluorouracil 400 mg/m2 (bolus) then 2400 mg/m2 (46-h continuous infusion) every 2 weeks]. The primary endpoint was progression-free survival (PFS). All analyses were exploratory.ResultsTwo hundred and forty patients with BRAF/PIK3CA wild-type (n = 196) or BRAF- and/or PIK3CA-mutated tumours (n = 44) were enrolled. Median PFS was 12.7 and 8.8 months in patients with BRAF/PIK3CA wild-type and BRAF/PIK3CA-mutated tumours, respectively [hazard ratio (HR) = 1.22; 95% confidence interval (CI) 0.80-1.85; P = 0.3602]. In the BRAF- and/or PIK3CA-mutated cohort, median PFS was 2.8, 8.8 and 15.0 months in patients with BRAF/PI3KCA-mutated (n = 8), BRAF-mutated/PI3KCA wild-type (n = 16) and BRAF wild-type/PI3KCA-mutated (n = 20) tumours, respectively (P = 0.0002). PFS was similar with bevacizumab plus FOLFIRI versus cetuximab plus FOLFIRI in BRAF/PIK3CA wild-type (HR = 0.99; 95% CI 0.67-1.45; P = 0.9486) and BRAF/PIK3CA-mutated tumours (HR = 1.11; 95% CI 0.53-2.35; P = 0.7820). The most common grade 3/4 treatment-related adverse events were neutropenia, diarrhoea and asthenia in both treatment groups.ConclusionsBRAF/PIK3CA status influences outcomes in patients with RAS wild-type mCRC but does not appear to assist with the selection of first-line targeted therapy.

Assessment of the Circulating Tumor Cells and Microsatellite Instability in Colorectal Cancer Patients: Prognostic and Diagnostic Value.

BackgroundMicrosatellite instability (MSI) and circulating tumor cells (CTCs) play important roles in the diagnosis, prognosis and management of colorectal cancer (CRC) patients.MethodsCTCs and MSI were assessed in the blood and representative tumor tissues of 100 CRC patients by flow cytometry (FCM) and PCR amplification. The data were correlated to relevant clinicopathological features of the patients, progression-free survival (PFS) and overall survival (OS) rates.ResultsMSI-high was detected in 44 (44.0%) patients, MSI-low in 37 (37%), and microsatellite stable (MSS) in 19 (19.0%) patients (P=0.007). The baseline CTCs count (<4 cells/7mL blood) was reported in 39% of the patients, and CTCs ≥4 cells/7mL blood in 61% of the patients (P=0.028). Improved PFS and OS rates were associated significantly with MSI-high (P<0.001), decreased CTC levels during the course of treatment (P<0.001) and post-treatment CTCs (P=0.008). There was no significant association between MSI-high and PFS or OS in early-stage patients (P=0.187 and P=0.187; respectively); however, it was associated significantly with better PFS and OS in late-stage patients (P<0.001). Multivariate analysis showed that only a change in serial CTC levels is considered an independent prognostic factor for OS (P<0.012). Post-treatment CTCs level, serial CTCs level changes during the course of treatment, lymph nodes and distant metastasis were independent prognostic factors for PFS (P<0.001, P= 0.047, P=0.001 and P<0.001; respectively).ConclusionMSI and CTCs could be used as accurate, reliable and sensitive diagnostic and prognostic biomarkers for CRC patients’ survival rates and outcomes.

Baseline and post-treatment circulating tumor cell (CTC) counts with prostate-specific membrane antigen (PSMA)-targeted radionuclide therapy (TRT) in men with metastatic castration-resistant prostate cancer (mCRPC).

158 Background: CTC counts are an independent prognostic factor in men with mCRPC; certain changes following treatment (conversion from detectable to undetectable or unfavorable to favorable) are associated with improved overall survival. Most PSMA-TRT efficacy data have focused on PSA or imaging changes. Here, we describe baseline and post-treatment CTC counts from subjects receiving PSMA-TRT. Methods: Men with mCRPC treated on prospective clinical trials of PSMA-TRT and with available CTC counts (CellSearch) were included in our analysis. Depending upon the era of the trial, post-treatment counts were performed at 4-6 (initial era) or 12 weeks (recent era) after a single cycle of PSMA-TRT (individual trial data reported elsewhere). We describe CTC counts at baseline and compare pre-treatment counts to those after PSMA-TRT. Results: 116 men treated with PSMA-TRT had baseline CTC count (90 with both pre- and post-treatment CTC). Forty-four patients (37.9%) received 177Lu-J951, 46 (39.7%) received 177Lu-PSMA-617, and 26 (22.4%) received 225Ac-J591. Median age was 71.5. Fifty-eight patients (50%) had previously received taxane chemotherapy, median PSA was 82.98 ng/mL, and 66 (56.9%) were in the high-risk Halabi (CALGB) prognostic group. Eighty-nine out of one hundred sixteen (76.7%) had detectable baseline CTC and 58/116 (50%) had unfavorable baseline CTC count. Forty-nine out of seventy (70%) had post-treatment CTC count decline, 23/70 (32.9%) converted from detectable to undetectable, and 17/47 (36.2%) converted from unfavorable to favorable. CTC changes stratified by type of PSMA-TRT are reported in the table. Conclusions: This is the largest analysis of CTC changes in patients who have received PSMA-TRT. In addition to PSA changes and other previously reported outcomes, even when low doses of radionuclide therapy as part of dose-escalation studies are included, the majority with detectable CTC counts have post-treatment CTC count decline. A significant portion of patients experience favorable CTC changes. [Table: see text]

Association of circulating tumor cells (CTC) with survival outcomes in patients (pts) with metastatic castration-sensitive prostate cancer (mCSPC) in a real-world cohort.

59 Background: In mCSPC, baseline CTC counts have been shown to correlate with PSA responses and progression free survival (PFS) in small studies in the context of androgen deprivation therapy (ADT) without modern intensification with docetaxel or novel hormonal therapy. Similar correlation of CTC count with PSA responses and PFS was recently reported from an ongoing phase 3 trial in mCSPC setting (SWOG1216) without reporting the association in the context of ADT intensification. Furthermore, none of these studies correlated CTCs with overall survival (OS). Herein we evaluated whether CTCs were associated with outcomes including OS in a real world mCPSC population treated with intensified as well as non-intensified ADT. Methods: Eligibility criteria: new mCSPC receiving ADT with or without intensification and enumeration of baseline CTCs by FDA cleared Cell Search CTC assay. The relationship between CTC counts (categorized as: 0, 1-4, and ≥5/7.5 ml) and both PFS and OS was assessed in the context of Cox proportional hazards models, both unadjusted and adjusted for age, Gleason, PSA at ADT initiation, de novo vs. non-de novo status, and ADT intensification vs. non-intensification therapy. Results: Overall 99 pts were identified. Baseline characteristics are summarized in Table. In unadjusted analyses, CTC counts of ≥5 as compared to 0 were strongly associated with inferior PFS (hazard ratio [HR] 3.38, 95% CI 1.85-6.18; p &lt; 0.001) and OS (HR 4.44 95% CI 1.63-12.10; p = 0.004). In multivariate analyses, CTC counts of ≥5 as compared to 0 continued to be associated with inferior PFS (HR 5.49, 95% CI 2.64-11.43; p &lt; 0.001) and OS (HR 4.00, 95% CI 1.31-12.23; p = 0.015). Within the ADT intensification subgroup also, high CTC counts were associated with poor PFS and OS. For PFS, the univariate HR for CTC ≥5 vs. 0 was 4.87 (95% CI 1.66-14.30; p = 0.004) and multivariate HR for CTC ≥5 vs. 0 was 7.43 (95% CI 1.92-28.82; p = 0.004). For OS, the univariate HR for CTC ≥5 vs. 0 was 15.88 (95% CI 1.93-130.58; p = 0.010) and multivariate HR for CTC ≥5 vs. 0 was 24.86 (95% CI 2.03-304.45; p = 0.012). Conclusions: To best of our knowledge this is the first study to show that high baseline CTC counts are strongly associated with inferior PFS as well as OS in pts with newly diagnosed mCSPC, even in those who received intensified ADT therapy. Identifying these pts at highest risk of progression and death can help with counselling and prognostication in clinics as well as design and enrollment in future clinical trials. [Table: see text]

CTC counts as a biomarker of prognosis and response in metastatic castration-resistant prostate cancer (mCRPC) from the CARD trial.

161 Background: In the prospective CARD trial (NCT02485691), cabazitaxel significantly improved radiographic progression-free survival (rPFS) and overall survival (OS) versus abiraterone or enzalutamide in patients with metastatic castration-resistant prostate cancer who had received docetaxel and progressed within 12 months with the alternative androgen-signaling-targeted inhibitor. Here we analyzed circulating tumor cell (CTC) counts as a biomarker of prognosis and response to therapy. Methods: Blood samples collected at screening (SC), Cycle 2 (C2) Day 1, and end of therapy were sent to Epic Sciences for CTC analysis. The association between CTC counts, defined as any cytokeratin-positive, CD45-negative cell, and clinical outcomes were pre-specified and analyzed using a multivariate Cox model for time-to-events (rPFS, OS) or a Χ2 test for categorical outcomes (objective tumor response). Results: Of the 255 patients randomized, 237 (92.9%) had evaluable samples at SC and 213 (83.5%) at C2. CTCs ≥ 1 were detected in 204 samples at SC (86%) and 178 samples at C2 (84%), with a median (interquartile range) count per mL of blood of 4.1 (1.3–14.2) and 5.2 (1.5–17.3), respectively. At baseline, higher CTC counts were associated with higher values of lactate dehydrogenase (P = 0.014), alkaline phosphatase (P &lt; 0.001), and prostate-specific antigen (P &lt; 0.001). High CTC counts, measured as a continuous variable at SC (all arms combined) were associated with a shorter OS (P = 0.03), but not rPFS (P = 0.7). However, favorable changes in absolute CTC count from SC to C2, all arms combined, were associated with longer rPFS (P = 0.03). Conversion from high (≥ 3) to low (&lt; 3) CTC count at C2 or maintenance of a low CTC count at C2 was associated with objective tumor response (P = 0.007). Analysis of the associations of CTC androgen receptor variant 7, chromosomal instability, heterogeneity status (Shannon Index), single-cell genotypes (e.g. retinoblastoma/phosphatase and tensin homolog copy-loss), and the presence of CTCs with small-cell/neuroendocrine morphology is ongoing. Conclusions: This preplanned analysis of the CARD trial confirms that baseline CTC counts, measured by Epic Sciences platform, are prognostic. Moreover, early favorable changes in CTC counts from baseline to C2 are associated with response to therapy. Funding: Sanofi. Clinical trial information: NCT02485691.

Abstract PS2-20: Prognostic value of baseline circulating tumor cells (CTCs) enumerations is for stage III and stage IV breast cancer

Abstract Introduction: Prognosis of metastatic breast cancer (MBC) is initially predicted by the cancer’s characteristics based on AJCC TNM system, including the size of the cancer tumor, invasion into nearby tissue, lymph nodes and other parts of the body beyond the breast. Although additional information including hormone-receptor status, HER2 status, and possibly Oncotype DX score contributed to improve prognostic evaluation, predicting clinical outcomes and treatment benefit for MBC is still a challenge in clinic because of the clinical and biologically heterogeneous condition. We recently reported that CTCs enumeration can classify MBC in two distinct prognostic groups independently of clinical and molecular characteristics (Crit Rev Oncol Hematol. 2019). Moreover, our group reported that CTCs is associated with HER2 expression in MBC which may indicate more aggressive tumor (2019 AACR #1919). Here we compared CTCs enumeration of Stage III and Stage IV, which would be helpful to evaluate the MBC metastasis capability and treatment in clinic. Methods: The study included 38 specimens prospectively collected under IRB-approved protocol from 38 patients with Stage III MBC, and 254 specimens from 254 patients with stage IV MBC who received standard systemic treatments based on disease subtypes at NMH (2016-2020). Duplicate whole blood samples (7.5ml/each) were collected in EDTA tubes from these patients who were longitudinally characterized for CTCs before therapy (baseline). CTCs enrichment and enumeration were performed in FDA approved semi-automated fluorescence CELLTRACKS ANALYZERII® System (Menarini) by using CELLSEARCH® CXC Kit contains antibodies targeting the Epithelial Cell Adhesion Molecule (EpCAM) antigen for capturing CTCs, anti-CK-PE which is specific for the intracellular protein cytokeratin in epithelial cells, DAPI for staining the cell nucleus, anti-CD45-APC is specific for leukocytes (2019 ASCO #1036). The CTCs were classified based on morphology and correct phenotype as CK+, DAPI+ and CD45-. Kruskal-Wallis test was used for statistics. Results: Patients were classified as Luminal, HER2 positive and TNBC disease subtypes in 46.6%, 46.7% and 6.7% respectively in Stage III patients, and 54%, 18% and 28% respectively in Stage IV patients. The patients at age above 50 were 26.% in Stage III group and 68% in Stage IV group respectively. IBC patients represented 61.5% and 33.5% of Stage III and Stage IV patients respectively. Metastasis in liver, lung and bone were diagnosed in 40.7%, 40.2% and 62.8% in Stage IV patients. CTC negative (&amp;lt;5 CTCs) and positive (≥5CTCs) patients were identified in 32/38 (84.22%, group 1) and 6/38 (15.78%, group 2) respectively in Stage III patients, and 149/254 (59%, Stage IV indolent ) and 105/254 (41%, Stage IV aggressive ) respectively in Stage IV patients. Patients in Group 1 have a significantly less recurrence probability than patients in Group 2 (p=0.015). Correspondingly, patients with Stage IV indolent also had significantly longer survival than patients with Stage aggressive disease (p=0.0021). When comparing the all population, Group 1 patients still have the highest survival probability (p=0.00057) within 47 months follow-up survey. More interesting, there was no any CTC-clusters found in all Stage III patients when there were 28 out of 254 stage IV patients (11.02%) were detected with CTC-clusters, who had the worst prognosis in compared to either Stage IV patients without CTC-clusters or Stage III patients (p=0.00035). Conclusions: In this study, we showed that enumeration of baseline CTC and CTC-clusters correlated with worse prognosis even the patients were pathologically diagnosed for the same stage, which provided an additional measure to predict disease recurrence after systemic therapies especially for Stage III MBC patients. Citation Format: Qiang Zhang, Zheng Cai, Lorenzo Gerratana, Paolo D'Amico, Andrew A. Davis, Saya Liz Jacob, Elena Vagia, Ami N Shah, Lisa Flaum, Youbin Zhang, Wenan Qiang, Firas Wehbe, Amir Behdad, William Gradishar, Leonidas C Platanias, Massimo Cristofanilli. Prognostic value of baseline circulating tumor cells (CTCs) enumerations is for stage III and stage IV breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS2-20.

Phase II/III study of SM-88 in patients with metastatic pancreatic cancer.

437 Background: SM-88 (racemetyrosine, Tyme Inc) is a dysfunctional tyrosine derivative used with MPS (methoxsalen 10mg, phenytoin 50mg and sirolimus 0.5mg). SM-88 was well tolerated with improvement in survival among select heavily pretreated PDAC patients who achieved stable disease (HR 0.08, p = 0.02) (Noel et al. Annal Oncol 2019). Circulating tumor cells (CTCs) were prognostic in identifying a PDAC subgroup that may be more likely to benefit from SM-88. Preliminary radiomic analysis of the largest metastases at baseline correlated with baseline CTCs (Ocean et al, Annal Oncol 2019). Here we describe the subsequent randomized portion of the trial in third-line patients only, of SM-88 vs physician/patient choice chemotherapy, to evaluate the potential role of SM-88 in metastatic PDAC through analysis of CTCs and passively acquired biometrics data from a wearable device. Methods:Prospective open-label RCT (Tyme 88 Panc Part 2, NCT03512756) after 2 prior lines for metastatic PDAC. A cell adhesion matrix (CAM) was used to enrich solitary CTCs and cells in clusters floating in the medium after 24 hour culture. Isolated CTCs were collected each cycle on day 1, isolated, and enumerated by flow cytometry using the epithelial cell surface marker Epi+ and cellular uptake of green fluorescent labeled CAM (GCAM+). Results:As of Sept 15, 67 subjects were consented. Randomized and evaluable subjects (n=38) included: mean age 65y (45-86); BMI 24.6 (18.8-38.7); female 39.5%; White 76.3%. Of treated subjects 65.8% (25/38) had 166 AEs, with 25.7% (26/101) being at least possibly SM-88-related, with 1 Grade 3. Four CTC subpopulations defined by GCAM, Epi+ and cluster status, were enumerated and correlated to each other (r=0.03-0.71). At least one CTC subpopulation was detected at baseline (mean 33.8 cells/2mL) in all subjects (n=27). The longest metastatic lesion diameter at baseline correlated with baseline CTCs (r=0.55 for Epi+ cluster; r=0.52 for GCAM+ cluster). CTCs were successfully separated and enumerated at each cycle for correlation with survival, response and other parameters. The median baseline daily step count during the first two weeks on treatment was 3993.8 (IQR: 2745.6 - 5078) for those alive vs. 689.3 (IQR: 630.0-2083.6) among deaths in evaluable subjects (p = NS). Passively acquired mean heart rate during week 3 on trial was 89.3 (SD 10.5) among those who died vs. 78.0 (SD 9.2) among those living; medians are 87.0 for deaths vs. 79.2 for alive (p= NS). Conclusions: In a preliminary exploratory analysis, passively acquired biometrics from a wearable device can be collected for correlation with other clinical outcomes. CTC collection and enumeration is also feasible for correlation with traditional trial outcomes. Given that the longest lesion diameter is correlated with CTCs at baseline, additional radiologic feature analysis (eg radiomics) may be important predictor of CTCs. SM-88 was well tolerated with no treatment-related Grade 4 or 5 events. Clinical trial information: NCT03512756.

The role of circulating tumor cells and K-ras mutations in patients with locally advanced rectal cancer: a prospective study.

Rectal cancer is a common malignancy with a relatively poor prognosis. We assessed the possible prognostic and predictive role(s) of circulating tumor cells (CTCs) and K-ras mutations in locally advanced rectal carcinoma (LARC) patients. CTCs number and K-ras mutation status were assessed in the Peripheral blood and tumor tissue samples of 60 patients with LARC compared to control group (normal rectal mucosa). Data were correlated to relevant clinico-pathological features, response to treatment, disease free (DFS) and overall survival (OS) rates. K-ras mutations were present in 24/60 (40%) patients. Baseline CTCs (< 5 cells/7ml blood) were detected in 23/60 (38.3%) patients, and 37 (61.7%) had baseline CTCs (≥ 5 cells/7ml) blood (P = 0.071). Serial sampling showed a decrease in CTCs levels in 40 (66.7%) patients and increase in 20 (33.3%) patients (P = 0.01). Patients with K-ras mutations had a significantly poor response to treatment, with reduced DFS and OS rates (P = 0.001, 0.004, and 0.001; respectively). Similarly, decreased CTCs levels during treatment associated significantly with better pathological responses (P = 0.003). Multivariate analysis demonstrated that K-ras mutation and baseline CTCs are independent prognostic factors for DFS (P = 0.014 and 0.045; respectively) and OS (P = 0.002 and 0.045; respectively). The presence of mutant K-ras and baseline CTCs ≥ 5 cells associated significantly with poor pathological response, shorter DFS and OS rates compared to those with either K-ras mutation or CTCs ≥ 5 cells only (P = 0.014, 0.005 and 0.001, respectively). K-ras mutations, baseline and serial CTCs changes represent good prognostic and predictive factors for LARC patients.