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Purpose:We previously demonstrated that high levels of circulating methylated DNA are associated with subsequent disease progression in women with metastatic breast cancer (MBC). In this study, we evaluated the clinical utility of a novel liquid biopsy-breast cancer methylation (LBx-BCM) prototype assay using the GeneXpert cartridge system for early assessment of disease progression in MBC.Experimental Design:The 9-marker LBx-BCM prototype assay was evaluated in TBCRC 005, a prospective biomarker study, using plasma collected at baseline, week 4, and week 8 from 144 patients with MBC.Results:At week 4, patients with MBC with high cumulative methylation (CM) had a significantly shorter median PFS (2.88 months vs. 6.60 months, P = 0.001) and OS (14.52 months vs. 22.44 months, P = 0.005) compared with those with low CM. In a multivariable model, high versus low CM was also associated with shorter PFS (HR, 1.90; 95% CI, 1.20–3.01; P = 0.006). Change in CM from baseline to week 4 (OR, 4.60; 95% CI, 1.77–11.93; P = 0.002) and high levels of CM at week 4 (OR, 2.78; 95% CI, 1.29–5.99; P = 0.009) were associated with progressive disease at the time of first restaging. A robust risk model based on week 4 circulating CM levels was developed to predict disease progression as early as 3 months after initiating a new treatment.Conclusions:The automated LBx-BCM prototype assay is a promising clinical tool for detecting disease progression a month after initiating treatment in women with MBC undergoing routine care. The next step is to validate its clinical utility for specific treatments.

The pharmacokinetics (PK) and safety of ofatumumab and bendamustine alone and in combination were evaluated in patients with treatment-naive or relapsed indolent B-cell non-Hodgkin lymphoma (iNHL). Patients were randomly assigned to ofatumumab and bendamustine or ofatumumab alone. Ofatumumab PK concentration profiles and parameters were similar, alone or in combination with bendamustine. A decrease of 14% in the maximum observed plasma concentration (C

Background: Clinical trials reported 25-30% pathologic complete response (pCR) rates in HER2+ breast cancer (BC) patients treated with anti-HER2 therapies without chemotherapy. We hypothesize that a multiparameter classifier can identify patients with HER2 “addicted” tumors who may benefit from a chemotherapy-sparing strategy. Patients and Methods: Baseline HER2+ BC specimens from TBCRC023 and PAMELA trials of neoadjuvant lapatinib+trastuzumab (plus endocrine therapy in ER+ tumors) were used. HER2 protein and gene amplification (ratio), HER2-enriched (HER2-E), and PIK3CA mutation status were assessed by dual gene protein assay (GPA), research-based PAM50, and targeted DNA-sequencing. GPA cutoffs and classifier of response were constructed in TBCRC023 using a decision tree algorithm, then validated in PAMELA. Results: In TBCRC023, 72 BCs had GPA, PAM50, and sequencing data, of which 15 had pCR. Recursive partitioning identified cutoffs of HER2 ratio≥4.6 and %3+ IHC-staining≥97.5%. With PAM50 and sequencing data, the model added HER2-E and PIK3CA wild-type (wt). For clinical implementation, the classifier was locked as HER2 ratio≥4.5 and %3+ IHC-staining≥90% and PIK3CA-wt and HER2-E, yielding 55% and 94% positive (PPV) and negative (NPV) predictive values, respectively. Independent validation using 44 PAMELA cases with all three biomarkers yielded 47% PPV and 82% NPV. Importantly, our classifier’s high NPV signifies its strength in accurately identifying patients who may not be good candidates for treatment de-escalation. Conclusions: Our multiparameter classifier differentially identifies patients who may benefit from HER2-targeted therapy alone from those who need chemotherapy and predicts pCR to anti-HER2 therapy alone comparable to chemotherapy plus dual anti-HER2 therapy in unselected patients.

PDF file - 209KB, Supplementary Figure S2. Niclosamide effect on Wnt/beta-catenin signaling. Activity of TCF/LEF plasmid reporter in the TOPflash assay was evaluated on adherent 2LMP, SUM159, HCC143 and HCC1187 cells. Cell lines were treated with 1 microM niclosamide for 24 hours (SUM159, HCC1143 and HCC1187) or with 0.25 microM niclosamide (2LMP). Each cell line was also treated with Wnt3A ligand (5 ng/mL) alone and in combination with niclosamide. The experiment was performed in triplicate. The bars represent means plus-minus SE. Treatment was compared to control or Wnt3A. Niclosamide vs. control (*P < 0.05), niclosamide vs. control (**P < 0.01), niclosamide vs. Wnt3A (#P < 0.05), niclosamide + Wnt3A vs. Wnt3A (##P < 0.01).

PDF file - 96KB, Supplementary Figure S4. Wnt/beta-catenin signaling proteins in LRP6 KD cells. Western blot for expression of Wnt/beta-catenin signaling proteins in adherent 2LMP shRNA LRP6 knockdown cells compared to shRNA control cells.

PDF file - 59KB, Supplementary Figure S5. In vivo toxicity study. Athymic nude mice were treated with niclosamide (20 mg/kg), TRA-8 (200 ng) or the combination for two weeks. Mice were weighed twice a week for two weeks.

Basal-like breast cancers (BLBC) are poorly differentiated and display aggressive clinical behavior. These tumors become resistant to cytotoxic agents, and tumor relapse has been attributed to the presence of cancer stem cells (CSC). One of the pathways involved in CSC regulation is the Wnt/β-catenin signaling pathway. LRP6, a Wnt ligand receptor, is one of the critical elements of this pathway and could potentially be an excellent therapeutic target. Niclosamide has been shown to inhibit the Wnt/β-catenin signaling pathway by causing degradation of LRP6. TRA-8, a monoclonal antibody specific to TRAIL death receptor 5, is cytotoxic to BLBC cell lines and their CSC-enriched populations. The goal of this study was to examine whether niclosamide is cytotoxic to BLBCs, specifically the CSC population, and if in combination with TRA-8 could produce increased cytotoxicity. Aldehyde dehydrogenase (ALDH) is a known marker of CSCs. By testing BLBC cells for ALDH expression by flow cytometry, we were able to isolate a nonadherent population of cells that have high ALDH expression. Niclosamide showed cytotoxicity against these nonadherent ALDH-expressing cells in addition to adherent cells from four BLBC cell lines: 2LMP, SUM159, HCC1187, and HCC1143. Niclosamide treatment produced reduced levels of LRP6 and β-catenin, which is a downstream Wnt/β-catenin signaling protein. The combination of TRA-8 and niclosamide produced additive cytotoxicity and a reduction in Wnt/β-catenin activity. Niclosamide in combination with TRA-8 suppressed growth of 2LMP orthotopic tumor xenografts. These results suggest that niclosamide or congeners of this agent may be useful for the treatment of BLBC. Mol Cancer Ther; 13(4); 800–11. ©2014 AACR.

Supplementary Methods

PDF file - 128KB, Supplementary Figure S1. Sensitivity of MF10A mammary epithelial cells to niclosamide and TRA-8 mediated cytotoxicity. (A) Attached MCF10A cell line was treated for 48 hours with niclosamide (0.08, 0.1, 0.25 and 0.5 microM) and analyzed for cell viability using ATPlite assay.

HER2 is a transmembrane tyrosine kinase receptor that mediates cell growth, differentiation, and survival. HER2 is overexpressed in approximately 20% of breast cancers and in subsets of gastric, colorectal, and esophageal cancers. Both antibody and small-molecule drugs that target HER2 and block its tyrosine kinase activity are effective in treating HER2-driven cancers. In this article, we describe the preclinical properties of tucatinib, an orally available, reversible HER2-targeted small-molecule tyrosine kinase inhibitor. In both biochemical and cell signaling experiments, tucatinib inhibits HER2 kinase activity with single-digit nanomolar potency and provides exceptional selectivity for HER2 compared with the related receptor tyrosine kinase EGFR, with a >1,000-fold enhancement in potency for HER2 in cell signaling assays. Tucatinib potently inhibits signal transduction downstream of HER2 and HER3 through the MAPK and PI3K/AKT pathways and is selectively cytotoxic in HER2-amplified breast cancer cell lines in vitro. In vivo, tucatinib is active in multiple HER2+ tumor models as a single agent and shows enhanced antitumor activity in combination with trastuzumab or docetaxel, resulting in improved rates of partial and complete tumor regression. These preclinical data, taken together with the phase-I tucatinib clinical trial results demonstrating preliminary safety and activity, establish the unique pharmacologic properties of tucatinib and underscore the rationale for investigating its utility in HER2+ cancers.

PDF file - 89KB, Supplementary Table S1. Sensitivity of BLBC cell lines to niclosamide mediated cytotoxicity. SUM159, HCC1187, HCC1143 and 2LMP BLBC NAAE and adherent cell lines were treated with niclosamide for 48 hours and analyzed for viability using the ATPlite assay. Individual experiments were assayed in quadruplicate and values represent the mean and SD of three independent experiments. SUM159 P < 0.05, HCC1187 P < 0.05, HCC1143 P < 0.0005, 2LMP P > 0.05.

PDF file - 72KB, Supplementary Table S2. Immunohistochemistry of mouse tumors treated for two weeks with vehicle control, niclosamide (20 mg/kg), TRA-8 (200 micro-g) or the combination of both agents. Tumors were stained and analyzed (H-score) for p-STAT3, cytosolic beta-catenin, nuclear beta-catenin and cleaved caspase 3. The values represent an average of three tumors per group (plus-minus SE).

Supplementary Figure S1: Comparative potency of tucatinib, neratinib, and lapatinib in cellular HER2 and EGFR phosphorylation assays; Supplementary Figure S2: Effect of tucatinib, trastuzumab, or combination treatment on mouse body weight in HER2+ PDX models; Supplementary Table S1: Tucatinib activity correlates with HER2 surface receptor density; Supplementary Table S2. HER2 copy number variation in PDX models

PDF file - 99KB, Supplementary Figure S3. ATPlite viability of cells treated with niclosamide. 2LMP, SUM159, HCC1143 and HCC1187 adherent (A) or NAAE (B) cells were treated with niclosamide for 24 hours and analyzed for viability using ATPlite assay. Individual experiments were assayed in triplicate and values represent the mean plus-minus SE.

Study Schema

Purpose:We previously demonstrated that high levels of circulating methylated DNA are associated with subsequent disease progression in women with metastatic breast cancer (MBC). In this study, we evaluated the clinical utility of a novel liquid biopsy-breast cancer methylation (LBx-BCM) prototype assay using the GeneXpert cartridge system for early assessment of disease progression in MBC.Experimental Design:The 9-marker LBx-BCM prototype assay was evaluated in TBCRC 005, a prospective biomarker study, using plasma collected at baseline, week 4, and week 8 from 144 patients with MBC.Results:At week 4, patients with MBC with high cumulative methylation (CM) had a significantly shorter median PFS (2.88 months vs. 6.60 months, P = 0.001) and OS (14.52 months vs. 22.44 months, P = 0.005) compared with those with low CM. In a multivariable model, high versus low CM was also associated with shorter PFS (HR, 1.90; 95% CI, 1.20–3.01; P = 0.006). Change in CM from baseline to week 4 (OR, 4.60; 95% CI, 1.77–11.93; P = 0.002) and high levels of CM at week 4 (OR, 2.78; 95% CI, 1.29–5.99; P = 0.009) were associated with progressive disease at the time of first restaging. A robust risk model based on week 4 circulating CM levels was developed to predict disease progression as early as 3 months after initiating a new treatment.Conclusions:The automated LBx-BCM prototype assay is a promising clinical tool for detecting disease progression a month after initiating treatment in women with MBC undergoing routine care. The next step is to validate its clinical utility for specific treatments.

Association between week 4 CM and disease status at first restaging after adjusting for CEA.

Everolimus and Cisplatin Modulation of p73 and p63 Expression In Vivo.

Purpose:To evaluate safety and efficacy outcomes for subjects on the ECHELON-1 study treated in North America (NA).Patients and Methods:ECHELON-1 is a global, open-label, randomized phase III study comparing doxorubicin, vinblastine, and dacarbazine in combination with brentuximab vedotin (A+AVD) versus ABVD (AVD + bleomycin) as first-line therapy in subjects with stage III or IV classical Hodgkin lymphoma (cHL; NCT01712490). Subjects were randomized 1:1 to receive A+AVD or ABVD intravenously on days 1 and 15 of each 28-day cycle for up to 6 cycles.Results:The NA subgroup consisted of 497 subjects in the A+AVD (n = 250) and ABVD (n = 247) arms. Similar to the primary analysis based on the intent-to-treat population, the primary endpoint [modified progression-free survival (PFS) per independent review] demonstrated an improvement among subjects who received A+AVD compared with ABVD (HR = 0.60; P = 0.012). For PFS, the risk of progression or death was also reduced (HR = 0.50; P = 0.002). Subsequent anticancer therapies were lower in the A+AVD arm. Grade 3 or 4 adverse events (AEs) were more common, but there were fewer study discontinuations due to AEs in the A+AVD arm as compared with ABVD. Noted differences between arms included higher rates of febrile neutropenia (20% vs. 9%) and peripheral neuropathy (80% vs. 56%), but lower rates of pulmonary toxicity (3% vs. 10%) in subjects treated with A+AVD versus ABVD.Conclusions:The efficacy benefit and manageable toxicity profile observed in the NA subgroup of ECHELON-1 support A+AVD as a frontline treatment option for patients with stage III or IV cHL.

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Everolimus Inhibited mTOR Activity as Assessed by pS6 Expression without Significant Correlation with Clinical Activity

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Purpose: Tigatuzumab (TIG), an agonistic anti-DR5 antibody, triggers apoptosis in DR5+ human tumor cells without crosslinking. TIG has strong in vitro/in vivo activity against basal-like breast cancer cells enhanced by chemotherapy agents. This study evaluates activity of TIG and chemotherapy in patients with metastatic triple-negative breast cancer (TNBC).Experimental Design: Randomized 2:1 phase II trial of albumin-bound paclitaxel (nab-PAC) ± TIG in patients with TNBC stratified by prior chemotherapy. Patients received nab-PAC weekly × 3 ± TIG every other week, every 28 days. Primary objective was within-arm objective response rate (ORR). Secondary objectives were safety, progression-free survival (PFS), clinical benefit, and TIG immunogenicity. Metastatic research biopsies were required.Results: Among 64 patients (60 treated; TIG/nab-PAC n = 39 and nab-PAC n = 21), there were 3 complete remissions (CR), 8 partial remissions (PR; 1 almost CR), 11 stable diseases (SD), and 17 progressive diseases (PD) in the TIG/nab-PAC arm (ORR, 28%), and no CRs, 8 PRs, 4 SDs, and 9 PDs in the nab-PAC arm (ORR, 38%). There was a numerical increase in CRs and several patients had prolonged PFS (1,025+, 781, 672, 460, 334) in the TIG/nab-PAC arm. Grade 3 toxicities were 28% and 29%, respectively, with no grade 4–5. Exploratory analysis suggests an association of ROCK1 gene pathway activation with efficacy in the TIG/nab-PAC arm.Conclusions: ORR and PFS were similar in both. Preclinical activity of TIG in basal-like breast cancer and prolonged PFS in few patients in the combination arm support further investigation of anti-DR5 agents. ROCK pathway activation merits further evaluation. Clin Cancer Res; 21(12); 2722–9. ©2015 AACR.See related article by Paoletti et al., p. 2771

Purpose: Because of inherent disease heterogeneity, targeted therapies have eluded triple-negative breast cancer (TNBC), and biomarkers predictive of treatment response have not yet been identified. This study was designed to determine whether the mTOR inhibitor everolimus with cisplatin and paclitaxel would provide synergistic antitumor effects in TNBC.Methods: Patients with stage II/III TNBC were enrolled in a randomized phase II trial of preoperative weekly cisplatin, paclitaxel and daily everolimus or placebo for 12 weeks, until definitive surgery. Tumor specimens were obtained at baseline, cycle 1, and surgery. Primary endpoint was pathologic complete response (pCR); secondary endpoints included clinical responses, breast conservation rate, safety, and discovery of molecular features associated with outcome.Results: Between 2009 and 2013, 145 patients were accrued; 36% of patients in the everolimus arm and 49% of patients in the placebo arm achieved pCR; in each arm, 50% of patients achieved complete responses by imaging. Higher rates of neutropenia, mucositis, and transaminase elevation were seen with everolimus. Clinical response to therapy and long-term outcome correlated with increased frequency of DNA damage response (DDR) gene mutations, Basal-like1 and Mesenchymal TNBC-subtypes, AR-negative status, and high Ki67, but not with tumor-infiltrating lymphocytes.Conclusions: The paclitaxel/cisplatin combination was well tolerated and active, but addition of everolimus was associated with more adverse events without improvement in pCR or clinical response. However, discoveries made from correlative studies could lead to predictive TNBC biomarkers that may impact clinical decision-making and provide new avenues for mechanistic exploration that could lead to clinical utility. Clin Cancer Res; 23(15); 4035–45. ©2017 AACR.

Seven supplemental tables referenced in the body of the manuscript. Supplemental Table 1: Progression free survival outcomes by region Supplemental Table 2 Summary of response rates to frontline therapy in North America Supplemental Table 3 Summary of dose modification for brentuximab vedotin and bleomycin in North America, Europe, and Asia Supplemental Table 4 Summary of TEAEs Supplemental Table 5 Most common (>15%) TEAEs in either treatment arm Supplemental Table 6 Rates of peripheral neuropathy in North America, Europe, and Asia Supplemental Table 7 CTCAE Definition and staging of peripheral neuropathy and brentuximab vedotin dose modification guidance

Presence of Intratumoral and Stromal Tumor-Infiltrating Lymphocytes (TILs) did not Correlate with Pathological Responses

Alterations in the PI3K/AKT/mTOR Pathway are not Enriched in Clinical Responders

SUPPLEMENTARY APPENDIX TABLES

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Purpose:Resistance to treatment with endocrine therapy in patients with HR+, HER2− advanced breast cancer (ABC) is common and dual inhibition of CDK4/6 and PI3K pathways may delay the development of resistance. This phase Ib trial evaluates the safety and tolerability of triple and double regimens containing the CDK4/6 inhibitor ribociclib.Patients and Methods:In this open-label, multicenter, phase Ib study, 70 postmenopausal women with HR+, HER2− ABC were enrolled into one of four treatment combinations: ribociclib (once daily, 3 weeks on, 1 week off) plus fulvestrant; ribociclib (continuous dosing) plus fulvestrant; ribociclib plus alpelisib plus fulvestrant; or ribociclib plus buparlisib plus fulvestrant.Results:The recommended phase II dose (RP2D) of ribociclib was confirmed to be 600 mg (3 weeks on, 1 week off) and 400 mg (continuous dosing) plus fulvestrant 500 mg. For the triple combination with buparlisib, the RP2D was ribociclib 400 mg plus buparlisib 30 mg plus fulvestrant 500 mg. Enrollment for the triple combinations was stopped due to unexpected toxicity. No RP2D was determined for the alpelisib combination. The safety profiles of the ribociclib plus fulvestrant combinations were consistent with those in previous studies. There was no marked difference in ribociclib exposure in the presence of triple-combination partners. The highest overall response rate was seen in the buparlisib triple combination (25.0%; 95% confidence interval, 9.8–46.7).Conclusions:Ribociclib plus fulvestrant demonstrated safety in the treatment of patients with HR+, HER2− ABC. Triple combinations with alpelisib or buparlisib plus fulvestrant are not recommended for phase II investigation.See related commentary by Clark et al., p. 371

Purpose: The antibody–drug conjugate (ADC) brentuximab vedotin comprises a CD30-directed antibody covalently attached to the potent antimicrotubule agent monomethyl auristatin E (MMAE) via a protease-cleavable linker. This study explored the safety, maximum-tolerated dose (MTD), and activity of weekly dosing of brentuximab vedotin in patients with relapsed or refractory CD30-positive hematologic malignancies.Experimental Design: In this phase I dose-escalation study, brentuximab vedotin was administered intravenously on Days 1, 8, and 15, of each 28-day cycle at doses ranging from 0.4 to 1.4 mg/kg. Forty-four patients were enrolled: 38 with Hodgkin lymphoma, five with systemic anaplastic large cell lymphoma, and one with peripheral T-cell lymphoma not otherwise specified. Doses were escalated in increments of 0.2 mg/kg until dose-limiting toxicity (DLT) was observed. Patients were monitored for antitherapeutic antibodies and pharmacokinetic parameters. Antitumor assessments were carried out every two cycles.Results: The MTD was 1.2 mg/kg. The most common adverse events were peripheral sensory neuropathy, fatigue, nausea, diarrhea, arthralgia, and pyrexia; and the majority of events were mild to moderate in severity. Tumor regression occurred in 85% of patients and the overall objective response rate was 59% (n = 24), with 34% (n = 14) complete remissions. The median duration of response was not reached at a median follow-up of 45 weeks on study.Conclusions: Weekly administration of brentuximab vedotin resulted in tumor regression and durable remissions in patients with CD30-positive malignancies. This ADC was associated with manageable toxicity, including peripheral neuropathy. Further study in CD30-positive malignancies is warranted. Clin Cancer Res; 18(1); 248–55. ©2011 AACR.

Purpose: AME-133v is a humanized monoclonal antibody engineered to have increased affinity to CD20 and mediate antibody-dependent cell-mediated cytotoxicity (ADCC) better than rituximab. Safety, pharmacokinetics, and efficacy were assessed in a phase 1/2 trial in patients with previously treated follicular lymphoma (FL).Patients and Methods: AME-133v was characterized in vitro by ADCC and cell binding assays. A phase 1 study was conducted in which 23 previously treated patients with FL were assigned sequentially to one of five dose-escalation cohorts of AME-133v at 2, 7.5, 30, 100, or 375 mg/m2 weekly × 4 doses.Results: AME-133v showed a 13- to 20-fold greater binding affinity for CD20 and was 5- to 7-fold more potent than rituximab in ADCC assays. Cell binding assays showed AME-133v and rituximab competed for an overlapping epitope on the CD20 antigen, and AME-133v inhibited binding of biotinylated rituximab to CD20 in a concentration-dependent manner. AME-133v was well tolerated by patients and common related adverse events included chills and fatigue. One patient experienced a dose-limiting toxicity of neutropenia. AME-133v showed nonlinear pharmocokinetics with properties similar to rituximab. Selective reduction of B cells during and after AME-133v treatment was shown by flow cytometry of peripheral blood. A partial or complete response was observed in 5 of 23 (22%) patients and the median progression-free survival was 25.4 weeks.Conclusions: AME-133v was safe and well tolerated at the doses tested. AME-133v showed encouraging results as an anti-CD20 therapy in heavily pretreated FL patients with the less favorable FcγRIIIa F-carrier genotype. Clin Cancer Res; 18(5); 1395–403. ©2012 AACR.

Breast Cancer Gene Panel (GeneWiz)

Purpose: Circulating tumor cells (CTC) are prognostic in metastatic breast cancer (MBC). We tested whether EpCAM-based capture system (CellSearch) is effective in patients with triple-negative (TN) MBC, and whether CTC apoptosis and clustering enhances the prognostic role of CTC.Experimental Design: CTC enumeration and apoptosis were determined using the CXC CellSearch kit at baseline and days 15 and 29 in blood drawn from TN MBC patients who participated in a prospective randomized phase II trial of nanoparticle albumin-bound paclitaxel (nab-paclitaxel) with or without tigatuzumab. Association between levels of CTC and patient outcomes was assessed using logistic regression, Kaplan–Meier curves, and Cox proportional hazards modeling.Results: Nineteen of 52 (36.5%), 14 of 52 (26.9%), and 13 of 49 (26.5%) patients who were evaluable had elevated CTC (≥5 CTC/7.5 mL whole blood) at baseline and at days 15 and 29, respectively. Patients with elevated versus not elevated CTC at each time point had worse progression-free survival (PFS; P = 0.005, 0.0003, 0.0002, respectively). The odds of clinical benefit response for those who had elevated versus low CTC at baseline and days 15 and 29 were 0.25 (95% CI: 0.08–0.84; P = 0.024), 0.19 (95% CI: 0.05–0.17; P = 0.014), and 0.06 (95% CI: 0.01–0.33; P = 0.001), respectively. There was no apparent prognostic effect comparing CTC apoptosis versus non-apoptosis. Presence of CTC cluster at day 15 and day 29 was associated with shorter PFS.Conclusions: CTC were detected using CellSearch assay in approximately one-third of TN MBC patients. Elevated CTC at baseline and days 15 and 29 were prognostic, and reductions in CTC levels reflected response. Clin Cancer Res; 21(12); 2771–9. ©2015 AACR.See related article by Forero-Torres et al., p. 2722

Supplementary Figure 1-5. Supplementary Figure 1. Example of cluster image using the CellSearch® system. Supplementary Figure 2. REMARK diagram of the 19 patients who had elevated CTC (Group B/C) at baseline that were used to study "CTC-response". Supplementary Figure 3. PFS for TIG-treated patients versus the control group according to baseline CTC levels: CTC

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Supplementary legend