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Malignancies of the peritoneal cavity are associated with a dismal prognosis. Systemic chemotherapy is the gold standard for patients with unresectable peritoneal disease, but its intraperitoneal effect is hampered by the peritoneal-plasma barrier. Intraperitoneal chemotherapy, which is administered repeatedly into the peritoneal cavity through a peritoneal implanted port, could provide a novel treatment modality for this patient population. This review provides a systematic overview of intraperitoneal used drugs, the performed clinical studies so far, and the complications of the peritoneal implemental ports. Several anticancer drugs have been studied for intraperitoneal application, with the taxanes paclitaxel and docetaxel as the most commonly used drug. Repeated intraperitoneal chemotherapy, mostly in combination with systemic chemotherapy, has shown promising results in Phase I and Phase II studies for several tumor types, such as gastric cancer, ovarian cancer, colorectal cancer, and pancreatic cancer. Two Phase III studies for intraperitoneal chemotherapy in gastric cancer have been performed so far, but the results regarding the superiority over standard systemic chemotherapy alone, are contradictory. Pressurized intraperitoneal administration, known as PIPAC, is an alternative way of administering intraperitoneal chemotherapy, and the first prospective studies have shown a tolerable safety profile. Although intraperitoneal chemotherapy might be a standard treatment option for patients with unresectable peritoneal disease, more Phase II and Phase III studies focusing on tolerability profiles, survival rates, and quality of life are warranted in order to establish optimal treatment schedules and to establish a potential role for intraperitoneal chemotherapy in the approach to unresectable peritoneal disease.

Multiple prognostic biomarkers, including circulating tumor cell (CTC) counts, exist in metastatic castration-resistant prostate cancer (mCRPC) patients, but none of them have been implemented into daily clinical care. The modified fast aneuploidy screening test-sequencing system (mFast-SeqS), which yields a genome-wide aneuploidy score, is able to reflect the fraction of cell-free tumor DNA (ctDNA) within cell-free DNA (cfDNA) and may be a promising biomarker in mCRPC. In this study, we investigated the prognostic value of dichotomized aneuploidy scores (

Clinicopathological characteristics, CTC and CEC counts, and primary tumor EpCAM and MCAM staining specified per patient.

Legends to supplementary table 1 and figure 1

Circulating tumor cells (CTC) are detected by the CellSearch System in 20% to 25% of patients with primary breast cancer (pBC). To improve CTC detection, we investigated melanoma cell adhesion molecule (MCAM) as enrichment marker next to epithelial cell adhesion molecule (EpCAM) and tested the clinical relevance of MCAM-positive CTCs in patients with HER2-negative stage II/III pBC starting neoadjuvant chemotherapy (NAC) in the NEOZOTAC trial. Using the CellSearch System, EpCAM-positive and MCAM-positive CTCs were separately enriched from 7.5 mL blood, at baseline and after the first NAC cycle. Circulating endothelial cells (CEC) were measured using flow cytometry. Primary objective was to improve the CTC detection rate to ≥40% combining EpCAM/MCAM. Correlations of CTC and CEC counts and pathologic complete response (pCR) were also explored. At baseline, we detected EpCAM-positive and MCAM-positive CTCs in 12 of 68 (18%) and 8 of 68 (12%) patients, respectively. After one cycle, this was 7 of 44 (16%) and 7 of 44 (16%) patients, respectively. The detection rate improved from 18% at baseline and 16% after one cycle with EpCAM to 25% (P = 0.08) and 30% (P = 0.02), respectively, with EpCAM/MCAM. No patients with MCAM-positive CTCs versus 23% of patients without MCAM-positive CTCs at baseline achieved pCR (P = 0.13). EpCAM-positive CTCs and CEC counts were not correlated to pCR. Combined EpCAM/MCAM CellSearch enrichment thus increased the CTC detection rate in stage II/III pBC. We found no associations of CTC and CEC counts with pCR to NAC. The clinical relevance of MCAM-positive CTCs deserves further study. Mol Cancer Ther; 14(3); 821–7. ©2014 AACR.

Examples of EpCAM and MCAM staining in primary tumor tissues from core needle biopsies taken before start of neoadjuvant chemotherapy

Supplementary Figures S1-S2 from mRNA and microRNA Expression Profiles in Circulating Tumor Cells and Primary Tumors of Metastatic Breast Cancer Patients

Supplementary Tables S1-S2 from mRNA and microRNA Expression Profiles in Circulating Tumor Cells and Primary Tumors of Metastatic Breast Cancer Patients

Purpose: Molecular characterization of circulating tumor cells (CTC) holds great promise. Unfortunately, routinely isolated CTC fractions currently still contain contaminating leukocytes, which makes CTC-specific molecular characterization extremely challenging. In this study, we determined mRNA and microRNA (miRNA) expression of potentially CTC-specific genes that are considered to be clinically relevant in breast cancer.Experimental Design: CTCs were isolated with the epithelial cell adhesion molecule–based CellSearch Profile Kit. Selected genes were measured by real-time reverse transcriptase PCR in CTCs of 50 metastatic breast cancer patients collected before starting first-line systemic therapy in blood from 53 healthy blood donors (HBD) and in primary tumors of 8 of the patients. The molecular profiles were associated with CTC counts and clinical parameters and compared with the profiles generated from the corresponding primary tumors.Results: We identified 55 mRNAs and 10 miRNAs more abundantly expressed in samples from 32 patients with at least 5 CTCs in 7.5 mL of blood compared with samples from 9 patients without detectable CTCs and HBDs. Clustering analysis resulted in 4 different patient clusters characterized by 5 distinct gene clusters. Twice the number of patients from cluster 2 to 4 had developed both visceral and nonvisceral metastases. Comparing transcript levels in CTCs with those measured in corresponding primary tumors showed clinically relevant discrepancies in estrogen receptor and HER2 levels.Conclusions: Our study shows that molecular profiling of low numbers of CTCs in a high background of leukocytes is feasible and shows promise for further studies on the clinical relevance of molecular characterization of CTCs. Clin Cancer Res; 17(11); 3600–18. ©2011 AACR.

Background Patients with unresectable intrahepatic cholangiocarcinoma (iCCA) have poor survival. This systematic review describes the survival outcomes of hepatic arterial infusion pump (HAIP) chemotherapy with floxuridine for patients with unresectable iCCA. Patients and Methods A literature search was conducted using the electronic databases PubMed, Medline (Ovid), Embase, Web of Science, Google Scholar, and Cochrane to find studies that reported data on the survival of patients with unresectable iCCA treated with HAIP chemotherapy using floxuridine. The quality of the studies was assessed using the Newcastle–Ottawa quality assessment Scale (NOS). Overall survival (OS) was the primary outcome measure, and progression-free survival (PFS), response rates, resection rates, and toxicity were defined as secondary outcome measures. Results After removing duplicates, 661 publications were assessed, of which nine studies, representing a total of 478 patients, met the inclusion criteria. Three out of nine studies were phase II clinical trials, one study was a prospective dose-escalation study, and the remaining five studies were retrospective cohort studies. After accounting for overlapping cohorts, 154 unique patients were included for pooled analysis. The weighted median OS of patients with unresectable iCCA treated with HAIP chemotherapy with floxuridine was 29.0 months (range 25.0–39 months). The pooled 1-, 2-, 3-, and 5-year OS were 86.4, 55.5, 39.5, and 9.7%, respectively. Conclusion HAIP chemotherapy with floxuridine for patients with unresectable iCCA was associated with a 3-year OS of 39.5%, which is favorable compared with systemic chemotherapy for which no 3-year survivors were reported in the Advanced Biliary Cancer (ABC) trials.

Simple Summary The treatment outcomes in patients with head and neck cancer vary greatly, and serious side effects are often observed. Being able to predict therapy effects is therefore crucial for choosing the best treatment option for each patient. In this study, we developed an assay to evaluate how head and neck tumor cells respond to radiation and chemotherapy. Treatment of thin patient-derived cancer tissue slices in the laboratory (in vitro) resulted in large differences in individual tumor's reactions to treatment. In the sensitive tumors, cancer cells repaired the DNA damage inflicted by therapy only partially, stopped multiplying, and showed increased levels of cell death. On the other hand, resistant tumors were able to recover from the damage caused by the treatment. The next crucial step is to investigate whether the differences we observed in vitro can indeed predict the treatment outcomes; this is currently being tested in an ongoing clinical trial. Background: Head and neck squamous cell carcinoma (HNSCC) displays a large heterogeneity in treatment response, and consequently in patient prognosis. Despite extensive efforts, no clinically validated model is available to predict tumor response. Here we describe a functional test for predicting tumor response to radiation and chemotherapy on the level of the individual patient. Methods: Resection material of 17 primary HNSCC patients was cultured ex vivo, irradiated or cisplatin-treated, after which the effect on tumor cell vitality was analyzed several days after treatment. Results: Ionizing radiation (IR) affected tumor cell growth and viability with a clear dose-response relationship, and marked heterogeneity between tumors was observed. After a single dose of 5Gy, proliferation in IR-sensitive tumors dropped below 30% of the untreated level, while IR-resistant tumors maintained at least 60% of proliferation. IR-sensitive tumors showed on average a twofold increase in apoptosis, as well as an increased number and size of DNA damage foci after treatment. No differences in the homologous recombination (HR) proficiency between IR-sensitive and -resistant tumors were detected. Cisplatin caused a decrease in proliferation, as well as induction of apoptosis, again with marked variation between the samples. Conclusions: Our functional ex vivo assay discriminated between IR-sensitive and IR-resistant HNSCC tumors, and may also be suitable for predicting response to cisplatin. Its predictive value is currently under investigation in a prospective clinical study.

Active surveillance instead of standard surgery after neoadjuvant chemoradiotherapy (nCRT) has been proposed for patients with oesophageal cancer. Circulating tumour DNA (ctDNA) may be used to facilitate selection of patients for surgery. We show that detection of ctDNA after nCRT seems highly suggestive of major residual disease. Tumour biopsies and blood samples were taken before, and 6 and 12 weeks after, nCRT. Biopsies were analysed with regular targeted next-generation sequencing (NGS). Circulating cell-free DNA (cfDNA) was analysed using targeted NGS with unique molecular identifiers and digital polymerase chain reaction. cfDNA mutations matching pre-treatment biopsy mutations confirmed the presence of ctDNA. In total, 31 patients were included, of whom 24 had a biopsy mutation that was potentially detectable in cfDNA (77%). Pre-treatment ctDNA was detected in nine of 24 patients (38%), four of whom had incurable disease progression before surgery. Pre-treatment ctDNA detection had a sensitivity of 47% (95% CI 24-71) (8/17), specificity of 85% (95% CI 42-99) (6/7), positive predictive value (PPV) of 89% (95% CI 51-99) (8/9), and negative predictive value (NPV) of 40% (95% CI 17-67) (6/15) for detecting major residual disease (10% residue in the resection specimen or progression before surgery). After nCRT, ctDNA was detected in three patients, two of whom had disease progression. Post-nCRT ctDNA detection had a sensitivity of 21% (95% CI 6-51) (3/14), specificity of 100% (95% CI 56-100) (7/7), PPV of 100% (95% CI 31-100) (3/3), and NPV of 39% (95% CI 18-64) (7/18) for detecting major residual disease. The addition of ctDNA to the current set of diagnostics did not lead to more patients being clinically identified with residual disease. These results indicate that pre-treatment and post-nCRT ctDNA detection may be useful in identifying patients at high risk of disease progression. The addition of ctDNA analysis to the current set of diagnostic modalities may not improve detection of residual disease after nCRT. © 2022 The Authors. The Journal of Pathology published by John WileySons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Background: Metastatic castration-resistant prostate cancer (mCRPC) patients with positive AR-V7 expression in their circulating tumour cells (CTCs) rarely derive benefit from abiraterone and enzalutamide. Design: We performed a prospective, multicenter, single arm phase II clinical trial (CABA-V7) in mCRPC patients previously treated with docetaxel and androgen deprivation therapy. Objective: In this trial, we investigated whether cabazitaxel treatment resulted in clinically meaningful PSA response rates in patients with positive CTC-based AR-V7 expression and collected liquid biopsies for genomic profiling. Results: Cabazitaxel was found to be modestly effective, with only 12% of these patients obtaining a PSA response. Genomic profiling revealed that CTC-based AR-V7 expression was not associated with other known mCRPC-associated alterations. CTC-based AR-V7 status and dichotomised CTC counts were observed as independent prognostic markers at baseline. Conclusions: AR-V7 positivity predicted poor overall survival (OS). However, cabazitaxel-treated AR-V7 positive patients and those lacking AR-V7 positivity, who received cabazitaxel as standard of care, appeared to have similar OS. Therefore, despite the low response rate, cabazitaxel may still be an effective treatment in this poor prognosis, AR-V7 positive patient population.

Hepatic arterial infusion pump (HAIP) chemotherapywith floxuridine has been explored as a means to control cancerin the liver. In this systematic review (https://doi.org/10.1245/s10434-022-11439-x), 478 patients with unresectable intrahepatic cholangiocarcinoma (iCCA) were identified from ninestudies; survival outcomes of 154 patients were used in themeta-analysis. HAIP chemotherapy with floxuridine forpatients with unresectable iCCA was associated with a pooled3-year overall survival (OS) of 39.5% (95% confidence interval(CI) 31.5–47.4%) and a weighted median OS of 29.0 months(range 25.0–39 months).

Background: Management of Ras wild-type colorectal cancer (CRC) patients upon disease progression after the successful use of targeted treatment with anti-EGFR monoclonal antibodies and backbone chemotherapy remains a clinical challenge. Summary: Development of treatment resistance with prevalence of preexisting RAS mutated clones, RAS mutation conversion, truncation of extracellular receptor domains as well as HER2 and MET amplification are molecular events that can be difficult to follow without the use of sophisticated laboratory techniques. The clinical hurdle of re-biopsy and tumor heterogeneity can be overcome by the implementation of next-generation sequencing (NGS) to analyze circulating tumor DNA (ctDNA) and identify druggable mutations or recovery of RAS-wildness. In this opinion paper, we summarize with critical thinking the clinical approach to be followed after the failure of first-line treatment in Ras wild-type CRC tumors with the use of NGS. Rechallenge with anti-EGFR inhibitors, in case of persistent or recovery of RAS-wildness, and targeted approach of specific mutations (BRAF inhibitors), amplifications (anti-Her2 treatment), or fusion proteins (NTRK inhibitors) can by guided by the use of NGS. The use of NGS platforms for serial analysis of ctDNA is an important step to better understand the molecular landscape of metastatic CRC and guide clinical decisions. Key Messages: NGS should be considered a mainstay in clinical practice for the management of CRC patients and health authorities should consider reimbursing its use in the appropriate clinical settings.

309 Background: Treatment of locally advanced and metastatic esophagogastric cancer (EGC) largely depends on systemic treatment including taxanes or platinum compounds – agents which are known to cause chemotherapy induced peripheral neuropathy (CIPN). To date, the extend of CIPN in EGC has not been investigated in real-world data. Methods: We identified patients with EGC from the Netherlands Cancer Registry (NCR) and Prospective Observational Cohort study of Oesophageal-gastric cancer Patients (POCOP) who underwent systemic treatment between 2016 and 2018 and completed at least one EORTC QLQ-CIPN20 questionnaire. Total CIPN scores and scores for the motor, sensory and autonomic scales (ranged 0-100) were calculated, and analyzed over time for patients receiving chemoradiation (CRT), peri-operative chemotherapy (POC) and patients primarily treated with palliative chemotherapy (PC). To gain insight in the differences between CIPN score at baseline and after 3, 6, 9, 12, 18 and 24 months we constructed linear mixed effect models. Results: We included 1052 EGC patients. 769 patients received CRT (response: 76% at baseline, 30% after 24 months), 143 received POC (response: 79% at baseline, 33% after 24 months) and 140 received PC (response: 74% at baseline, 28% after 12 months). Over time, neuropathy scores increased in all scales for patients receiving POC and PC, with a maximum increase of 16.5 points in total CIPN score after 6 months in the PC group compared to baseline. For patients receiving CRT, increase in neuropathy scores was more subtle, with a maximum increase in total CIPN score of 4.3 points after 24 months compared to baseline. Results of mixed effect models showed a significant increase of CIPN scores over time in all three groups compared to baseline, persisting for as long as two years after start of initial treatment. Conclusions: CIPN frequently occurs in EGC patients treated with chemotherapy, especially with PC. Due to the possible irreversibility of CIPN, it is important that clinicians inquire patients about neurotoxicity often, adjust the dose of neurotoxic agents as needed and that more research is done into strategies possibly preventing the occurrence of CIPN. Results of mixed model analysis to assess changes in total CIPN score over time. Each model was adjusted for age, sex, performance status and number of comorbidities. The model for POC and PC was also adjusted for use of oxaliplatin.[Table: see text] *p-value aChemoradiotherapy bPeri-operative chemotherapy cPalliative chemotherapy, no estimates were calculated for 18 and 24 months due to the low number of responses.

Patients with unresectable intrahepatic cholangiocarcinoma (iCCA) have poor survival. This systematic review describes the survival outcomes of hepatic arterial infusion pump (HAIP) chemotherapy with floxuridine for patients with unresectable iCCA.A literature search was conducted using the electronic databases PubMed, Medline (Ovid), Embase, Web of Science, Google Scholar, and Cochrane to find studies that reported data on the survival of patients with unresectable iCCA treated with HAIP chemotherapy using floxuridine. The quality of the studies was assessed using the Newcastle-Ottawa quality assessment Scale (NOS). Overall survival (OS) was the primary outcome measure, and progression-free survival (PFS), response rates, resection rates, and toxicity were defined as secondary outcome measures.After removing duplicates, 661 publications were assessed, of which nine studies, representing a total of 478 patients, met the inclusion criteria. Three out of nine studies were phase II clinical trials, one study was a prospective dose-escalation study, and the remaining five studies were retrospective cohort studies. After accounting for overlapping cohorts, 154 unique patients were included for pooled analysis. The weighted median OS of patients with unresectable iCCA treated with HAIP chemotherapy with floxuridine was 29.0 months (range 25.0-39 months). The pooled 1-, 2-, 3-, and 5-year OS were 86.4, 55.5, 39.5, and 9.7%, respectively.HAIP chemotherapy with floxuridine for patients with unresectable iCCA was associated with a 3-year OS of 39.5%, which is favorable compared with systemic chemotherapy for which no 3-year survivors were reported in the Advanced Biliary Cancer (ABC) trials.

Metastatic and locally-advanced neuroendocrine neoplasms (aNEN) form clinically and genetically heterogeneous malignancies, characterized by distinct prognoses based upon primary tumor localization, functionality, grade, proliferation index and diverse outcomes to treatment. Here, we report the mutational landscape of 85 whole-genome sequenced aNEN. This landscape reveals distinct genomic subpopulations of aNEN based on primary localization and differentiation grade; we observe relatively high tumor mutational burdens (TMB) in neuroendocrine carcinoma (average 5.45 somatic mutations per megabase) with TP53, KRAS, RB1, CSMD3, APC, CSMD1, LRATD2, TRRAP and MYC as major drivers versus an overall low TMB in neuroendocrine tumors (1.09). Furthermore, we observe distinct drivers which are enriched in somatic aberrations in pancreatic (MEN1, ATRX, DAXX, DMD and CREBBP) and midgut-derived neuroendocrine tumors (CDKN1B). Finally, 49% of aNEN patients reveal potential therapeutic targets based upon actionable (and responsive) somatic aberrations within their genome; potentially directing improvements in aNEN treatment strategies., Metastatic and locally-advanced neuroendocrine neoplasms (aNEN) display heterogeneous clinical and genetic characteristics. Here, the authors investigate the mutational landscape of 85 aNEN by whole genome sequencing and identify distinct subpopulations, tumour mutational burden patterns, drivers and actionable somatic alterations.

As the majority of patients experiences locoregional relapse and/or distant metastasis even after radical resection of esophageal cancer, many efforts have been made and are ongoing to identify the optimal multimodality treatment strategy. The true benefit and harm of neoadjuvant therapy including chemotherapy, radiotherapy or the combination, is still difficult to interpret given the heterogeneity in patient and tumor characteristics. Nonetheless, neoadjuvant chemoradiation with weekly carboplatin and paclitaxel (the CROSS regimen) is considered standard of care for squamous cell carcinoma in Europe. Definitive chemoradiation is considered an equal alternative in the United States. For adenocarcinoma, preoperative chemoradiation with a platinum and 5FU or the CROSS regimen and peri-operative chemotherapy with a platinum and 5FU or the FLOT (fluorouracil, leukovorin, oxaliplatin and docetaxel) regimen are all options. New developments in systemic anti-tumor therapy will most likely involve dual anti-HER2 inhibition or novel anti-HER2 antibody-drug conjugates for adenocarcinoma. Immunotherapy monotherapy in an unselected patient population does not seem to be as effective in esophageal cancer as it is in other cancer types. However, when we can correctly identify the subset of patients which does benefit from this treatment by employing new predictive markers, or find an effective synergistic combination of immunotherapy with chemotherapy and/or radiotherapy, immunotherapy could still improve patient outcome in the future.

Background The interpretation of the presence of AR-V7 in circulating tumour cells (CTCs) in men with metastatic castration-resistant prostate cancer (mCRPC) remains to be elucidated. AR-V7 may hold promise as a predictive biomarker, but there may be prognostic impact of AR-V7 positivity as well. To investigate the clinical value of AR-V7, we determined whether AR-V7 detection in CTCs in patients with mCRPC is associated with CTC counts and survival. Methods Between December 2011 and January 2019, three prospective clinical trials collected clinical data of patients with mCRPC, who progressed after docetaxel and/or enzalutamide or abiraterone. Baseline (and follow-up) blood samples were withdrawn determining CTC count and AR-V7 expression. The majority of patients started cabazitaxel as the next line of treatment after AR-V7 characterisation. Results A total of 127 samples were evaluable for the analysis of CTC count versus AR-V7 status. Although an association was observed between AR-V7 and CTC count in all patients with mCRPC (p = 0.017), no such association was found in the prognostic unfavourable subgroup of patients with ≥5 CTCs. After adjusting for clinical prognostic factors, AR-V7 expression in CTCs was not associated with overall survival (hazard ratio = 1.33, 95% confidence interval = 0.81–2.15, p = 0.25). Conclusion We found that AR-V7 expression in CTCs had no additional prognostic value in patients with mCRPC, mostly treated with cabazitaxel. In patients with mCRPC with a predefined worse prognosis of a higher CTC count (≥5), a predictive biomarker is an important unmet medical need. Prospective trials should investigate whether AR-V7 detection in CTCs may guide treatment selection for these adverse prognosis patients.

IntroductionThe locoregional failure (LRF) rate in human papilloma virus (HPV)-negative oropharyngeal squamous cell carcinoma (OPSCC) remains disappointingly high and toxicity is substantial. Response prediction prior to or early during treatment would provide opportunities for personalised treatment. Currently, there are no accurate predictive models available for correct OPSCC patient selection. Apparently, the pivotal driving forces that determine how a OPSCC responds to treatment, have yet to be elucidated. Therefore, the holistiC early respOnse assessMent for oroPharyngeaL cancer paTiEnts study focuses on a holistic approach to gain insight in novel potential prognostic biomarkers, acquired before and early during treatment, to predict response to treatment in HPV-negative patients with OPSCC.Methods and analysisThis single-centre prospective observational study investigates 60 HPV-negative patients with OPSCC scheduled for primary radiotherapy (RT) with cisplatin or cetuximab, according to current clinical practice. A holistic approach will be used that aims to map the macroscopic (with Intra Voxel Incoherent Motion Diffusion Kurtosis Imaging (IVIM-DKI); before, during, and 3 months after RT), microscopic (with biopsies of the primary tumour acquired before treatment and irradiated ex vivo to assess radiosensitivity), and molecular landscape (with circulating tumour DNA (ctDNA) analysed before, during and 3 months after treatment). The main end point is locoregional control (LRC) 2 years after treatment. The primary objective is to determine whether a relative change in the mean of the diffusion coefficient D (an IVIM-DKI parameter) in the primary tumour early during treatment, improves the performance of a predictive model consisting of tumour volume only, for 2 years LRC after treatment. The secondary objectives investigate the potential of other IVIM-DKI parameters, ex vivo sensitivity characteristics, ctDNA, and combinations thereof as potential novel prognostic markers.Ethics and disseminationThe study was approved by the Medical Ethical Committee of Erasmus Medical Center. The main results of the trial will be presented in international meetings and medical journals.Trial registration numberNL8458.

Background: Metastatic neuroendocrine tumors (mNET) form a clinically and genetically heterogeneous malignancy, characterized by distinct prognosis based upon primary tumor localization, functionality, grade and proliferation index, and a wide variation in treatment outcome. To get a better insight into this heterogeneity in prognosis and outcome, and also to reveal putative novel treatment targets, we here analyzed the mutational landscape of 91 metastatic mNET biopsies derived from whole-genome sequencing (WGS). Material and Methods: Between May 2016 and July 2018, 91 mNET patients from 14 Dutch hospitals were included in the study protocol (NCT01855477) of the Center for Personalized Cancer Treatment (CPCT). Patients with mNET were categorized by primary location into midgut (N = 44), pancreas (N = 26), lung (N = 5), unknown (N = 12) and other (N = 4). The WGS of matched peripheral blood and tumor tissues was performed on a HiSeq X Ten system to sequencing depths of 38x and 105x, respectively, and subsequently aligned to the human reference genome (GRCh37) and analyzed for somatic mutations, mutational signatures, copy-number alterations and structural variants with the GRIDSS, PURPLE, LINX suite, GISTIC2 and in-house algorithms. Results: The overall mutational landscape of mNET is hallmarked by a relative stable diploid tumor genome and low median tumor mutational burden (TMB) of 1.32 (IQR: 0.91 - 2.1) mutations per genomic Mb. Differences in median TMB relating to primary location were observed; ranging from 1.1 (midgut), 1.49 (pancreas), 2.87 (lung) to 4.18 (unknown); six mNET had high tumor mutational burden (TMB ≥ 10), but without microsatellite-instability signatures. In addition, we observed striking evidence of somatic aberrations due to alkylating agents (sig. 11), combined with the highest cohort-wide TMB (39.6) for a single patient treated with streptozocin prior to biopsy. Three mNETs revealed MUTYH-related alterations (sig. 18) and furthermore, we detected chromothripsis (n = 6) and APOBEC-related regional hypermutation (kataegis; n = 11). Midgut-derived mNET (n = 44) predominantly harbored somatic alterations in CDKN1B (n = 11) and CDKN2A/B (n = 7). Strikingly, no somatic driver mutation was seen in almost half of these midgut-derived mNET (n = 21). Pancreatic mNET (n = 26) predominantly harbored alterations in MEN1 (n = 8), TP53 (n = 8), ATRX (n = 6), CDKN2A/B (n = 6) and DAXX (n = 5). In total, 39 mNET (43%) showed clinically-actionable somatic alterations for current on- and off-label NET therapies. Conclusion: This study comprises the largest WGS repository of mNET to date, and demonstrates the genetic heterogeneity of mNET linked to primary localization. Several potential therapeutic targets were identified which are worthwhile to explore for their clinical value in the treatment of mNET patients. Citation Format: Job van Riet, Harmen J. van de Werken, Edwin Cuppen, Ferry A. L. M. Eskens, Margot E. Tesselaar, Linde M. van Veenendaal, Heinz-Josef Klümpen, Wouter Dercksen, Gerlof D. Valk, Martijn P. J. K. Lolkema, Stefan Sleijfer, Bianca Mostert. In-depth analysis of the genomic landscape of 91 metastatic neuroendocrine tumors reveals subtype-heterogeneity and potential therapeutic targets [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5673.

The detection of mutated genes in cell-free DNA (cfDNA) in plasma has emerged as an important minimally invasive way to obtain detailed information regarding tumor biology. Reliable determination of circulating tumor–derived DNA, often present at a low quantity amidst an excess of normal DNA in plasma, would be of added value for screening and monitoring of cancer patients and for hypothesis-generating studies in valuable retrospective cohorts. Our aim was to establish a workflow to simultaneously assess four hotspot estrogen receptor mutations (mESR1) in cfDNA isolated from only 200 μL of plasma by means of uniplex or multiplex pre-amplification combined with digital PCR. This workflow was then applied in metastatic breast cancer (MBC) patients receiving systemic therapies for MBC. In accordance with previous studies, estrogen receptor mutations were more frequently detected in endocrine-treated MBC patients at progressive disease [34.1% (15/44)] than before the start of endocrine therapy [3.9% (2/51); P = 0.001]. For a subset of samples, results were compared with analysis of these mutations by Oncomine-targeted next-generation sequencing, which, although requiring a higher cfDNA input, yielded concordant results. The data establish development and validation of a digital PCR workflow for the simultaneous detection of several tumor-derived mutations in minute amounts of cfDNA and show the potential of this workflow for use on archived volume-limited blood samples.

Recent reports have emphasized the clinical relevance of detecting AR-V7 in circulating tumor cells (CTCs). Our aim was to set up a validated multicenter pipeline to measure AR-V7 by quantitative RT-PCR (RT-qPCR) in RNA isolated from CellSearch-enriched CTCs to provide an AR-V7–positive or AR-V7–negative score in a clinically acceptable time range. CellSearch-enirched CTCs from patients with metastatic castration–resistant prostate cancer were characterized by RT-qPCR. After optimization, it was prospectively tested whether it was possible to report the AR-V7 status within 11 days (PRELUDE study). In the range of the RNA equivalent of 0.2 to 12 VCaP cells, the CV for AR-V7 was 9% (n = 37). The limit of detection was 0.3, and the limit of quantitation was 3 cells in the final RT-qPCR. No differences were observed between AR-V7 data generated by five technicians or in two different laboratories. For the 45 patients in PRELUDE, 13 patients were ineligible, 22 patients were AR-V7 negative, and 10 were AR-V7 positive. The median time to inform the physician of the test result was 7 days (range, 2 to 11 days). This assay can establish the AR-V7 status in CTCs from patients with metastatic castration–resistant prostate cancer. Furthermore, it was possible to provide an AR-V7 outcome within 11 days, indicating that it may be used to choose between an anti–androgen receptor or taxane-based cabazitaxel treatment.

Before using circulating tumor cells (CTCs) as liquid biopsy, insight into molecular discrepancies between CTCs and primary tumors is essential. We characterized CellSearch-enriched CTCs from 62 metastatic breast cancer (MBC) patients with >= 5 CTCs starting first-line systemic treatment. Expression levels of 35 tumor-associated, CTC-specific genes, including ESR1, coding for the estrogen receptor (ER), were measured by reverse transcription quantitative polymerase chain reaction and correlated to corresponding primary tumors. In 30 patients (48%), gene expression profiles of 35 genes were discrepant between CTCs and the primary tumor, but this had no prognostic consequences. In 15 patients (24%), the expression of ER was discrepant Patients with ER-negative primary tumors and ER-positive CTCs had a longer median TTS compared to those with concordantly ER-negative CTCs (8.5 versus 2.1 months, P = 0.05). From seven patients, an axillary lymph node metastasis was available. In two patients, the CTC profiles better resembled the lymph node metastasis than the primary tumor. Our findings suggest that molecular discordances between CTCs and primary tumors frequently occur, but that this bears no prognostic consequences. Alterations in ER-status between primary tumors and CTCs might have prognostic implications. (C) 2015 Elsevier Ireland Ltd. All rights reserved.

BACKGROUND: Despite good outcomes for many, a substantial group of patients undergoing metastasectomy for isolated liver metastases from colorectal cancer (CRC) experience early recurrence. We have investigated whether circulating tumour cell (CTC) detection can identify patients developing disease recurrence within 1 year after liver metastasectomy.METHODS: In CRC patients undergoing liver metastasectomy, 30 ml peripheral blood was withdrawn preoperatively. CTCs were detected by the CellSearch system after a density-gradient-based enrichment step.RESULTS: One hundred and seventy-three samples from 151 individual patients were analysed. In 75 samples (43%), CTCs were detected, 16% had ⩾3 CTCs/7.5 ml of blood. Eighty-two patients (47%) experienced early disease recurrence (CONCLUSIONS: The presence of CTCs in preoperative peripheral blood samples does not identify patients at risk for early disease recurrence after curative resection of colorectal liver metastases. Other parameters are needed to better identify patients at high risk to relapse after liver metastasectomy for CRC.

Allele-specific amplification, combined with TaqMan probe real-time polymerase chain reaction (real-time AS-PCR), has been widely used for detecting genetic variants, single nucleotide polymorphisms, and genetic mutations. In addition, several probe-blocking methods have been introduced in real-time AS-PCR to block amplification of wild-type templates and to increase detection sensitivity and specificity. However, most of these methods provide a limited sensitivity of no better than 1% and are complex in design of blockers, and thus cannot be readily adapted for different mutation assays. We have developed a modified non-extendable primer blocker (NEPB) for real-time AS-PCR (AS-NEPB-PCR). The NEPB method provides an easy design of allele-specific primer and corresponding primer blocker that can be used in any single nucleotide polymorphism or mutation detection, specifically in the detection of low-frequency mutations. The method is straight-forward in assay optimization and can achieve 0.1% sensitivity with 100% specificity (95% confidence interval, 92-100%) in detecting K-Ras, B-Raf, and EGFR mutations in cancer cells. (J Mol Diagn 2013, 15: 62-69; http://dx.doi.org/10.1016/j.jmoldx.2012.08.007)

// Wendy Onstenk 1 , Anieta M. Sieuwerts 1 , Bianca Mostert 1 , Zarina Lalmahomed 2 , Joan B. Bolt-de Vries 1 , Anne van Galen 1 , Marcel Smid 1 , Jaco Kraan 1 , Mai Van 1 , Vanja de Weerd 1 , Raquel Ramirez-Moreno 1 , Katharina Biermann 3 , Cornelis Verhoef 2 , Dirk J. Grunhagen 2 , Jan N.M. IJzermans 2 , Jan W. Gratama 1 , John W.M. Martens 1 , John A. Foekens 1 , Stefan Sleijfer 1 1 Erasmus MC Cancer Institute, Department of Medical Oncology and Cancer Genomics Netherlands, Rotterdam, The Netherlands 2 Department of Surgery, Erasmus University Medical Center, Rotterdam, The Netherlands 3 Department of Pathology, Erasmus University Medical Center, Rotterdam, The Netherlands Correspondence to: Wendy Onstenk, email: w.onstenk@erasmusmc.nl Keywords: circulating tumor cells, CTCs, CellSearch, colorectal cancer, gene expression profiling Received: April 06, 2016 Accepted: May 29, 2016 Published: June 20, 2016 ABSTRACT Background: CTCs are a promising alternative for metastatic tissue biopsies for use in precision medicine approaches. We investigated to what extent the molecular characteristics of circulating tumor cells (CTCs) resemble the liver metastasis and/or the primary tumor from patients with metastatic colorectal cancer (mCRC). Results: The CTC profiles were concordant with the liver metastasis in 17/23 patients (74%) and with the primary tumor in 13 patients (57%). The CTCs better resembled the liver metastasis in 13 patients (57%), and the primary tumor in five patients (22%). The strength of the correlations was not associated with clinical parameters. Nine genes ( CDH1 , CDH17 , CDX1 , CEACAM5 , FABP1 , FCGBP , IGFBP3 , IGFBP4 , and MAPT ) displayed significant differential expressions, all of which were downregulated, in CTCs compared to the tissues in the 23 patients. Patients and Methods: Patients were retrospectively selected from a prospective study. Using the CellSearch System, CTCs were enumerated and isolated just prior to liver metastasectomy. A panel of 25 CTC-specific genes was measured by RT-qPCR in matching CTCs, primary tumors, and liver metastases. Spearman correlation coefficients were calculated and considered as continuous variables with r=1 representing absolute concordance and r=-1 representing absolute discordance. A cut-off of r>0.1 was applied in order to consider profiles to be concordant. Conclusions: In the majority of the patients, CTCs reflected the molecular characteristics of metastatic cells better than the primary tumors. Genes involved in cell adhesion and epithelial-to-mesenchymal transition were downregulated in the CTCs. Our results support the use of CTC characterization as a liquid biopsy for precision medicine.

Purpose: Molecular characterization of circulating tumor cells (CTC) holds great promise. Unfortunately, routinely isolated CTC fractions currently still contain contaminating leukocytes, which makes CTC-specific molecular characterization extremely challenging. In this study, we determined mRNA and microRNA (miRNA) expression of potentially CTC-specific genes that are considered to be clinically relevant in breast cancer. Experimental Design: CTCs were isolated with the epithelial cell adhesion molecule–based CellSearch Profile Kit. Selected genes were measured by real-time reverse transcriptase PCR in CTCs of 50 metastatic breast cancer patients collected before starting first-line systemic therapy in blood from 53 healthy blood donors (HBD) and in primary tumors of 8 of the patients. The molecular profiles were associated with CTC counts and clinical parameters and compared with the profiles generated from the corresponding primary tumors. Results: We identified 55 mRNAs and 10 miRNAs more abundantly expressed in samples from 32 patients with at least 5 CTCs in 7.5 mL of blood compared with samples from 9 patients without detectable CTCs and HBDs. Clustering analysis resulted in 4 different patient clusters characterized by 5 distinct gene clusters. Twice the number of patients from cluster 2 to 4 had developed both visceral and nonvisceral metastases. Comparing transcript levels in CTCs with those measured in corresponding primary tumors showed clinically relevant discrepancies in estrogen receptor and HER2 levels. Conclusions: Our study shows that molecular profiling of low numbers of CTCs in a high background of leukocytes is feasible and shows promise for further studies on the clinical relevance of molecular characterization of CTCs. Clin Cancer Res; 17(11); 3600–18. ©2011 AACR.

Most assays to detect circulating tumor cells (CTCs) rely on EpCAM expression on tumor cells. Recently, our group reported that in contrast to other molecular breast cancer subtypes, "normal-like" cell lines lack EpCAM expression and are thus missed when CTCs are captured with EpCAM-based technology [J Natl Cancer Inst 101(1):61-66, 2009]. Here, the use of CD146 is introduced to detect EpCAM-negative CTCs, thereby improving CTC detection. CD146 and EpCAM expression were assessed in our panel of 41 breast cancer cell lines. Cells from 14 cell lines, 9 of which normal-like, were spiked into healthy donor blood. Using CellSearch (TM) technology, 7.5 ml whole blood was enriched for CTCs by adding ferrofluids loaded with antibodies against EpCAM and/or CD146 followed by staining for Cytokeratin and DAPI. Hematopoietic cells and circulating endothelial cells (CECs) were counterstained with CD45 and CD34, respectively. A similar approach was applied for blood samples of 20 advanced breast cancer patients. Eight of 9 normal-like breast cancer cell lines lacked EpCAM expression but did express CD146. Five of these 8 could be adequately recovered by anti-CD146 ferrofluids. Of 20 advanced breast cancer patients whose CTCs were enumerated with anti-EpCAM and anti-CD146 ferrofluids, 9 had CD146+ CTCs. Cells from breast cancer cell lines that lack EpCAM expression frequently express CD146 and can be recovered by anti-CD146 ferrofluids. CD146+ CTCs are present in the peripheral blood of breast cancer patients with advanced disease. Combined use of anti-CD146 and anti-EpCAM is likely to improve CTC detection in breast cancer patients.

Recently, miRNA-expression profiling in primary tumors has yielded promising results. However, establishing miRNA expression in the circulation probably has advantages over determination in primary tumor tissue, further augmenting the potential applications of miRNA determination in oncology. Circulating tumor cells (CTCs) have rapidly developed as important prognostic and therapy-monitoring biomarkers in metastatic breast, colorectal and prostate cancer when enumerated, and their isolation enables subsequent analysis using various molecular applications, including miRNA-expression analysis. In addition to CTC-associated miRNAs, free circulating miRNAs have been identified in whole blood, plasma and serum. Determination of miRNAs in peripheral blood, either cell-free or CTC-associated, is expected to become important in oncology, especially when linked to and interpreted together with epithelial CTCs. In this article, we will discuss miRNA-expression profiling in primary tumors, depict the potential applications of measuring miRNA in the circulation and review the literature on cell-free circulating miRNAs, as well as offering some methodological and technical considerations on the measurement of circulating miRNAs.

Introduction: The presence of the androgen receptor splice variant 7 (AR-V7) in circulating tumor cells (CTCs) has shown to be associated with resistance to anti-AR treatment with abiraterone or enzalutamide, but not to chemotherapy containing taxanes such as cabazitaxel in patients with metastatic castration-resistant prostate cancer (mCRPC). The primary objective of this project was to set up a validated multi-center pipeline to measure AR-V7 by RT-qPCR in RNA isolated from CellSearch-enriched CTCs. This pipeline included a) the pre-analytical phase (appropriate anonymized sample collection and sample preservation), b) the analytical phase (assay performance with high analytical sensitivity, precision and specificity and c) the post-analytical phase (data evaluation and reporting). Materials and Methods: CellSearch-enirched CTCs from metastatic castration-resistant prostate cancer (mCRPC) patients were characterized by RT-qPCR. To validate the pipeline, we determined the proportion of blood samples from mCRPC patients having three or more CTCs per 7.5 mL of blood for whom an AR-V7 status can be assayed and returned to the clinician within 11 days (design PRELUDE study protocol). Results: In the range of the RNA equivalent of 0.2-12 AR-V7 positive VCaP cells the coefficient of variation (CV) for AR-V7 was 9% (n=37). The limit of detection (LOD) was 0.3 and the limit of quantification (LOQ) 3 cells in the final RT-qPCR. In none of 17 healthy blood donors an AR-V7 signal was detected, showing high diagnostic specificity (100%). No differences were observed between AR-V7 data generated by four different technicians or in two different laboratories. For the 45 patients in the PRELUDE study, thirteen patients were not eligible due to poor RNA quality (n=1) or because the blood sample did not contain enough epithelial signal (n=12). Twenty-two patients were AR-V7 negative and ten AR-V7 positive. The median, 75th and 90th percentile reporting times from blood draw to dissemination of the test results were 7, 8 and 9 days, respectively. Conclusion: This AR-V7 test with validated cut-offs, a strong intra- and inter-laboratory performance and the ability to report the outcome within a clinically acceptable time frame for the large majority of patients, met our pre-specified objectives. The AR-V7 test is now ready to be used in prospective studies (including randomized controlled trials) to test its predictive value for outcome on post-docetaxel (anti-AR of cabazitaxel) treatment. Citation Format: Anieta M. Sieuwerts, Bianca Mostert, Michelle van der Vlugt-Daane, Jaco Kraan, Corine Beaufort, Mai Van, Wendy J. Prager, Bram De Laere, Nick Beije, Paul Hamberg, Hans M. Westgeest, Metin Tascilar, Luc Y. Dirix, Wendy Onstenk, Ronald de Wit, Martijn P. Lolkema, Ron H. Mathijssen, John W. Martens, Stefan Sleijfer. A multicenter project to test the validity and logistics surrounding the testing of AR-V7 mRNA expression in circulating tumor cells [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 2617.

The enumeration of circulating tumor cells has long been regarded as an attractive diagnostic tool, as circulating tumor cells are thought to reflect aggressiveness of the tumor and may assist in therapeutic decisions in patients with solid malignancies. However, implementation of this assay into clinical routine has been cumbersome, as a validated test was not available until recently. Circulating tumor cells are rare events which can be detected specifically only by using a combination of surface and intracellular markers, and only recently a number of technical advances have made their reliable detection possible. Most of these new techniques rely on a combination of an enrichment and a detection step. This review addresses the assays that have been described so far in the literature, including the enrichment and detection steps and the markers used in these assays. We have focused on breast cancer as most clinical studies on CTC detection so far have been done in these patients. (C) 2009 Elsevier Ltd. All rights reserved.

Identification of specific subtypes of circulating tumor cells in peripheral blood of cancer patients can provide information about the biology of metastasis and improve patient management. However, to be effective, the method used to identify circulating tumor cells must detect all tumor cell types. We investigated whether the five subtypes of human breast cancer cells that have been defined by global gene expression profiling-025EFnormal-like, basal, HER2-positive, and luminal A and B-025EFwere identified by CellSearch, a US Food and Drug Administration-approved test that uses antibodies against the cell surface-expressed epithelial cell adhesion molecule (EpCAM) to isolate circulating tumor cells. We used global gene expression profiling to determine the subtypes of a well-defined panel of 34 human breast cancer cell lines (15 luminal, nine normal-like, five basal-like, and five Her2-positive). We mixed 50-150 cells from 10 of these cell lines with 7.5 mL of blood from a single healthy human donor, and the mixtures were subjected to the CellSearch test to isolate the breast cancer cells. We found that the CellSearch isolation method, which uses EpCAM on the surface of circulating tumor cells for cell isolation, did not recognize, in particular, normal-like breast cancer cells, which in general have aggressive features. New tests that include antibodies that specifically recognize normal-like breast tumor cells but not cells of hematopoietic origin are needed.

Detection of circulating tumor cells (CTCs) in whole blood from metastatic cancer patients by the Cell-Search (TM) CTC Test (Veridex LLC, Warren, NJ, USA) has been shown to have clinical relevance. In addition to enumeration, there is great interest in molecular characterization of these CTCs. We aimed to establish a robust method to perform mRNA expression analysis of multiple genes by a real-time reverse transcriptase (RT)-PCR on small numbers of CTCs enriched from whole blood by the CellSearch (TM) system. Despite the 4 log depletion of leukocytes after CellSearch enrichment, the CTC-enriched fractions still contained leukocytes, in particular B-lymphocytes, which severely interfered with our CTC-specific gene expression profiling. After extensive washing and leukocyte-specific depletion by anti-CD45 coated magnetic beads prior to CellSearch TM enrichment, the number of leukocytes present in the enriched fraction was still high (range 60-929). However, by using a set of genes with no or minor expression by leukocytes, we succeeded to perform quantitative gene expression profiling specific for as little as one breast cancer CTC present in a CTC-enriched environment typically containing over 800 contaminating leukocytes. Our method allows molecular characterization specific for as little as one CTC, and can be used to expand the understanding of the biology of metastasis and, potentially, to improve patient management.

Background Molecular characterization of circulating tumor cells (CTC) is promising for personalized medicine. We aimed to identify a CTC gene expression profile predicting outcome to first-line aromatase inhibitors in metastatic breast cancer (MBC) patients. Methods: CTCs were isolated from 78 MBC patients before treatment start. mRNA expression levels of 96 genes were measured by quantitative reverse transcriptase polymerase chain reaction. After applying predefined exclusion criteria based on lack of sufficient RNA quality and/or quantity, the data from 45 patients were used to construct a gene expression profile to predict poor responding patients, defined as disease progression or death

Circulating tumor cells (CTC) are detected by the CellSearch System in 20% to 25% of patients with primary breast cancer (pBC). To improve CTC detection, we investigated melanoma cell adhesion molecule (MCAM) as enrichment marker next to epithelial cell adhesion molecule (EpCAM) and tested the clinical relevance of MCAM-positive CTCs in patients with HER2-negative stage II/III pBC starting neoadjuvant chemotherapy (NAC) in the NEOZOTAC trial. Using the CellSearch System, EpCAM-positive and MCAM-positive CTCs were separately enriched from 7.5 mL blood, at baseline and after the first NAC cycle. Circulating endothelial cells (CEC) were measured using flow cytometry. Primary objective was to improve the CTC detection rate to ≥40% combining EpCAM/MCAM. Correlations of CTC and CEC counts and pathologic complete response (pCR) were also explored. At baseline, we detected EpCAM-positive and MCAM-positive CTCs in 12 of 68 (18%) and 8 of 68 (12%) patients, respectively. After one cycle, this was 7 of 44 (16%) and 7 of 44 (16%) patients, respectively. The detection rate improved from 18% at baseline and 16% after one cycle with EpCAM to 25% (P = 0.08) and 30% (P = 0.02), respectively, with EpCAM/MCAM. No patients with MCAM-positive CTCs versus 23% of patients without MCAM-positive CTCs at baseline achieved pCR (P = 0.13). EpCAM-positive CTCs and CEC counts were not correlated to pCR. Combined EpCAM/MCAM CellSearch enrichment thus increased the CTC detection rate in stage II/III pBC. We found no associations of CTC and CEC counts with pCR to NAC. The clinical relevance of MCAM-positive CTCs deserves further study. Mol Cancer Ther; 14(3); 821–7. ©2014 AACR.

Introduction: The molecular characterization of circulating tumor cells (CTCs) is a promising tool for the repeated and non-invasive evaluation of predictive and prognostic factors. Challenges associated with CTC characterization using the only FDA approved method for CTC enumeration, the CellSearch technique, include the presence of an excess of leukocytes in CTC-enriched blood fractions. Here we aimed to identify colorectal tumor-specific gene expression levels in the blood of patients with and without detectable CTCs according to CellSearch criteria. Materials and methods: Blood of 30 healthy donors (HDs) and 142 metastatic colorectal cancer (mCRC) patients was subjected to CellSearch CTC enumeration and isolation. In all samples, 95 mRNAs were measured by reverse transcriptase quantitative PCR (RT-qPCR). HD blood samples and patient samples with three or more CTCs were compared to identify CTC-specific mRNAs. Patient samples without detectable CTCs were separately analyzed. Results: Thirty-four CTC-specific mRNAs were higher expressed in patients with >= 3 CTCs compared with HDs (Mann Whitney U-test P < 0.05). Among patients without detectable CTCs, a HD-unlike subgroup was identified which could be distinguished from HDs by the expression of epithelial genes such as KRT19, KRT20 and AGR2. Also, in an independent patient set, a similar HD-unlike group could be identified among the patients without detectable CTCs according to the CellSearch system. Conclusion: Extensive molecular characterization of colorectal CTCs is feasible and a subgroup of patients without detectable CTCs according to CellSearch criteria bears circulating tumor load, which may have clinical consequences. This CTC-specific gene panel for mCRC patients may enable the exploration of CTC characterization as a novel means to further individualize cancer treatment. (C) 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Background: A circulating tumor cell (CTC) count is an established prognostic factor in metastatic breast cancer (MBC). Besides enumeration, CTC characterization promises to improve outcome prediction and treatment guidance. Having shown the feasibility of quantifying clinically relevant mRNA transcripts in CTCs, we determined the prognostic value of CTC gene expression in MBC. Patients and methods: CTCs were isolated and enumerated from blood of 197 MBC patients who were about to start first-line systemic therapy. Of these, 180 were assessable for quantification of mRNA expression by RT-qPCR in relation to time-to-treatment failure (TTF). A prognostic CTC gene profile was generated by leave-one-out cross validation in a 103 patient discovery set and validated in 77 patients. Additionally, all 180 patients were randomly divided into two equal sets to discover and validate a second prognostic profile. Results: CTC count predicted for TTF at baseline {>= 5 versus

Aims In most pregnancy reports, atrioventricular septal defects (AVSD) are not differentiated from more simple septal defects, thus underestimating the risks of pregnancy. To investigate the magnitude and determinants of risk during pregnancy in female patients with balanced AVSD.Methods and results Using a nation-wide registry (CONCOR), 79 female patients with balanced/isolated AVSD were identified. A total of 29 patients had 62 pregnancies, including 12 miscarriages (19%) and two elective abortions. Detailed recordings of each completed (greater-than 20 weeks gestation) pregnancy (n=48, 26 women) were obtained. Cardiovascular events complicated almost 40% of the completed pregnancies. In particular, post-partum persistence of pregnancy-related New York Heart Association (NYHA) class deterioration [23% mainly patients with residual atrial septal defects (ASD)] and deterioration of pre-existing left AV-valvular regurgitation (17%) were frequently recorded. Additional cardiac complications were arrhythmias (19%) and symptomatic heart failure (2%). Congenital heart disease (CHD) recurred in six children (12%): AVSD (n=4, three with left-sided hypoplasia), patent ductus arteriosus (n=1), and ASD (n=1). Three children died including two children with left-sided hypoplasia.Conclusion Pregnancy is not always well tolerated in women with AVSD, predominantly due to NYHA class deterioration and worsening of pre-existing AV-valvular regurgitation. Offspring mortality is high (6.3%), primarily due to recurrence of complex CHD.

11017 Background: Before considering circulating tumor cells (CTCs) as a liquid biopsy in metastatic breast cancer (MBC), the degree of molecular discrepancy between primary tumors (PTs) and CTCs h...