Florian Eisner

Complex Tumor Genomes Inferred from Single Circulating Tumor Cells by Array-CGH and Next-Generation Sequencing

Circulating tumor cells (CTC) released into blood from primary cancers and metastases reflect the current status of tumor genotypes, which are prone to changes. Here, we conducted the first comprehensive genomic profiling of CTCs using array-comparative genomic hybridization (CGH) and next-generation sequencing. We used the U.S. Food and Drug Administration-cleared CellSearch system, which detected CTCs in 21 of 37 patients (range, 1-202/7.5 mL sample) with stage IV colorectal carcinoma. In total, we were able to isolate 37 intact CTCs from six patients and identified in those multiple colorectal cancer-associated copy number changes, many of which were also present in the respective primary tumor. We then used massive parallel sequencing of a panel of 68 colorectal cancer-associated genes to compare the mutation spectrum in the primary tumors, metastases, and the corresponding CTCs from two of these patients. Mutations in known driver genes [e.g., adenomatous polyposis coli (APC), KRAS, or PIK3CA] found in the primary tumor and metastasis were also detected in corresponding CTCs. However, we also observed mutations exclusively in CTCs. To address whether these mutations were derived from a small subclone in the primary tumor or represented new variants of metastatic cells, we conducted additional deep sequencing of the primary tumor and metastasis and applied a customized statistical algorithm for analysis. We found that most mutations initially found only in CTCs were also present at subclonal level in the primary tumors and metastases from the same patient. This study paves the way to use CTCs as a liquid biopsy in patients with cancer, providing more effective options to monitor tumor genomes that are prone to change during progression, treatment, and relapse.

Increased neutrophil-lymphocyte ratio is a poor prognostic factor in patients with primary operable and inoperable pancreatic cancer

Background:The neutrophil-lymphocyte ratio (NLR) has been proposed as an indicator of systemic inflammatory response. Previous findings from small-scale studies revealed conflicting results about its independent prognostic significance with regard to different clinical end points in pancreatic cancer (PC) patients. Therefore, the aim of our study was the external validation of the prognostic significance of NLR in a large cohort of PC patients.Methods:Data from 371 consecutive PC patients, treated between 2004 and 2010 at a single centre, were evaluated retrospectively. The whole cohort was stratified into two groups according to the treatment modality. Group 1 comprised 261 patients with inoperable PC at diagnosis and group 2 comprised 110 patients with surgically resected PC. Cancer-specific survival (CSS) was assessed using the Kaplan–Meier method. To evaluate the independent prognostic significance of the NLR, the modified Glasgow prognostic score (mGPS) and the platelet-lymphocyte ratio univariate and multivariate Cox regression models were applied.Results:Multivariate analysis identified increased NLR as an independent prognostic factor for inoperable PC patients (hazard ratio (HR)=2.53, confidence interval (CI)=1.64–3.91, P<0.001) and surgically resected PC patients (HR=1.61, CI=1.02–2.53, P=0.039). In inoperable PC patients, the mGPS was associated with poor CSS only in univariate analysis (HR=1.44, CI=1.04–1.98).Conclusion:Risk prediction for cancer-related end points using NLR does add independent prognostic information to other well-established prognostic factors in patients with PC, regardless of the undergoing therapeutic modality. Thus, the NLR should be considered for future individual risk assessment in patients with PC.

Establishment of tumor‐specific copy number alterations from plasma DNA of patients with cancer

With the increasing number of available predictive biomarkers, clinical management of cancer is becoming increasingly reliant on the accurate serial monitoring of tumor genotypes. We tested whether tumor‐specific copy number changes can be inferred from the peripheral blood of patients with cancer. To this end, we determined the plasma DNA size distribution and the fraction of mutated plasma DNA fragments with deep sequencing and an ultrasensitive mutation‐detection method, i.e., the Beads, Emulsion, Amplification, and Magnetics (BEAMing) assay. When analyzing the plasma DNA of 32 patients with Stage IV colorectal carcinoma, we found that a subset of the patients (34.4%) had a biphasic size distribution of plasma DNA fragments that was associated with increased circulating tumor cell numbers and elevated concentration of mutated plasma DNA fragments. In these cases, we were able to establish genome‐wide tumor‐specific copy number alterations directly from plasma DNA. Thus, we could analyze the current copy number status of the tumor genome, which was in some cases many years after diagnosis of the primary tumor. An unexpected finding was that not all patients with progressive metastatic disease appear to release tumor DNA into the circulation in measurable quantities. When we analyzed plasma DNA from 35 patients with metastatic breast cancer, we made similar observations suggesting that our approach may be applicable to a variety of tumor entities. This is the first description of such a biphasic distribution in a surprisingly high proportion of cancer patients which may have important implications for tumor diagnosis and monitoring.

Changes in Colorectal Carcinoma Genomes under Anti-EGFR Therapy Identified by Whole-Genome Plasma DNA Sequencing

Monoclonal antibodies targeting the Epidermal Growth Factor Receptor (EGFR), such as cetuximab and panitumumab, have evolved to important therapeutic options in metastatic colorectal cancer (CRC). However, almost all patients with clinical response to anti-EGFR therapies show disease progression within a few months and little is known about mechanism and timing of resistance evolution. Here we analyzed plasma DNA from ten patients treated with anti-EGFR therapy by whole genome sequencing (plasma-Seq) and ultra-sensitive deep sequencing of genes associated with resistance to anti-EGFR treatment such as KRAS, BRAF, PIK3CA, and EGFR. Surprisingly, we observed that the development of resistance to anti-EGFR therapies was associated with acquired gains of KRAS in four patients (40%), which occurred either as novel focal amplifications (n = 3) or as high level polysomy of 12p (n = 1). In addition, we observed focal amplifications of other genes recently shown to be involved in acquired resistance to anti-EGFR therapies, such as MET (n = 2) and ERBB2 (n = 1). Overrepresentation of the EGFR gene was associated with a good initial anti-EGFR efficacy. Overall, we identified predictive biomarkers associated with anti-EGFR efficacy in seven patients (70%), which correlated well with treatment response. In contrast, ultra-sensitive deep sequencing of KRAS, BRAF, PIK3CA, and EGFR did not reveal the occurrence of novel, acquired mutations. Thus, plasma-Seq enables the identification of novel mutant clones and may therefore facilitate early adjustments of therapies that may delay or prevent disease progression.

External Validation of the Derived Neutrophil to Lymphocyte Ratio as a Prognostic Marker on a Large Cohort of Pancreatic Cancer Patients

Background With growing evidence on the role of inflammation in cancer biology, the presence of a systemic inflammatory response has been postulated as having prognostic significance in a wide range of cancer types. The derived neutrophil to lymphocyte ratio (dNLR), which represents an easily determinable potential prognostic marker in daily practise and clinical trials, has never been externally validated in pancreatic cancer (PC) patients. Methods Data from 474 consecutive PC patients, treated between 2004 and 2012 at a single centre, were evaluated retrospectively. Cancer-specific survival (CSS) was assessed using the Kaplan-Meier method. To evaluate the prognostic relevance of dNLR, univariate and multivariate Cox regression models were applied. Results We calculated by ROC analysis a cut-off value of 2.3 for the dNLR to be ideal to discriminate between patients’ survival in the whole cohort. Kaplan-Meier curve reveals a dNLR≥2.3 as a factor for decreased CSS in PC patients (p<0.001, log-rank test). An independent significant association between high dNLR≥2.3 and poor clinical outcome in multivariate analysis (HR = 1.24, CI95% = 1.01–1.51, p = 0.041) was identified. Conclusion In the present study we confirmed elevated pre-treatment dNLR as an independent prognostic factor for clinical outcome in PC patients. Our data encourage independent replication in other series and settings of this easily available parameter as well as stratified analysis according to tumor resectability.

Asymptomatic deep vein thrombosis and superficial vein thrombosis in ambulatory cancer patients: impact on short-term survival

Background:Asymptomatic venous thrombotic events (VTEs) are possible findings in ambulatory cancer patients. Data regarding the incidence and clinical impact of asymptomatic VTEs are conflicting. We therefore conducted a study to evaluate the occurrence of asymptomatic VTEs of the lower limbs in ambulatory cancer patients to further evaluate the association of these asymptomatic VTEs on survival during a 9-month follow-up period.Methods:In our prospective cohort, we included 150 consecutive ambulatory cancer patients who were free of any clinical symptoms for VTEs. Compression ultrasound to detect deep vein thrombosis (DVT) and superficial venous thrombosis (SVT) of the lower limbs was performed by a vascular specialist in all patients at baseline. In case of pathological findings the patients were treated with low molecular weight heparin (LMWH) because of current established guidelines. The occurrence of death was investigated during a 9-month follow-up period.Results:A total of 27 (18%) patients with VTEs were detected, which included 13 patients (8.7%) with a SVT and 16 patients (10.7%) showing a DVT. Two patients had both, a SVT and a DVT as well. During the 9-month follow-up period the occurrence of a VTE at baseline was associated with a 2.4-fold increased risk for death (HR 2.4 (1.2–5.3); P=0.03).Conclusion:Asymptomatic VTEs of the lower limbs in ambulatory cancer patients are frequently occurring concomitant features and are associated with poor survival during a 9-month follow-up period despite anticoagulation with LMWH.

Low expression of the putative tumour suppressor spinophilin is associated with higher proliferative activity and poor prognosis in patients with hepatocellular carcinoma

Background:Spinophilin, a multifunctional intracellular scaffold protein, is reduced in certain types of cancer and is regarded as a novel putative tumour suppressor protein. However, the role of spinophilin in hepatocellular carcinoma (HCC) has never been explored before.Methods:In this study, we determined for the first time the expression pattern of spinophilin in human HCC by immunohistochemistry and quantitative reverse transcriptase–PCR analysis. In addition, we performed immunohistochemical analysis of p53, p14ARF and the proliferation marker Ki-67. Kaplan–Meier curves and multivariate Cox proportional models were used to study the impact on clinical outcome. Small interfering RNA (siRNA) was used to silence spinophilin and to explore the effects of reduced spinophilin expression on cellular growth.Results:In our study, complete loss of spinophilin immunoreactivity was found in 44 of 104 HCCs (42.3%) and reduced levels were found in an additional 37 (35.6%) cases. After adjusting for other prognostic factors, multivariate Cox regression analysis identified low expression of spinophilin as an independent prognostic factor with respect to disease-free (hazard ratio (HR)=1.8; 95% confidence interval (CI)=1.04–3.40; P=0.043) and cancer-specific survival (HR=2.0; CI=1.1–3.8; P=0.025). Reduced spinophilin expression significantly correlated with higher Ki-67 index in HCC (P=0.014). Reducing spinophilin levels by siRNA induced a higher cellular growth rate and increased cyclin D2 expression in tumour cells (P<0.05).Conclusion:This is the first study of the expression pattern and distribution of spinophilin in HCC. According to our data, the loss of spinophilin is associated with higher proliferation and might be useful as a prognostic marker in patients with HCC.

Clinico-pathological characteristics and clinical outcome of different histological types of pancreatic cancer in a large Middle European series

Aims Pancreatic cancer (PC) is a heterogeneous disease in terms of histological and molecular subtypes. The aim of this study was to evaluate the prognostic impact of different histological subtypes on cancer-specific survival (CSS) in a large single-centre Middle European cohort. Methods We retrospectively studied the records of 400 consecutive PC patients who were treated from 2004 to 2010 at a single tertiary academic centre. The association of histological subtypes and parameters such as tumour stage, tumour grade, levels of tumour markers carcinoembryonic antigen and CA19-9 at diagnosis, was studied. CSS was calculated using the Kaplan-Meier method, and the influence of each parameter on CSS was assessed with univariate and multivariable Cox proportional models. Results The survival time was significantly shorter in the ductal adenocarcinoma and acinar histological subtypes compared to neuroendocrine differentiation (p<0.001). No survival difference was observed between ductal adenocarcinomas and patients with a histological variant of ductal adenocarcinoma, namely, mucinous non-cystic adenocarcinoma (p=0.7). In multivariable analysis, ductal adenocarcinoma (HR=3.1, CI 1.6 to 6.1, p=0.001) and acinar carcinoma (HR=3.2, CI 1.3 to 8.5, p=0.016) were identified as independent predictors for CSS. Conclusions Our findings suggest that the main histological subtype is an independent predictor of CSS in patients with PC. Thus, our data underline the importance of routine assessment of histological type in PC for individual risk assessment. However, no clinical rationale for the subdivision of ductal adenocarcinoma and mucinous non-cystic adenocarcinoma can be supported by our study.

A glyco-engineered anti-HER2 monoclonal antibody (TrasGEX) induces a long-lasting remission in a patient with HER2 overexpressing metastatic colorectal cancer after failure of all available treatment options

Colorectal cancer (CRC) is one of the most common forms of malignancies and the second leading cause of cancer-related death in Western countries.1 Although chemotherapy in combination with novel antiangiogenic or antiepidermal growth factor receptor (EGFR)-directed agents has significantly improved the survival time of patients with metastatic CRC (mCRC), patients with metastatic disease still face a very poor 5-year survival rate less than 10%.2 ,3 The human EGFR 2 (HER2) has proven as an effective drug target in breast and gastric cancers. In CRC, data about the therapeutic utility of anti-HER2 agents are sparse, and had been only reported for treatment-naive or second-line-treated patients.4 There is only one clinical phase II study reporting the use of the first-generation HER2/neu-directed trastuzumab in combination with irinotecan in first-line or second-line setting. Although some activity of this drug combination has been observed, the study was terminated early due to insufficient patient recruitment.5 In this patient case, we report about the successful use of a novel glyco-engineered anti-HER2 monoclonal antibody (TrasGEX) with 10-fold to 140-fold enhanced antibody dependent cellular cytotoxicity (ADCC) activity in a female patient with HER2/neu overexpressing mCRC and FcγRIII status F/V after failure of all other available treatment options. A 61-year-old female Caucasian patient was newly diagnosed with CRC in December 2002, and consequently underwent a left hemicolectomy at the Department of Surgery, Medical University of Graz. A stage II pathological T4N0 CRC was …

SOX9 is a proliferation and stem cell factor in hepatocellular carcinoma and possess widespread prognostic significance in different cancer types

SOX9 has been previously shown to be involved in hepatocellular carcinoma (HCC) and other types of cancer. However, prognostic studies so far involved rather small cohorts or lack external validation and experimental data. In this study, we firstly determined the histological expression pattern of SOX9 in human HCC by immunohistochemistry (n = 84) and evaluated its prognostic value. External cohorts of publicly available datasets were used to validate its prognostic relevance in HCC (n = 359) and other types of cancer including breast (n = 3951), ovarian (n = 1306), lung (n = 1926) and gastric cancer (n = 876). Functional SOX9 knock-down studies using siRNA and cancer stem cell models were generated in a panel of liver and breast cancer cell lines. High level of SOX9 was associated with poor survival even after adjustment for other prognostic factors in multivariate analysis (HR = 2.103, 95%CI = 1.064 to 4.156, p = 0.021). SOX9 prevailed a poor prognostic factor in all cancer validation cohorts (p<0.05). Reduced SOX9 expression by siRNA decreased the growth of liver cancer cells (p<0.05). SOX9 expression was associated with stem cell features in all tested cell lines (p<0.05). In conclusion, this study demonstrated in a large number of patients from multiple cohorts that high levels of SOX9 are a consistent negative prognostic factor.