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. 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. 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). 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.

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. Cancer Res; 73(10); 2965–75. ©2013 AACR.

Tertiary lymphoid structures (TLS) are associated with favorable outcome in non-metastatic colorectal carcinoma (nmCRC), but the dynamics of TLS maturation and its association with effective anti-tumor immune surveillance in nmCRC are unclear. Here, we hypothesized that not only the number of TLS but also their composition harbors information on recurrence risk in nmCRC. In a comprehensive molecular, tissue, laboratory, and clinical analysis of 109 patients with stage II/III nmCRC, we assessed TLS numbers and degree of maturation in surgical specimens by multi-parameter immunofluorescence of follicular dendritic cell (FDC) and germinal center (GC) markers. TLS formed in most tumors and were significantly more prevalent in highly-microsatellite-instable (MSI-H) and/or BRAF-mutant nmCRC. We could distinguish three sequential TLS maturation stages which were characterized by increasing prevalence of FDCs and mature B-cells: [1] Early TLS, composed of dense lymphocytic aggregates without FDCs, [2] Primary follicle-like TLS, having FDCs but no GC reaction, and [3] Secondary follicle-like TLS, having an active GC reaction. A simple integrated TLS immunoscore reflecting these parameters identified a large subgroup of nmCRC patients with a very low risk of recurrence independently of clinical co-variables such as ECOG performance status, age, stage, and adjuvant chemotherapy. We conclude that (1) mismatch repair and BRAF mutation status are associated with the formation of TLS in nmCRC, (2) TLS formation in nmCRC follows sequential maturation steps, culminating in germinal center formation, and (3) this maturation process harbors important prognostic information on the risk of disease recurrence.

Background: Patients with prostate cancer may present with metastatic or recurrent disease despite initial curative treatment. The propensity of metastatic prostate cancer to spread to the bone has limited repeated sampling of tumor deposits. Hence, considerably less is understood about this lethal metastatic disease, as it is not commonly studied. Here we explored whole-genome sequencing of plasma DNA to scan the tumor genomes of these patients non-invasively. Methods: We wanted to make whole-genome analysis from plasma DNA amenable to clinical routine applications and developed an approach based on a benchtop high-throughput platform, that is, Illuminas MiSeq instrument. We performed whole-genome sequencing from plasma at a shallow sequencing depth to establish a genome-wide copy number profile of the tumor at low costs within 2 days. In parallel, we sequenced a panel of 55 high-interest genes and 38 introns with frequent fusion breakpoints such as the TMPRSS2-ERG fusion with high coverage. After intensive testing of our approach with samples from 25 individuals without cancer we analyzed 13 plasma samples derived from five patients with castration resistant (CRPC) and four patients with castration sensitive prostate cancer (CSPC). Results: The genome-wide profiling in the plasma of our patients revealed multiple copy number aberrations including those previously reported in prostate tumors, such as losses in 8p and gains in 8q. High-level copy number gains in the AR locus were observed in patients with CRPC but not with CSPC disease. We identified the TMPRSS2-ERG rearrangement associated 3-Mbp deletion on chromosome 21 and found corresponding fusion plasma fragments in these cases. In an index case multiregional sequencing of the primary tumor identified different copy number changes in each sector, suggesting multifocal disease. Our plasma analyses of this index case, performed 13 years after resection of the primary tumor, revealed novel chromosomal rearrangements, which were stable in serial plasma analyses over a 9-month period, which is consistent with the presence of one metastatic clone. Conclusions: The genomic landscape of prostate cancer can be established by non-invasive means from plasma DNA. Our approach provides specific genomic signatures within 2 days which may therefore serve as 'liquid biopsy'.

PURPOSE: Previous studies suggested that serum levels of microRNA (miR)-371a-3p (so-called M371 test) have a much higher sensitivity and specificity than the classic markers of testicular germ cell tumors (GCTs) and are applicable toward both seminoma and nonseminoma. We sought to confirm the usefulness of this test as a novel biomarker for GCT. PATIENTS AND METHODS: In a prospective, multicentric study, serum samples of 616 patients with testicular GCTs and 258 male controls were examined for serum levels of miRNA-371a-3p (miR levels) by quantitative polymerase chain reaction. The GCT population encompassed 359 patients with seminoma and 257 with nonseminoma; 371 had clinical stage I disease, 201 had systemic disease, and 46 had relapses. Paired measurements before and after orchiectomy were performed in 424 patients; 118 with systemic disease had serial measurements during treatment. miR levels were compared with those of β-human chorionic gonadotropin, α-fetoprotein, and lactate dehydrogenase. RESULTS: For the primary diagnosis of GCT, the M371 test showed a sensitivity of 90.1%, a specificity of 94.0%, an area under the curve of 0.966 upon receiver operating characteristic analysis, and a positive predictive value of 97.2%. α-Fetoprotein, β-human chorionic gonadotropin, and lactate dehydrogenase had sensitivities of less than 50% in seminoma and slightly higher sensitivities in nonseminomas. miR levels were significantly associated with clinical stage, primary tumor size, and response to treatment. Relapses had elevated miR levels that subsequently dropped to normal upon remission. Teratoma did not express miR-371a-3p. CONCLUSION: The M371 test outperforms the classic markers of GCT with both a sensitivity and a specificity greater than 90%. All histologic subgroups, except teratoma, express this marker. The test could be considered for clinical implementation after further validation.

Background: Methods: Results: Conclusion: