T-regulatory cells predict clinical outcome in soft tissue sarcoma patients: a clinico-pathological study.

BACKGROUND: Soft tissue sarcomas (STS) are generally considered non-immunogenic, although specific subtypes respond to immunotherapy. Antitumour response within the tumour microenvironment relies on a balance between inhibitory and activating signals for tumour-infiltrating lymphocytes (TILs). This study analysed TILs and immune checkpoint molecules in STS, and assessed their prognostic impact regarding local recurrence (LR), distant metastasis (DM), and overall survival (OS). METHODS: One-hundred and ninety-two surgically treated STS patients (median age: 63.5 years; 103 males [53.6%]) were retrospectively included. Tissue microarrays were constructed, immunohistochemistry for PD-1, PD-L1, FOXP3, CD3, CD4, and CD8 performed, and staining assessed with multispectral imaging. TIL phenotype abundance and immune checkpoint markers were correlated with clinical and outcome parameters (LR, DM, and OS). RESULTS: Significant differences between histology and all immune checkpoint markers except for FOXP3+ and CD3-PD-L1+ cell subpopulations were found. Higher levels of PD-L1, PD-1, and any TIL phenotype were found in myxofibrosarcoma as compared to leiomyosarcoma (all p < 0.05). The presence of regulatory T cells (Tregs) was associated with increased LR risk (p = 0.006), irrespective of margins. Other TILs or immune checkpoint markers had no significant impact on outcome parameters. CONCLUSIONS: TIL and immune checkpoint marker levels are most abundant in myxofibrosarcoma. High Treg levels are independently associated with increased LR risk, irrespective of margins.

Volume Computed Tomography Perfusion Imaging: Evaluation of the Significance in Oncologic Follow-up of Metastasizing Renal Cell Carcinoma in the Early Period of Targeted Therapy - Preliminary Results.

INTRODUCTION: The aim of this study was to assess the significance of volume computed tomography perfusion imaging of metastasizing renal cell carcinoma (mRCC) in the early period after the initiation of targeted therapy. METHODS: Blood flow (BF), blood volume, and clearance (CL) were calculated in 10 patients with histologically verified mRCC before and 1 month after initiation of targeted therapy using compartmental analysis algorithms. In addition, the longest diameter of tumor was measured for both time points and compared. Correlation test was performed between perfusion parameters and size changes with time to progression (TTP). RESULTS: Blood flow and CL were significantly lower after therapy initiation, whereas blood volume and the long diameter remained unchanged. Median values before and after 4 weeks of therapy were 144.2 versus 99.4 mL/min/100 mL for BF (P = 0.009) and 115.5 versus 46.8 mL/min/100 mL for CL (P = 0.007). Changes in BF and CL showed very strong negative correlation with TTP (r = -0.838, P = 0.009 and r = -0.826, P = 0.011, respectively). CONCLUSIONS: Our preliminary study results indicate that volume computed tomography perfusion may assess targeted therapy response of mRCC earlier than the currently used Response Evaluation Criteria in Solid Tumors. In addition, changes in BF and CL may be a promising parameter for prediction of TTP.

In Situ Detection and Quantification of AR-V7, AR-FL, PSA, and KRAS Point Mutations in Circulating Tumor Cells.

BACKGROUND: Liquid biopsies can be used in castration-resistant prostate cancer (CRPC) to detect androgen receptor splice variant 7 (AR-V7), a splicing product of the androgen receptor. Patients with AR-V7-positive circulating tumor cells (CTCs) have greater benefit of taxane chemotherapy compared with novel hormonal therapies, indicating a treatment-selection biomarker. Likewise, in those with pancreatic cancer (PaCa), KRAS mutations act as prognostic biomarkers. Thus, there is an urgent need for technology investigating the expression and mutation status of CTCs. Here, we report an approach that adds AR-V7 or KRAS status to CTC enumeration, compatible with multiple CTC-isolation platforms. METHODS: We studied 3 independent CTC-isolation devices (CellCollector, Parsortix, CellSearch) for the evaluation of AR-V7 or KRAS status of CTCs with in situ padlock probe technology. Padlock probes allow highly specific detection and visualization of transcripts on a cellular level. We applied padlock probes for detecting AR-V7, androgen receptor full length (AR-FL), and prostate-specific antigen (PSA) in CRPC and KRAS wild-type (wt) and mutant (mut) transcripts in PaCa in CTCs from 46 patients. RESULTS: In situ analysis showed that 71% (22 of 31) of CRPC patients had detectable AR-V7 expression ranging from low to high expression [1-76 rolling circle products (RCPs)/CTC]. In PaCa patients, 40% (6 of 15) had KRAS mut expressing CTCs with 1 to 8 RCPs/CTC. In situ padlock probe analysis revealed CTCs with no detectable cytokeratin expression but positivity for AR-V7 or KRAS mut transcripts. CONCLUSIONS: Padlock probe technology enables quantification of AR-V7, AR-FL, PSA, and KRAS mut/wt transcripts in CTCs. The technology is easily applicable in routine laboratories and compatible with multiple CTC-isolation devices.

Patterns of venous thromboembolism risk in patients with localized colorectal cancer undergoing adjuvant chemotherapy or active surveillance: an observational cohort study.

BACKGROUND: Venous thromoboembolism (VTE) is a frequent and burdensome complication of metastatic colorectal cancer (CRC). However, the epidemiology of VTE in patients with localized CRC after surgery in curative intent is incompletely understood. In this single-center observational cohort study, we investigate patterns of VTE risk in localized CRC, and define its relationship with baseline risk factors, adjuvant chemotherapy and CRC recurrence. METHODS: Five-hundred-sixteen patients with stage II/III CRC were included retrospectively at the time of surgery, and followed until the occurrence of VTE, CRC recurrence, or death (median age = 65.1 years, stage II and III: n = 151 (29.5%), n = 361 (70.5%); adjCTX: n = 339 (65.7%)). RESULTS: During a median follow-up of 2.7 years, 15 VTEs (2.7%) and 116 recurrences (22.5%) occurred, and 46 patients (8.9%) died. Six-month, 1-year, and 5-year VTE risks were 1.6%, 2.0% and 3.2%, respectively. In competing risk time-to-VTE regression, adjCTX was not associated with an increased risk of VTE (Subdistribution hazard ratio = 0.98, 95% CI:0.33-2.88, p = 0.97). The occurrence of disease recurrence strongly increased the risk of VTE (Multi-state model: Transition hazard ratio (THR) = 13.03, 95% CI:4.39-38.74, p < 0.0001)). Conversely, the onset of VTE did not predict for recurrence (THR = 1.95, 95% CI: 0.62-6.16, p = 0.25). CONCLUSION: VTE risk is very low in localized CRC and does not appear to be increased by adjuvant chemotherapy. Thus, primary thromboprophylaxis is unlikely to result in clinical benefit in this population. The strongest determinant of VTE risk appears to be disease recurrence.

Benefit of Metastasectomy in Renal Cell Carcinoma: A Propensity Score Analysis.

INTRODUCTION: To quantify the magnitude of benefit of metastasectomy as compared to medical treatment alone in patients with metastatic renal cell carcinoma (mRCC). PATIENTS AND METHODS: We therefore conducted a propensity score analysis of overall survival (OS) in 106 mRCC patients with metachronous metastasis, of whom 36 (34%) were treated with metastasectomy, and 70 (66%) with medical therapy alone. RESULTS: The most frequent metastasectomy procedures were lung resections (n = 13) and craniotomies (n = 6). Median time-to-progression after metastasectomy was 0.7 years (25th-75th percentile: 0.3-2.7). After a median follow-up of 6.2 years and 63 deaths, 5-year OS estimates were 41% and 22% in the metastasectomy and medical therapy group, respectively (log-rank P = .00007; Hazard ratio (HR) = 0.38, 95%CI: 0.21-0.68). Patients undergoing metastasectomy had a significantly higher prevalence of favorable prognostic factors, such as fewer bilateral lung metastases and longer disease-free intervals between nephrectomy and metastasis diagnosis. After propensity score weighting for these differences and adjusting for immortal time bias, the favorable association between metastasectomy and OS became much weaker (HR = 0.62, 95%CI: 0.39-1.00, P = .050). Propensity-score-weighted 5-year OS estimates were 24% and 20% in the metastasectomy and medical therapy group, respectively (log-rank P = .001). In exploratory analyses, the benefit of metastasectomy was confined to patients who achieved complete resection of all known metastases. CONCLUSION: Within the limitations of an observational study, these findings support the concept of metastasectomy being associated with an OS benefit in mRCC patients. Metastasectomies not achieving complete resection of all known lesions are likely without OS benefit.

Gemcitabine/nab-Paclitaxel versus FOLFIRINOX for palliative first-line treatment of advanced pancreatic cancer: A propensity score analysis.

BACKGROUND: Gemcitabine/nab-paclitaxel (GN) and FOLFIRINOX are standard first-line treatment options for advanced pancreatic ductal adenocarcinoma (aPDAC), but currently no prospective randomised head-to-head comparison between these treatments has yet been performed. METHODS: We conducted a comparative propensity score (PS) analysis of overall (OS) and progression-free survival (PFS) in a tri-centre cohort of patients with aPDAC undergoing palliative first-line treatment with either GN or FOLFIRINOX. RESULTS: In unadjusted analysis, OS and PFS were highly similar between patients treated with GN (n = 297) and FOLFIRINOX (n = 158). In detail, median, 1- and 2-year OS estimates were 10.1 months, 42% and 18% in the GN group, as compared to 11.2 months, 45% and 12% in the FOLFIRINOX group, respectively (log-rank p = 0.783). Accordingly, median (4.6 versus 4.8 months), 6-month (40% versus 43%) and 1-year (9% versus 9%) PFS estimates did not significantly differ (log-rank p = 0.717). However, patients treated with FOLFIRINOX were significantly younger, had fewer comorbidities, and a better Eastern Cooperative Oncology Group performance status. These imbalances were accounted for by weighting the data with the PS. In PS analysis of survival outcomes, OS and PFS remained comparable between the two treatment groups. In detail, PS-weighted median, 1- and 2-year OS estimates were 10.1 months, 42% and 18% in the GN group, as compared to 10.1 months, 40% and 13% in the FOLFIRINOX group (PS-weighted log-rank p = 0.449). PS-weighted PFS estimates again did not differ (PS-weighted log-rank p = 0.329). CONCLUSION: This real-world comparative effectiveness study indicates that FOLFIRINOX and GN have similar effectiveness in the palliative first-line treatment of aPDAC.

The use and diagnostic value of testing myositis-specific and myositis-associated autoantibodies by line immuno-assay: a retrospective study.

AIMS: Line immune-assays (LIA) for the detection of myositis-specific antibodies (MSA) are used widely for characterization of idiopathic inflammatory myopathies (IIM). Their current use and significance for the diagnosis of IIM remains unclear. METHODS: In this retrospective analysis, we retrieved clinical diagnoses of patients tested for MSA and myositis-associated antibodies (MAA) Jo-1, Mi-2α, Mi-2ß, TIF1γ, SRP, MDA-5, NXP-2, SAE, PL-7, PL-12, EJ, OJ, PM-Scl100, PM-Scl75 and Ku. We calculated clinical specificity, clinical sensitivity, negative- and positive predictive values (PPV) as well as positive and negative likelihood ratios. RESULTS: In total, we analyzed 3167 samples. After exclusion of repeated measurements and patients with insufficient clinical information, data of 1118 patients were available for analysis. A total of 242 patients tested positive for at least one antibody, of which 45 patients had a diagnosis of IIM; 25 IIM patients were negative for all MSA/MAA. Clinical specificity of MSA/MAA for the diagnosis of IIM ranged between 94.2% and 99.9%. Clinical sensitivity and PPV across all antibodies tested ranged from 0.0% to 12.9% and 0.0% to 72.7%, respectively. CONCLUSION: In clinical practice MSA/MAA are used widely for diagnostic work-up of IIM, resulting in a low pre-test probability. Clinicians should be aware that PPVs for most MSA/MAA are low.

C-Reactive Protein (CRP) Levels in Immune Checkpoint Inhibitor Response and Progression in Advanced Non-Small Cell Lung Cancer: A Bi-Center Study.

BACKGROUND: Biomarkers for predicting response to immune checkpoint inhibitors (ICI) are scarce and often lack external validation. This study provides a comprehensive investigation of pretreatment C-reactive protein (CRP) levels as well as its longitudinal trajectories as a marker of treatment response and disease outcome in patients with advanced non-small cell lung cancer (NSCLC) undergoing immunotherapy with anti PD-1 or anti PD-L1 agents. METHODS: We performed a retrospective bi-center study to assess the association between baseline CRP levels and anti PD-(L)1 treatment outcomes in the discovery cohort (n = 90), confirm these findings in an external validation cohort (n = 101) and explore the longitudinal evolution of CRP during anti PD-(L)1 treatment and the potential impact of dynamic CRP changes on treatment response and disease outcome in the discovery cohort. Joint models were implemented to evaluate the association of longitudinal CRP trajectories and progression risk. Primary treatment outcomes were progression-free survival (PFS) and overall survival (OS), while the objective response rate (ORR) was a secondary outcome, respectively. RESULTS: In the discovery cohort, elevated pretreatment CRP levels emerged as independent predictors of worse PFS (HR per doubling of baseline CRP = 1.37, 95% CI: 1.16-1.63, p < 0.0001), worse OS (HR per doubling of baseline CRP = 1.42, 95% CI: 1.18-1.71, p < 0.0001) and a lower ORR ((odds ratio (OR) of ORR per doubling of baseline CRP = 0.68, 95% CI: 0.51-0.92, p = 0.013)). In the validation cohort, pretreatment CRP could be fully confirmed as a predictor of PFS and OS, but not ORR. Elevated trajectories of CRP during anti PD-(L)1 treatment (adjusted HR per 10 mg/L increase in CRP = 1.22, 95% CI: 1.15-1.30, p < 0.0001), as well as a faster increases of CRP over time (HR per 10 mg/L/month faster increase in CRP levels = 13.26, 95% CI: 1.14-154.54, p = 0.039) were strong predictors of an elevated progression risk, whereas an early decline of CRP was significantly associated with a reduction in PFS risk (HR = 0.91, 95% CI: 0.83-0.99, p = 0.036), respectively. CONCLUSION: These findings support the concept that CRP should be further explored by future prospective studies as a simple non-invasive biomarker for assessing treatment benefit during anti PD-(L)1 treatment in advanced NSCLC.

Benefit of second-line systemic chemotherapy for advanced biliary tract cancer: A propensity score analysis.

Whether 2nd-line-chemotherapy (2LCTX) + best-supportive-care (BSC) benefits patients with advanced biliary tract cancer (aBTC) more than BSC alone is unclear. We therefore conducted a propensity-score-based comparative effectiveness analysis of overall survival (OS) outcomes in 80 patients with metastatic, recurrent, or inoperable aBTC, of whom 38 (48%) were treated with BSC + 2LCTX and 42 (52%) with BSC alone. After a median follow-up of 14.8 months and 49 deaths, the crude 6-, 12-, and 18-month Kaplan-Meier OS estimates were 77%, 53% and 23% in the BSC + 2LCTX group, and 29%, 21%, and 14% in patients in the BSC group (p = 0.0003; Hazard ratio (HR) = 0.36, 95%CI:0.20-0.64, p = 0.001). An inverse-probability-of-treatment-weighted (IPTW) analysis was conducted to rigorously account for the higher prevalence of favorable prognostic variables in the 2LCTX + BSC group. After IPTW-weighting, the favorable association between 2LCTX and OS prevailed (adjusted HR = 0.40, 95%CI: 0.17-0.95, p = 0.037). IPTW-weighted 6-, 12-, and 18-month OS estimates were 77%, 58% and 33% in the BSC + 2LCTX group, and 39%, 28% and 22% in the BSC group (p = 0.037). Moreover, the benefit of 2LCTX was consistent across several clinically-relevant subgroups. Within the limitations of an observational study, these findings support the concept that 2LCTX + BSC is associated with an OS benefit over BSC alone in aBTC.

Genetic profiling of putative breast cancer stem cells from malignant pleural effusions.

A common symptom during late stage breast cancer disease is pleural effusion, which is related to poor prognosis. Malignant cells can be detected in pleural effusions indicating metastatic spread from the primary tumor site. Pleural effusions have been shown to be a useful source for studying metastasis and for isolating cells with putative cancer stem cell (CSC) properties. For the present study, pleural effusion aspirates from 17 metastatic breast cancer patients were processed to propagate CSCs in vitro. Patient-derived aspirates were cultured under sphere forming conditions and isolated primary cultures were further sorted for cancer stem cell subpopulations ALDH1+ and CD44+CD24-/low. Additionally, sphere forming efficiency of CSC and non-CSC subpopulations was determined. In order to genetically characterize the different tumor subpopulations, DNA was isolated from pleural effusions before and after cell sorting, and compared with corresponding DNA copy number profiles from primary tumors or bone metastasis using low-coverage whole genome sequencing (SCNA-seq). In general, unsorted cells had a higher potential to form spheres when compared to CSC subpopulations. In most cases, cell sorting did not yield sufficient cells for copy number analysis. A total of five from nine analyzed unsorted pleura samples (55%) showed aberrant copy number profiles similar to the respective primary tumor. However, most sorted subpopulations showed a balanced profile indicating an insufficient amount of tumor cells and low sensitivity of the sequencing method. Finally, we were able to establish a long term cell culture from one pleural effusion sample, which was characterized in detail. In conclusion, we confirm that pleural effusions are a suitable source for enrichment of putative CSC. However, sequencing based molecular characterization is impeded due to insufficient sensitivity along with a high number of normal contaminating cells, which are masking genetic alterations of rare cancer (stem) cells.

Multigene methylation analysis of enriched circulating tumor cells associates with poor progression-free survival in metastatic breast cancer patients.

Blood-based biomarkers such as circulating tumor cells (CTCs) provide dynamic real-time assessment of molecular tumor characteristics beyond the primary tumor. The aim of this study was to evaluate the feasibility of a size-based microfilter to assess multigene methylation analysis of enriched CTCs in a prospective proof-of principle study. We examined the quantitative methylation status of nine genes (AKR1B1, BMP6, CST6, HOXB4, HIST1H3C, ITIH5, NEUROD1, RASSF1, SOX17) in enriched CTCs from metastatic breast cancer patients. Feasibility and clinical performance testing were assessed in a test set consisting of 37 patients and 25 healthy controls. With established cut-off values from the healthy control group, methylation of enriched CTCs was detected in at least one gene in 18/37 patients (48.6%), while 97.8% of all control samples were unmethylated. Patients with CTCs unmethylated for CST6, ITIH5, or RASSF1 showed significantly longer PFS compared to patients with corresponding enriched methylated CTCs. This proof-of-principle study shows the feasibility of a size-based microfilter to enrich and analyze multigene methylation profile of CTCs from metastatic breast cancer patients. For the first time, we report that multigene methylation analysis of enriched CTCs provides prognostic information in metastatic breast cancer patients.

Cancer Stem Cell Gene Variants in CD44 Predict Outcome in Stage II and Stage III Colon Cancer Patients.

BACKGROUND/AIM: Growing evidence suggests that human cancers are stem cell diseases and recent data support the existence of cancer stem cells (CSCs) in a variety of malignancies, including colon cancer. These CSCs were shown to be capable of initiating tumor development and progression. Several studies have suggested CD133, CD26 and CD44 as markers of tumor-initiating cells of colon cancer. The purpose of the present study was to assess the impact of single-nucleotide polymorphisms (SNPs) in stem cell-related genes on clinical outcome in a large cohort of colon cancer patients with clinical stage II and III. PATIENTS AND METHODS: Data from 599 consecutive patients with colon cancer stage II and III, treated between 1995 and 2011 at a single centre, were retrospectively evaluated. Genomic DNA was extracted from paraffin-embedded normal tissue distant from the tumor to obtain germline DNA. Allelic distribution of polymorphisms was tested for deviation from Hardy-Weinberg equilibrium using χ2-test. The association of polymorphisms with time to recurrence (TTR) and overall survival (OS) was analyzed using Kaplan-Meier curves and compared by the log-rank test. Case-wise deletion for missing polymorphisms was used in univariable and multivariable analyses. RESULTS: CD44 rs187115 showed a statistically significant association with TTR; patients carrying at least one G allele had a significant reduced risk of recurrence compared to patients with the homozygous A/A variant (hazard ratio (HR)=0.67, 95% confidence interval (CI)=0.48-0.94, p=0.019). CD44 rs13347 showed a statistically significant association with OS. Patients carrying at least one T allele in rs13347 had a significantly reduced risk of death compared to patients with the homozygous C/C variant (HR=0.61, 95% CI=0.41-0.92, p=0.019). None of the other investigated polymorphisms (CD44 rs187116, CD44 rs7116432, CD44 rs353639, DPP4 rs2268889, DPP4 rs3788979, DPP4 rs7608798 and CD133 rs2240688) were associated with either TTR or OS. CONCLUSION: Germline variants rs13347 and rs187115 in the stem cell gene CD44 are prognostically relevant in stage II and III colon cancer patients.