Robust optimization of the Gross Tumor Volume compared to conventional Planning Target Volume-based planning in photon Stereotactic Body Radiation Therapy of lung tumors.

BACKGROUND: Robust optimization has been suggested as an approach to reduce the irradiated volume in lung Stereotactic Body Radiation Therapy (SBRT). We performed a retrospective planning study to investigate the potential benefits over Planning Target Volume (PTV)-based planning. MATERIAL AND METHODS: Thirty-nine patients had additional plans using robust optimization with 5-mm isocenter shifts of the Gross Tumor Volume (GTV) created in addition to the PTV-based plan used for treatment. The optimization included the mid-position phase and the extreme breathing phases of the 4D-CT planning scan. The plans were compared for tumor coverage, isodose volumes, and doses to Organs At Risk (OAR). Additionally, we evaluated both plans with respect to observed tumor motion using the peak tumor motion seen on the planning scan and cone-beam CTs. RESULTS: Statistically significant reductions in irradiated isodose volumes and doses to OAR were achieved with robust optimization, while preserving tumor dose. The reductions were largest for the low-dose volumes and reductions up to 188 ccm was observed. The robust evaluation based on observed peak tumor motion showed comparable target doses between the two planning methods. Accumulated mean GTV-dose was increased by a median of 4.46 Gy and a non-significant increase of 100 Monitor Units (MU) was seen in the robust optimized plans. INTERPRETATION: The robust plans required more time to prepare, and while it might not be a feasible planning strategy for all lung SBRT patients, we suggest it might be useful for selected patients.

Phase II study of gemcitabine, oxaliplatin and capecitabine in patients with KRAS exon 2 mutated biliary tract cancers.

Background: Molecular markers may identify subgroups of patients with clinically distinct behavior and response to treatment. In some gastrointestinal tumors, KRAS has prognostic value and negative predictive value. This is the first prospective study to report the outcome of combination chemotherapy in biliary tract cancer patients with KRAS mutation.Methods: From 2009 to 2015, 25 patients were included from two Scandinavian centers. Main inclusion criteria were non-resectable biliary tract cancer, ECOG performance status 0-2 and tumor KRAS mutation. A bi-weekly cycle of chemotherapy was administered as gemcitabine 1000 mg/m2 and oxaliplatin 85 mg/m2 day 1, followed by 7 days of oral capecitabine 1000 mg/m2. Response evaluation was done every six treatment and the primary endpoint was the fraction with progression free survival (PFS) at 6 months. The study also included a non-preplanned analysis of circulating tumor specific DNA.Results: Chemotherapy was given for a median of 5 months (range 0-14) and among 17 patients evaluable for response, best responses were complete response (1), partial response (2), and stable disease (14). Eighteen patients had CT-verified progression, six died between evaluations and one patient is still progression-free. Median PFS was 6.8 months (95% CI 3.1-11.0) and median overall survival (OS) was 11.2 months (95% CI 6.6-14.3). The fraction with PFS at 6 months was 52% (95% CI 31-69%). Exploratory analyses found an improved survival in patients with a low level of plasma DNA.Conclusion: Pretreatment molecular characterization was feasible in BTC, but the rate of KRAS mutations was low. The study met its primary endpoint with a fraction of PFS at six months of 52%. The effect of combination chemotherapy with gemcitabine, oxaliplatin and capecitabine in this selected population was comparable to results from unselected groups with PFS and OS of 6.8 and 11.2 months, respectively. ClinicalTrials.gov NCT00779454.

Circulating miRNAs in localized/locally advanced prostate cancer patients after radical prostatectomy and radiotherapy.

BACKGROUND: Overtreatment is a well-known clinical challenge in local prostate cancer (PCa). Although risk assessment models have contributed to a better stratification of patients with local PCa, a tailored management is still in its infancy. Over the last few decades, microRNAs (miRNAs) have shown promising results as biomarkers in PCa. The aim of this study was to investigate circulating miRNAs after management of local PCa. METHODS: The relative expression of four miRNAs (miRNA-21, -93, -125b, and miRNA-221) was assessed in plasma from 149 newly diagnosed patients with local or locally advanced PCa. Real-time polymerase chain reaction was used for analysis. A baseline sample at time of diagnosis and a follow-up sample after 6 months were assessed. The patients were grouped in an interventional cohort (radical prostatectomy, curative intent radiotherapy, or androgen-deprivation therapy alone) and an observational cohort (watchful waiting or active surveillance). RESULTS: In the interventional cohort, levels of both miRNA-93 and miRNA-221 were significantly lower in the follow-up samples compared to baseline z = -2.738, P = 0.006, and z = -4.498, P < 0.001, respectively. The same observation was recorded for miRNA-125b in the observational cohort (z = -2.656, P = 0.008). Both miRNA-125b and miRNA-221 were correlated with risk assessment r = 0.23, P = 0.015, and r = 0.203, P = 0.016 respectively, while miRNA-93 showed tendency to significant correlation with the prostatectomy Gleason score (r = 0.276, P = 0.0576). CONCLUSIONS: The current results indicate a possible role of miRNA-93 and miRNA-221 in disease monitoring in localized and locally advanced PCa. Larger studies are warranted to assess the clinical impact of these biomarkers.

Programmed Death Ligand-1 expression in stage II colon cancer - experiences from a nationwide populationbased cohort.

BACKGROUND: Patients suffering from high risk stage II colon cancer (CC) may benefit from adjuvant onco-therapy, but additional prognostic markers are needed for better treatment stratification. We investigated the prognostic value of Programmed Death Ligand-1 (PD-L1) in a true population-based cohort of patients with stage II CC. METHODS: PD-L1 expression on tumour cells was evaluated by immunohistochemistry in 572 colon cancers. Whole sections from tumour blocks representing the deepest invasive front of the primary tumour were used for analysis. A cut-off of 5% positivity was used for dichotomizing the data. The prognostic value was investigated in Cox proportional hazard models for recurrence-free survival (RFS) and overall survival (OS). RESULTS: Overall, 6% of the tumours were classified as high PD-L1. High PD-L1 was related to female gender (p = 0.028), high malignancy grade (< 0.001), right side localization (p < 0.001) and microsatellite instability (MSI) (p < 0.001). Thirty-one (18%) of the MSI and 4 (1%) of the microsatellite stable tumours were classified as high PD-L1, respectively. PD-L1 expression provided no prognostic value as a single marker. In patients with MSI tumours, high PD-L1 expression had no significant impact regarding OS or RFS. CONCLUSIONS: PD-L1 expression in tumour cells of stage II CC did not provide any prognostic impact, neither in the entire population-based cohort nor in the group of MSI patients. Additional investigations of the immunogenic microenvironment are needed for evaluating the prognostic information in CC.

The prognostic value of tumour stroma ratio and tumour budding in stage II colon cancer. A nationwide population-based study.

PURPOSE: High-risk patients with stage II colon cancer (CC) may benefit from adjuvant chemotherapy, but additional prognostic markers are needed for better stratification. We investigated the prognostic value of tumour stroma ratio (TSR) and tumour budding (TB). METHODS: A nationwide population-based cohort of 573 patients with stage II CC was included. TSR was scored on hematoxylin and eosin sections as low TSR (> 50% stroma) and high TSR (≤ 50% stroma). TB was evaluated in hotspots on pan-cytokeratin stained sections in 10 high power fields (HPF) at the invasive front and classified by the mean number of buds per HPF as high grade budding (≥ 10 buds) or low-grade budding (< 10 buds). The prognostic value was investigated in Cox proportional hazard models for recurrence-free survival (RFS) and overall survival (OS). RESULTS: Low TSR was associated with worse RFS (HR = 1.342 (95% CI 1.006-1.791), p = 0.045) and OS (HR = 1.376 (95% CI 1.016-1.862), p = 0.039). Furthermore, an association was found between low TSR and microsatellite stabile tumours (p < 0.001). The mean number of buds per HPF was associated to TSR with increasing number of buds related to a lower TSR (p = 0.026). No statistically significant prognostic impact of TB regarding OS or RFS was detected. CONCLUSIONS: TSR provided valuable prognostic information, and adding TSR to the current risk stratification may contribute to better patient selection. The estimates of TSR and TB were found to be associated, but no prognostic value of TB was documented.

Cell-Free DNA in Metastatic Colorectal Cancer: A Systematic Review and Meta-Analysis.

BACKGROUND: Circulating DNA can be detected and quantified in the blood of cancer patients and used for detection of tumor-specific genetic alterations. The clinical utility has been intensively investigated for the past 10 years. The majority of reports focus on analyzing the clinical potential of tumor-specific mutations, whereas the use of total cell-free DNA (cfDNA) quantification is somehow controversial and sparsely described in the literature, but holds important clinical information in itself. The purpose of the present report was to present a systematic review and meta-analysis of the prognostic value of total cfDNA in patients with metastatic colorectal cancer (mCRC) treated with chemotherapy. In addition, we report on the overall performance of cfDNA as source for KRAS mutation detection. MATERIALS AND METHODS: A systematic literature search of PubMed and Embase was performed by two independent investigators. Eligibility criteria were (a) total cfDNA analysis, (b) mCRC, and (c) prognostic value during palliative treatment. The preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines were followed, and meta-analysis applied on both aggregate data extraction and individual patients' data. RESULTS: Ten eligible cohorts were identified, including a total of 1,076 patients. Seven studies used quantitative polymerase chain reaction methods, two BEAMing [beads, emulsification, amplification, and magnetics] technology, and one study digital droplet polymerase chain reaction. The baseline levels of cfDNA was similar in the presented studies, and all studies reported a clear prognostic value in favor of patients with lowest levels of baseline cfDNA. A meta-analysis revealed a combined estimate of favorable overall survival hazard ratio (HR) in patients with levels below the median cfDNA (HR = 2.39, 95% confidence interval 2.03-2.82, p < .0001). CONCLUSION: The total cfDNA levels are high in patients with mCRC and bear strong prognostic information, which should be tested prospectively by using a predefined cut-off value based on normal values in healthy cohorts. Finally, the potential use of cfDNA for detection of tumor-specific mutations was emphasized in a large individual patients' data meta-analysis. IMPLICATIONS FOR PRACTICE: Reliable prognostic markers could help to guide patients and treating physicians regarding the relevance and choice of systemic therapy. Small fragments of circulating cell-free DNA (cfDNA) can be measured in a simple blood sample. This report presents the first meta-analysis of the prognostic value of total cfDNA measurement in patients with metastatic colorectal cancer. Data from 1,076 patients confirmed that patients with the lowest pre-treatment levels of cfDNA had a significantly higher chance of longer survival than those with higher levels. Cell-free DNA analysis can also be used for detection of tumor-specific mutations, and hold potential as a valuable tool in colorectal cancer treatment.

Risk of recurrence in patients with colon cancer stage II and III: a systematic review and meta-analysis of recent literature.

BACKGROUND: Adjuvant chemotherapy is established routine therapy for colon cancer (CC) patients with radically resected stage III and 'high-risk' stage II disease. The decision on recommending adjuvant chemotherapy, however, is based on data from older patient cohorts not reflecting improvements in pre-operative staging, surgery, and pathological examination. The aim is to review the current risk of recurrence in stage II and III patients and second, to estimate the relative importance of routinely assessed clinico-pathological variables. METHODS: The PubMed/MEDLINE and the Cochrane databases were systematically searched for randomized controlled studies and observational studies published after 1 January 2005 with patients included after January 1995 on prognosis in surgically treated stage II and III CC patients. RESULTS: Of 2596 studies identified, 37 met the inclusion criteria and 25 provided data for meta-analysis. The total patient sample size in the 25 studies reporting either disease-free (DFS) or recurrence-free survival was 15 559 in stage II and 18 425 in stage III. Five-year DFS for stage II patients operated without subsequent adjuvant chemotherapy was 81.4% [95% confidence interval (CI) 75.4-87.4; in studies with good/very good quality of reporting 82.7%, (95% CI 80.8-84.6)]. For stage II patients treated with adjuvant chemotherapy, the five-year DFS was 79.3% (95% CI 75.6-83.1). For stage III patients without chemotherapy, five-year DFS was 49.0% (95% CI 23.2-74.8) and for those treated with adjuvant chemotherapy, 63.6% (95% CI 59.3-67.9). The prognostic impact of commonly investigated clinico-pathological parameters, (pT-stage, pN-stage, differentiation, number of lymph nodes studied, MMR-status, and emergency surgery) were confirmed. CONCLUSIONS: In this meta-analysis, studies with good quality of reporting show a five-year DFS of 82.7% for stage II CC without adjuvant chemotherapy, whereas the five-year DFS is 63.8% for stage III CC with adjuvant chemotherapy. Due to insufficient reporting on treatment quality the presented DFS is likely an under-estimation of what is achieved at high-quality centers today.

Description of an activity-based enzyme biosensor for lung cancer detection.

BACKGROUND: Lung cancer is associated with the greatest cancer mortality as it typically presents with incurable distributed disease. Biomarkers relevant to risk assessment for the detection of lung cancer continue to be a challenge because they are often not detectable during the asymptomatic curable stage of the disease. A solution to population-scale testing for lung cancer will require a combination of performance, scalability, cost-effectiveness, and simplicity. METHODS: One solution is to measure the activity of serum available enzymes that contribute to the transformation process rather than counting biomarkers. Protease enzymes modify the environment during tumor growth and present an attractive target for detection. An activity based sensor platform sensitive to active protease enzymes is presented. A panel of 18 sensors was used to measure 750 sera samples from participants at increased risk for lung cancer with or without the disease. RESULTS: A machine learning approach is applied to generate algorithms that detect 90% of cancer patients overall with a specificity of 82% including 90% sensitivity in Stage I when disease intervention is most effective and detection more challenging. CONCLUSION: This approach is promising as a scalable, clinically useful platform to help detect patients who have lung cancer using a simple blood sample. The performance and cost profile is being pursued in studies as a platform for population wide screening.

Lung cancer is responsible for more deaths worldwide than all other cancers. It is often detected with the appearance of symptoms when treatment is limited and outcomes for the patient are much worse. While imaging chest scans can detect disease, they are poorly used even in the United States where it is an approved screening method. When cancer is present, protease enzymes are responsible for making space and modifying the lung tissue for the growing tumor. This report describes a panel of 18 sensors that release a fluorescent signal when these enzymes are present in a blood sample. The signal acts like a fingerprint of activity that can be used to identify people with lung cancer. This sensor platform can detect patients with curable lung cancer and could provide a platform for screening very large populations of at-risk individuals.

Non-CpG island promoter hypomethylation and miR-149 regulate the expression of SRPX2 in colorectal cancer.

Gene silencing by DNA hypermethylation of CpG islands is a well-characterized phenomenon in cancer. The effect of hypomethylation in particular of non-CpG island genes is much less well described. By genome-wide screening, we identified 105 genes in microsatellite stable (MSS) colorectal adenocarcinomas with an inverse correlation (Spearman's ρ ≤ -0.40) between methylation and expression. Of these, 35 (33%) were hypomethylated non-CpG island genes and two of them, APOLD1 (Spearman's ρ = -0.82) and SRPX2 (Spearman's ρ = -0.80) were selected for further analyses. Hypomethylation of both genes were localized events not shared by adjacent genes. A set of 662 FFPE DNA samples not only confirmed that APOLD1 and SRPX2 are hypomethylated in CRC but also revealed hypomethylation to be significantly (p < 0.01) associated with tumors being localized in the left side, CpG island methylator phenotype negative, MSS, BRAF wt, undifferentiated and of adenocarcinoma histosubtype. Demethylation experiments supported SRPX2 being epigenetically regulated via DNA methylation, whereas other mechanisms in addition to DNA methylation seem to be involved in the regulation of APOLD1. We further identified miR-149 as a potential novel post-transcriptional regulator of SRPX2. In carcinoma tissue, miR-149 was downregulated and inversely correlated to SRPX2 (ρ = -0.77). Furthermore, ectopic expression of miR-149 significantly reduced SRPX2 transcript levels. Our study highlights that in colorectal tumors, hypomethylation of non-CpG island-associated promoters deregulate gene expression nearly as frequent as do CpG-island hypermethylation. The hypomethylation of SRPX2 is focal and not part of a large block. Furthermore, it often translates to an increased expression level, which may be modulated by miR-149.

Combining sCD163 with CA 19-9 Increases the Predictiveness of Pancreatic Ductal Adenocarcinoma.

The objective of this study was to evaluate the diagnostic and prognostic potential of soluble CD163 (sCD163) in patients with pancreatic ductal adenocarcinoma (PDAC). Preoperative serum samples from 255 patients with PDAC were analyzed for sCD163 using a commercially available enzyme-linked immunosorbent assay. The diagnostic value of sCD163 was evaluated using receiver operating characteristic (ROC) curves. The prognostic significance of sCD163 was evaluated by Cox regression analysis and Kaplan-Meier survival curves. sCD163 was significantly increased in patients with PDAC, across all stages, compared to healthy subjects (stage 1: p value = 0.033; stage 2-4: p value ≤ 0.0001). ROC curves showed that sCD163 combined with CA 19-9 had the highest diagnostic potential compared to sCD163 and CA 19-9 alone both in patients with local PDAC and patients with advanced PDAC. Univariate and multivariate analysis showed no association between sCD163 and overall survival. This study found elevated levels of circulating sCD163 in patients with PDAC, regardless of stage, compared to healthy subjects. This suggests that sCD163 may have a clinical value as a novel diagnostic biomarker in PDAC.

The Soluble Urokinase-Type Plasminogen Activator Receptor as a Biomarker for Survival and Early Treatment Effect in Metastatic Colorectal Cancer.

The soluble urokinase-type plasminogen activator receptor (suPAR) is prognostic for overall survival (OS) in colorectal cancer (CRC). Our study explored the association between baseline suPAR and OS and progression-free survival (PFS) in metastatic CRC (mCRC). It is also the first study to explore the association between the initial change in suPAR level and OS, PFS and the first CT response evaluation. The study included 132 patients with mCRC treated with chemotherapy (FOLFIRI) with or without an EGFR-inhibitor. Blood samples were drawn before the first treatment cycle and in between the first and second treatment cycle. suPAR levels were determined using an ELISA assay. Using the Kaplan-Meyer method, we demonstrated a significantly shorter OS for patients with suPAR levels above the median (HR = 1.79, 95%CI = 1.10-2.92, p = 0.01). We also showed association between plasma suPAR level, gender and performance status (PS). However, we could not show any association with PFS, and analysis on the change in suPAR level provided no significant results. The results showing association between baseline suPAR and OS are in line with previous findings.

Early ctDNA response to chemotherapy. A potential surrogate marker for overall survival.

AIM: The aim of the study was to compare ctDNA response rate and objective response rate as surrogate markers for overall survival (OS) in patients with metastatic cancer treated with chemotherapy. METHODS: The study included 420 patients distributed in five cohorts with colorectal, ovarian, and non-small cell lung cancer. It represents a retrospective analysis of patients enrolled in prospective biomarker studies and clinical trials. All patients had ctDNA measured before start of treatment and at the first evaluation of objective response. ctDNA response rate was defined as the fraction of patients converting from a measurable level at baseline to an unmeasurable level at the first evaluation of objective response. Aberrant, tumour specific, methylated DNA was measured in plasma. The method involves DNA isolation, bisulphite conversion and droplet digital PCR. The primary outcome measure was the correlation between ctDNA response rate, overall response rate (ORR) and median survival. RESULTS: There was moderate correlation between ctDNA response rate and objective response at first evaluation (R2 = 0.68). The same applied to ctDNA response rate and ORR (R2 = 0.57). ctDNA held prognostic information in all the investigated tumour types (p < 0.05). There was a high correlation between ctDNA response and median survival across the included tumour types and treatments (R2 = 0.99) clearly outperforming both response at first evaluation and ORR (R2 = 0.70 and 0.57, respectively). CONCLUSION: The results suggest that ctDNA response might serve as a surrogate marker for OS. If validated, it may have great implications on the approval of new drugs.

Comparison of Mutated KRAS and Methylated HOXA9 Tumor-Specific DNA in Advanced Lung Adenocarcinoma.

Circulating tumor DNA (ctDNA) has been suggested as a biomarker in non-small cell lung cancer. The optimal target for measuring ctDNA has not yet been established. This study aimed to investigate methylated Homeobox A9 (meth-HOXA9) as an approach to detect ctDNA in advanced lung adenocarcinoma and compare it with mutated Kirsten rat sarcoma viral oncogene homolog (mut-KRAS) in order to determine the mutual agreement. DNA was purified from formalin-fixed, paraffin-embedded non-malignant lung tissue and lung adenocarcinoma tissue, and plasma from healthy donors and lung adenocarcinoma patients, respectively. KRAS mutations in tumor tissue were identified by next-generation sequencing and quantified in tumor and plasma by droplet digital polymerase chain reaction (ddPCR). The meth-HOXA9 analysis was based on bisulfite-converted DNA from tumor and plasma and quantified by ddPCR. Samples consisted of 20 archival non-malignant lung tissues, 48 advanced lung adenocarcinomas with matched plasma samples, and 100 plasma samples from healthy donors. A KRAS mutation was found in the tumor in 34/48 (70.8%) adenocarcinoma patients. All tumors were positive for meth-HOXA9, while none of the non-malignant lung tissues were. Meth-HOXA9 was detected in 36/48 (75%) of plasma samples, and the median level was 0.7% (range of 0-46.6%, n = 48). Mut-KRAS was detected in 29/34 (85.3%) of the plasma samples, and the median level was 1.2% (range of 0-46.1%, n = 34). There was a good correlation between meth-HOXA9 and mut-KRAS in plasma (Spearman's rho 0.83, p < 0.001). Meth-HOXA9 is present in tissue from incurable lung adenocarcinoma but not in non-malignant lung tissue. It may be used as an approach for detecting ctDNA. The results demonstrated a high agreement between meth-HOXA9 and mut-KRAS in patients with advanced lung adenocarcinoma.

Early identification of treatment benefit by methylated circulating tumor DNA in metastatic colorectal cancer.

BACKGROUND: The early identification of treatment effect is wanted in several settings, including the management of metastatic colorectal cancer (mCRC). A potential universal marker is circulating tumor DNA (ctDNA). Our prospective study explored the association between progression-free survival (PFS) and overall survival (OS), and early change of ctDNA after one cycle of chemotherapy in patients with mCRC. METHODS: The study included mCRC patients receiving standard first line combination chemotherapy with 5-Fluorouracil (FU), oxaliplatin, and bevacizumab. Hypermethylated neuropeptide Y (NPY) ctDNA (meth-ctDNA) served as a marker analyzed by droplet digital polymerase chain reaction (PCR). The meth-ctDNA level was analyzed in plasma before treatment start and again before cycle two. The patients were divided into two groups according to the dynamics of meth-ctDNA. Low ctDNA (LctDNA) included patients with zero or values of meth-ctDNA decreasing to a level including zero in the 95% confidence interval. High ctDNA (HctDNA) included all other patients (stable, increasing, or slightly decreasing values). The two groups were compared as to PFS and OS. RESULTS: The study included 123 patients. The PFS in the two groups differed significantly with a median of 9.2 and 6.7 months in LctDNA and HctDNA, respectively (p = 0.0005). This translated into a 12-month difference in OS with a median of 25.4 and 13.5 months, respectively (p = 0.0001). CONCLUSIONS: Early therapeutic reconsideration is of utmost importance. A low level of meth-ctDNA after one cycle of chemotherapy in the first line setting is a potential marker for excellent clinical outcomes. The clinical utility should be confirmed in randomized clinical trials.

Correlation Between Tumor-Specific Mutated and Methylated DNA in Colorectal Cancer.

PURPOSE: Analysis of circulating tumor DNA (ctDNA) is a potential improvement in precision medicine. In colorectal cancer (CRC), somatic mutations such as RAS and RAF in the blood (mut-ctDNA) are investigated for prognostic and predictive purposes. However, they are only present in approximately 60% of patients. Recently, ctDNA has been detected in patients with RAS/RAF wild type (WT) by methylated ctDNA (meth-ctDNA). The aim of this study was to compare mutated DNA with methylated DNA in malignant and nonmalignant tissue and plasma from CRC cohorts to establish a universal biomarker for ctDNA in all patients with CRC. MATERIALS AND METHODS: Tissue (n = 170) and plasma (n = 147) samples were analyzed for RAS/RAF mutations and neuropeptide Y methylation by droplet digital polymerase chain reaction. Tissue originated from nonmalignant WT and RAS/RAF-mutated adenomas, tumor-adjacent colorectal tissue, and WT and RAS/RAF-mutated tumor tissue. Plasma samples represented healthy donors and localized and metastatic CRCs. RESULTS: The level of neuropeptide Y-methylated DNA in the tissue cohorts differed between nonmalignant and malignant/premalignant tissues with minimal overlap. Furthermore, meth-ctDNA was detected in plasma from 100% of patients with metastatic disease, compared with 67% of those with localized disease and 8% of healthy donors. Median fraction of meth-ctDNA in metastatic and localized cancers was 13.25% and 0.04%, respectively. Correlation between mut-ctDNA and meth-ctDNA was high (r = 0.77 and 0.80 in localized and metastatic settings, respectively). CONCLUSION: Mut-ctDNA is interchangeable with meth-ctDNA in patients with CRC. On the basis of our results, meth-ctDNA should be considered a universal biomarker in metastatic CRC, but additional investigations of clinical utility are warranted.

The Prognostic Value of Tumor-Infiltrating lymphocytes in Stage II Colon Cancer. A Nationwide Population-Based Study.

BACKGROUND: Additional prognostic markers are needed for better treatment stratification of stage II colon cancer (CC). We investigated the prognostic value of tumor-infiltrating lymphocytes (TILs) in a true population-based cohort of patients with stage II CC. MATERIAL AND METHODS: A total of 573 patients were included. Tumor blocks representing the deepest invasive part of the primary tumor were used for analysis. CD3+ and CD8+ TILs at the invasive front were evaluated by immunohistochemistry on whole tumor sections. The invasive area was manually outlined, and Visiopharm Integrator System software was used for quantification. Data were dichotomized for comparison with clinical data. The prognostic value was investigated in Cox proportional-hazard models for recurrence-free survival (RFS) and overall survival (OS). RESULTS: Low CD3+ or CD8+ TILs were significantly associated with poor RFS and OS (P = .0021 and P ≤ .0009, respectively, log-rank test). In multiple Cox regression analysis, low CD3+ and CD8+ TILs were associated with reduced RFS with hazard ratio (HR) = 1.386 (95% CI 1.039-1.850), P = .026, and HR = 1.394 (95% CI 1.029-1.890), P = .032, respectively, independent of age, T-stage, localization, perforation, and microsatellite instability (MSI). In the subgroups of patients with low CD3+ or CD8+ TILs, there was no difference in survival between patients with MSI and microsatellite-stable tumors, (P = .821 and P = .907, respectively). CONCLUSION: Low CD3+ and CD8+ TILs in the invasive area are both related to inferior prognosis of stage II CC, and we recommend either of these parameters to be considered as additional high-risk factor.

Does heterogeneity matter in the estimation of tumour budding and tumour stroma ratio in colon cancer?

BACKGROUND: Tumour budding (TB) and Tumour Stroma Ratio (TSR) may be rewarding in the treatment stratification of patients with stage II colon cancer. However, lack of standardization may exclude these parameters from being used in a clinical setting. The purpose of this methodologic study was to compare stereology with semi-quantitative estimations of TSR, to investigate the intra-tumoural heterogeneity of TB and TSR, and to assess the intra- and inter-observer agreement. METHODS: Three paraffin embedded tumour blocks, one of them representing the deepest invasive front, were selected from each of 43 patients treated for stage II colon cancer. TSR was estimated in H&E sections semi-quantitatively using conventional microscopy, and stereologically on scanned slides, using the newCAST stereology platform. TB was scored across 10 high power fields at the invasive front in cytokeratin AE1/AE3 stained sections. RESULTS: Subjective, semi-quantitative estimates of TSR significantly correlated to the stereological estimates, with the best correlation found for sections with the deepest invasive tumour penetration (σ = 0.621, p < 0.001). Inter-observer agreement was moderate to substantial for both TB (Κappa = 0.46-0.73) and TSR (Κappa = 0.70-0.75). The Intraclass correlation coefficient (ICC) for TSR varied from 0.322 based on stereological hotspot estimation to 0.648 for the semi-quantitative evaluation. For TB, ICC varied from 0.646 based on continuous data to 0.698 based on categorical data (cut-off: 10 buds). Thus, the intra-tumoural heterogeneity for both TB and the semi-quantitative estimation of TSR was low. CONCLUSION: We recommend using only one tissue section representing the deepest invasive tumour area for estimation of TSR. For TB we recommend using one tissue section; however due to low representation of high-budding tumours, results must be considered with caution.

Computer-assisted stereology and automated image analysis for quantification of tumor infiltrating lymphocytes in colon cancer.

BACKGROUND: Precise prognostic and predictive variables allowing improved post-operative treatment stratification are missing in patients treated for stage II colon cancer (CC). Investigation of tumor infiltrating lymphocytes (TILs) may be rewarding, but the lack of a standardized analytic technique is a major concern. Manual stereological counting is considered the gold standard, but digital pathology with image analysis is preferred due to time efficiency. The purpose of this study was to compare manual stereological estimates of TILs with automatic counts obtained by image analysis, and at the same time investigate the heterogeneity of TILs. METHODS: From 43 patients treated for stage II CC in 2002 three paraffin embedded, tumor containing tissue blocks were selected one of them representing the deepest invasive tumor front. Serial sections from each of the 129 blocks were immunohistochemically stained for CD3 and CD8, and the slides were scanned. Stereological estimates of the numerical density and area fraction of TILs were obtained using the computer-assisted newCAST stereology system. For the image analysis approach an app-based algorithm was developed using Visiopharm Integrator System software. For both methods the tumor areas of interest (invasive front and central area) were manually delineated by the observer. RESULTS: Based on all sections, the Spearman's correlation coefficients for density estimates varied from 0.9457 to 0.9638 (p < 0.0001), whereas the coefficients for area fraction estimates ranged from 0.9400 to 0.9603 (P < 0.0001). Regarding heterogeneity, intra-class correlation coefficients (ICC) for CD3+ TILs varied from 0.615 to 0.746 in the central area, and from 0.686 to 0.746 in the invasive area. ICC for CD8+ TILs varied from 0.724 to 0.775 in the central area, and from 0.746 to 0.765 in the invasive area. CONCLUSIONS: Exact objective and time efficient estimates of numerical densities and area fractions of CD3+ and CD8+ TILs in stage II colon cancer can be obtained by image analysis and are highly correlated to the corresponding estimates obtained by the gold standard based on stereology. Since the intra-tumoral heterogeneity was low, this method may be recommended for quantifying TILs in only one histological section representing the deepest invasive tumor front.

Tissue microRNAs as predictors of outcome in patients with metastatic colorectal cancer treated with first line Capecitabine and Oxaliplatin with or without Bevacizumab.

PURPOSE: We tested the hypothesis that expression of microRNAs (miRNAs) in cancer tissue can predict effectiveness of bevacizumab added to capecitabine and oxaliplatin (CAPEOX) in patients with metastatic colorectal cancer (mCRC). EXPERIMENTAL DESIGN: Patients with mCRC treated with first line CAPEOX and bevacizumab (CAPEOXBEV): screening (n = 212) and validation (n = 121) cohorts, or CAPEOX alone: control cohort (n = 127), were identified retrospectively and archival primary tumor samples were collected. Expression of 754 miRNAs was analyzed in the screening cohort using polymerase chain reaction (PCR) arrays and expression levels were related to time to disease progression (TTP) and overall survival (OS). Significant miRNAs from the screening study were analyzed in all three cohorts using custom PCR arrays. In situ hybridization (ISH) was done for selected miRNAs. RESULTS: In the screening study, 26 miRNAs were significantly correlated with outcome in multivariate analyses. Twenty-two miRNAs were selected for further study. Higher miR-664-3p expression and lower miR-455-5p expression were predictive of improved outcome in the CAPEOXBEV cohorts and showed a significant interaction with bevacizumab effectiveness. The effects were strongest for OS. Both miRNAs showed high expression in stromal cells. Higher expression of miR-196b-5p and miR-592 predicted improved outcome regardless of bevacizumab treatment, with similar effect estimates in all three cohorts. CONCLUSIONS: We have identified potentially predictive miRNAs for bevacizumab effectiveness and additional miRNAs that could be related to chemotherapy effectiveness or prognosis in patients with mCRC. Our findings need further validation in large cohorts, preferably from completed randomized trials.

Functional screening identifies miRNAs influencing apoptosis and proliferation in colorectal cancer.

MicroRNAs (miRNAs) play a critical role in many biological processes and are aberrantly expressed in human cancers. Particular miRNAs function either as tumor suppressors or oncogenes and appear to have diagnostic and prognostic significance. Although numerous miRNAs are dys-regulated in colorectal cancer (CRC) only a small fraction has been characterized functionally. Using high-throughput functional screening and miRNA profiling of clinical samples the present study aims at identifying miRNAs important for the control of cellular growth and/or apoptosis in CRC. The high-throughput functional screening was carried out in six CRC cell lines transfected with a pre-miR library including 319 synthetic human pre-miRs. Phenotypic alterations were evaluated by immunostaining of cleaved cPARP (apoptosis) or MKI67 (proliferation). Additionally, TaqMan Human MicroRNA Array Set v2.0 was used to profile the expression of 667 miRNAs in 14 normal colon mucosa and 46 microsatellite stable stage II CRC patients. Among the miRNAs that induced growth arrest and apoptosis in the CRC cell lines, and at same time were dys-regulated in the clinical samples, miR-375 was selected for further analysis. Independent in vitro analysis of transient and stable transfected CRC cell lines confirmed that miR-375 reduces cell viability through the induction of apoptotic death. We identified YAP1 as a direct miR-375 target in CRC and show that HELLS and NOLC1 are down-stream targets. Knock-down of YAP1 mimicked the phenotype induced by miR-375 over-expression indicating that miR-375 most likely exerts its pro-apoptotic role through YAP1 and its anti-apoptotic down-stream targets BIRC5 and BCL2L1. Finally, in vivo analysis of mouse xenograft tumors showed that miR-375 expression significantly reduced tumor growth. We conclude that the high-throughput screening successfully identified miRNAs that induce apoptosis and/or inhibit proliferation in CRC cells. Finally, combining the functional screening with profiling of CRC tissue samples we identified clinically relevant miRNAs and miRNA targets in CRC.