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Capsular contracture is a severe complication to breast implants, but the pathophysiology remains unclear, and consensus is lacking on how to analyze implant capsules histologically. In this study, we developed and validated a histological scoring system for analyzing breast implant capsules. Biopsies of breast implant capsules from 480 patients who had undergone breast augmentation or reconstruction were collected and stained with hematoxylin and eosin. First, biopsies from 100 patients were used to select parameters for the scoring system. Then, biopsies from the remaining 380 patients were used to determine intra- and interobserver agreement of two blinded non-pathologist-observers and agreement with a pathologist. Finally, we tested the association between the parameters and capsular contracture. The histological scoring system consisted of ten parameters assessed on a two-, three-, or four-point scale. Intra- and interobserver agreement was almost perfect (0.83 and 0.80), and agreement with the pathologist was substantial (0.67). Inflammatory infiltration (pppp

Inflammation is one of the hallmarks of cancer and plays a crucial role in the development and progression. The objective of the present study was to investigate if high serum YKL-40 is related to poor prognosis in cervical cancer (CC) patients. A prospective biomarker study of 116 patients with CC (FIGO stage Ia

Purpose The Prosigna-PAM50 risk of recurrence (ROR) score has documented clinical utility for the prediction of 10-year distant recurrence (DR). The present study investigated the value of Prosigna-PAM50 for predicting 10-year DR and overall survival after 5 years of endocrine treatment for postmenopausal patients with invasive lobular carcinoma. Patients and Methods Using the Danish Breast Cancer Group database, we identified patients with a diagnosis from 2000 to 2003 of estrogen receptor-positive, human epidermal growth factor receptor 2-negative invasive ductal (n = 1570) or lobular (n = 341) cancer > 20 mm or 1 to 3 positive lymph nodes and applied multivariate Cox models. Results The median follow-up for DR was 9.3 years and for overall survival 15.2 years. Of the 341 lobular and 1570 ductal cases, 140 (41%) and 349 (22%) were classified as low ROR, with a 10-year DR rate of 7.7% (95% confidence interval [CI], 3.7%-13.6%) and 3.5% (95% CI, 1.8%-6.2%), respectively. The 10-year DR rate for the intermediate ROR group for those with lobular cancer was 18% (95% CI, 10.1%-27.9%) compared with 9.7% (95% CI, 6.7%-13.4%) for those with ductal cancer. Luminal B tumors had a significantly worse outcome than luminal A tumors in both lobular (hazard ratio, 1.89; 95% CI, 1.03%-3.45%; P = .04) and ductal (hazard ratio, 3.18; 95% CI, 2.29%-4.43%; P Conclusion Prosigna PAM-50 provides significant prognostic information beyond the clinicopathologic factors in patients with invasive lobular breast cancer. Those with lobular cancer had worse 10-year DR rates compared with those with ductal cancer in the same ROR category. Our results could have an effect on the treatment decisions regarding the addition of chemotherapy for those in the intermediate ROR group.

PD-L1 expression is a predictive biomarker for anti-PD-L1 immunotherapy in triple negative breast cancer (TNBC). In the neoadjuvant setting, immunohistochemical (IHC) evaluation of PD-L1 expression can only be performed on small tissue biopsies. In our study we investigated heterogeneity of PD-L1 expression in TNBC, and how reliably PD-L1 expression in small tissue samples reflects PD-L1 expression in larger tumor sections in TNBC. Tissue microarrays (TMAs) were constructed from surgical specimens of 110 patients with TNBC. TMAs contained 4 cores (1 mm in diameter) per patient. To evaluate PD-L1 expression, TMAs were stained with PD-L1 IHC 22C3 PharmDx. Single-core PD-L1 expression was compared to overall PD-L1 expression of each patient’s tumor, to ascertain how often small samples of tumor tissue show the same PD-L1 expression as larger tumor samples. Our study found substantial heterogeneity of PD-L1 expression between different TMA cores from the same patient. Heterogeneity was greater in immune cells (ICs) than in tumor cells, in large part due to the uneven distribution of ICs in the tumor. For IC PD-L1 expression, we found that sensitivity can be as low as 0.81 for detecting PD-L1 expression at the 1% threshold most commonly used in breast cancer. Negative predictive value for ICs was 0.7. There is substantial heterogeneity of PD-L1 expression between small tissue samples from the same TNBC tumor, especially for IC expression. This poses challenges for evaluation of PD-L1 expression in the neoadjuvant setting. Negative biopsies should prompt further investigation, and multiple biopsies might be necessary.

Simple Summary Around 15% of breast cancer patients are diagnosed as triple-negative (TNBC), which have significantly lower 5-year survival rates (77%) than other types of breast cancer (93%). Our study aimed at developing an image analysis-based biomarker to assess how the immune system interacts with the tumor and investigate the potential added value of stromal tumor-infiltrating lymphocytes (sTIL) for the prognosis of overall survival compared to the manual approach. In a large retrospective cohort of 257 patients, we found that our fully automated hematoxylin and eosin (H&E) image analysis pipeline can quantify sTIL density showing both high concordance with manual scoring and association with the prognosis of patients with TNBC. It also overcomes natural limitations of manual assessment that hinder clinical adoption of the immune biomarker. We conclude that sTIL scoring by automated image analysis has prognostic potential comparable to manual scoring and should be further investigated for future use in a clinical setting. Abstract Triple-negative breast cancer (TNBC) is an aggressive and difficult-to-treat cancer type that represents approximately 15% of all breast cancers. Recently, stromal tumor-infiltrating lymphocytes (sTIL) resurfaced as a strong prognostic biomarker for overall survival (OS) for TNBC patients. Manual assessment has innate limitations that hinder clinical adoption, and the International Immuno-Oncology Biomarker Working Group (TIL-WG) has therefore envisioned that computational assessment of sTIL could overcome these limitations and recommended that any algorithm should follow the manual guidelines where appropriate. However, no existing studies capture all the concepts of the guideline or have shown the same prognostic evidence as manual assessment. In this study, we present a fully automated digital image analysis pipeline and demonstrate that our hematoxylin and eosin (H&E)-based pipeline can provide a quantitative and interpretable score that correlates with the manual pathologist-derived sTIL status, and importantly, can stratify a retrospective cohort into two significant distinct prognostic groups. We found our score to be prognostic for OS (HR: 0.81 CI: 0.72–0.92 p = 0.001) independent of age, tumor size, nodal status, and tumor type in statistical modeling. While prior studies have followed fragments of the TIL-WG guideline, our approach is the first to follow all complex aspects, where appropriate, supporting the TIL-WG vision of computational assessment of sTIL in the future clinical setting.

Background: The current standard method for locating nonpalpable breast lesions is wire guided localization (WGL) despite several methodological difficulties. Radioactive Seed Localization (RSL) has been developed to reduce these difficulties. The aim of this randomized trial was to compare the rate of positive resection margins between RSL and WGL in patients with nonpalpable invasive breast cancer (IBC) or ductal carcinoma in situ (DCIS). Material and Methods: Patients with nonpalpable IBC or DCIS visible on ultrasound were randomized to either of the two localization methods. Primary outcome was margin status at the final pathological evaluation. According to Danish standard in the study period margins were defined positive if cancer cells were found < 2mm from the inked margin. Secondary outcomes were duration of the surgical procedure, weight of the excised specimen and patient's pain perception. χ2-test, Fisher's exact test and Wilcoxon rank-sum test, respectively, were used to test differences between groups. Level of statistical significance was set to 5%. The average activity of seeds used in the trial was 1.70 MBq (range 0.7-3.27). Results: 413 cases representing 409 patients were randomized; 207 were allocated to RSL and 206 to WGL. 23 cases, who did not meet inclusion criteria, chose to withdraw, or had a change in surgical management, were excluded. The remaining 390 were included in the analysis. Patient, surgical and pathological characteristics between the two groups were alike, except for significantly more patients with DCIS in the WGL group (5.1% vs 0.5%). Significantly more cases in the WGL group (9.7%) needed additional localization compared to the RSL group (2.1%) (p=0.0014). In all cases but one in the RSL group, the index lesion was removed. Margins were positive in 23 cases (11.8%) in the RSL group compared to 26 cases (13.3%) in the WGL group. We were not able to detect a difference in margin status between the two groups (p=0.65). For IBC only, the number of positive margins was 22 (11.3%) in the RSL group and 21 (11.4%) in the WGL group (p=0.997). There was no difference between the two groups in the amount of tissue removed whether the analysis was done on the primary excision (p=0.18) or the total weight including intraoperative re-excisions (p=0.33). There was no difference in pain perception between the two groups whether patients who received local anesthesia were kept in the analysis (p=0.28) or excluded (p=0.91). Local anesthesia was used more frequently in the RSL group. Finally, there was no difference in the duration of the surgical procedure (p=0.12), the complication rate (p=0.89) or the identification rate for SN (p=1.0). Conclusions: We were not able to detect any differences considering positive margins, patient's pain perception or duration of the surgical procedure between the two localization methods. However, RSL offers a major logistic advantage, as the seed localization can be done several days before surgery without any risk or discomfort for the patient, with a low proportion of patients needing additional localization. So the RSL procedure has now been found preferable at our institutions. Citation Format: Langhans L, Tvedskov TF, Klausen TL, Jensen M-B, Talman M-L, Vejborg I, Benian C, Roslind A, Hermansen J, Oturai PS, Bentzon N, Kroman N. Radioactive seed localization versus wire guided localization of nonpalpable invasive and in situ breast cancer: A Danish multicenter randomized controlled trial [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr S3-08.

BackgroundTumor progression is accompanied by dramatic remodeling of the surrounding extracellular matrix leading to the formation of a tumor-specific ECM, which is often more collagen-rich and of increased stiffness. The altered ECM of the tumor supports cancer growth and metastasis, but it is unknown if this effect involves modulation of T cell activity. To investigate if a high-density tumor-specific ECM could influence the ability of T cells to kill cancer cells, we here studied how T cells respond to 3D culture in different collagen densities.MethodsT cells cultured in 3D conditions surrounded by a high or low collagen density were imaged using confocal fluorescent microscopy. The effects of the different collagen densities on T cell proliferation, survival, and differentiation were examined using flow cytometry. Cancer cell proliferation in similar 3D conditions was also measured. Triple-negative breast cancer specimens were analyzed for the number of infiltrating CD8+ T cells and for the collagen density. Whole-transcriptome analyses were applied to investigate in detail the effects of collagen density on T cells. Computational analyses were used to identify transcription factors involved in the collagen density-induced gene regulation. Observed changes were confirmed by qRT-PCR analysis.ResultsT cell proliferation was significantly reduced in a high-density matrix compared to a low-density matrix and prolonged culture in a high-density matrix led to a higher ratio of CD4+ to CD8+ T cells. The proliferation of cancer cells was unaffected by the surrounding collagen-density. Consistently, we observed a reduction in the number of infiltrating CD8+ T-cells in mammary tumors with high collagen-density indicating that collagen-density has a role in regulating T cell abundance in human breast cancer.Whole-transcriptome analysis of 3D-cultured T cells revealed that a high-density matrix induces downregulation of cytotoxic activity markers and upregulation of regulatory T cell markers. These transcriptional changes were predicted to involve autocrine TGF-B signaling and they were accompanied by an impaired ability of tumor-infiltrating T cells to kill autologous cancer cells.ConclusionsOur study identifies a new immune modulatory mechanism, which could be essential for suppression of T cell activity in the tumor microenvironment.

Introduction: There is extensive research into immune-oncology (IO) treatment targets for triple negative breast cancer (TNBC). However, little knowledge exits on variations in expression levels of IO targets between paired primary tumors (PT) and metastases (MT) in TNBC. Objectives: To compare expression levels of IO drug targets in paired PT and distant MT from TNBC patients. PD-L1 protein expression was examined immunohistochemically, as well as differences in levels of tumor infiltrating lymphocytes (TILs). Material and methods:552 patients were primarily operated for TNBC at 2 university hospitals in Denmark between 2004 and 2014. Of these, samples from 22 patients were available from both PT (22 samples) and distant MT (24 samples, with 2 chronologically separate metastases for two patients). Localizations of metastases included 9 brain, 1 skin and 1 lymph node (both not regional), 2 bone, 5 liver, 2 soft tissue, 1 lung, and 1 adrenal gland. Gene expression analyses were performed on the Ion Torrent S5XL system, using the Oncomine Immune Response Research Assay. PD-L1 expression was evaluated immunohistochemically using the 28-8 clone (pharmdX). Cut-off levels for PD-L1 expression were 1% positive tumor cells. TILs were evaluated in accordance with guidelines from the International Immuno-Oncology Biomarker Working Group. Results: Gene expression levels of 29 established IO drug targets were tested on 18 of the 22 TNBC patients with matched PTs and MTs (Table 1). 4 genes showed significant downregulation in MTs compared to PTs: IDO1 (p=0.01), CXCR4 (p=0.01), KIR2DL1 (p=0.05) and TNFRSF9 (p=0.03). However, other important IO targets such as CTLA4, STAT3, TIGIT, and TNSFRSF18 (GITR) were not significantly different between PT and MT. CD276 (B7-H3), PD-1, IL2 and LAG3 showed a tendency towards significance (p= 0.08, 0.07, 0.05, 0.08), with LAG3 being the only gene upregulated in MT compared to PT. TILs were evaluated for all PTs and MTs. TILs in PTs ranged from 1 to 40% (median=13%). TILs in MTs ranged from 1 to 60% (median=5%). There was no significant overall change in TILs from PT to MT (p=0.2). However, 9 patients had a difference in TILs between PT and MT of > ±10% (-20 - +30). PD-L1 immunohistochemical evaluation was possible for both PT and MT in 20 cases. There was no significant overall change in PD-L1 expression (p=0.39). However, 4 patients with positive PTs, had negative MTs, and 4 patients with PD-L1 negative PTs, had positive MTs. Discussion: This is, to our knowledge, one of the largest studies of gene expression of IO drug targets in TNBC matched PTs and MTs. We show significant differences in gene expression levels for IDO1, CXCR4, KIR2DL1 and TNFRSF9, pointing to a generally less immune-active environment in MTs. This was also corroborated by lower levels of TILs in MTs, although these findings were not significant. For PD-L1 protein expression, we found that PD-L1 expression can change between PT and MT. In the clinical setting, treatment decisions are often based upon expression levels in a single biopsy or surgical specimen. Intra-patient heterogeneity should be taken into account in the diagnostic setting, and when making treatment decisions. Table 1IO drug targetRatio (Primary/metastasis)P-valueIDO19.65 (1.68-54.95)0.01CXCR41.83 (1.17-2.87)0.01KIR2DL12.17 (1.01-4.69)0.05TNFRSF97.11 (1.21-41.93)0.03CD276 (B7H3)1.51 (0.94-2.39)0.08PDCD1 (PD1)3.32 (0.88-12.64)0.07IL22.87 (0.99-8.28)0.05LAG30.28 (0.07-1.16)0.08CTLA41.96 (0.45-8.51)0.35STAT31.21 (0.92-1.60)0.16TIGIT1.87 (0.25-14.03)0.53TNFRSF18 (GITR)0.38 (0.06-2.20)0.26 Citation Format: Elisabeth S Stovgaard, Tim S Poulsen, Eva Balslev, Dorte L Nielsen, Anne Roslind, Maj-Lis Talman, Iben Kümler, Ib J Christensen, Estrid Høgdall. Triple negative breast cancer: Expression of immuno-oncology treatment targets and prevalence of tumor infiltrating lymphocytes in paired primary tumors and metastases [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-13.

To compare the rate of positive resection margins between radioactive seed localization (RSL) and wire-guided localization (WGL) after breast conserving surgery (BCS).WGL is the current standard for localization of nonpalpable breast lesions in BCS, but there are several difficulties related to the method.From January 1, 2014 to February 4, 2016, patients with nonpalpable invasive breast cancer or DCIS visible on ultrasound were enrolled in this randomized, multicenter, open-label clinical trial, and randomly assigned to RSL or WGL. The primary outcome was margin status after BCS. Secondary outcomes were duration of the surgical procedure, weight of surgical specimen, and patients' pain perception. Analyses were performed by intention-to-treat (ITT) and per protocol.Out of 444 eligible patients, 413 lesions representing 409 patients were randomized; 207 to RSL and 206 to WGL. Twenty-three did not meet inclusion criteria, chose to withdraw, or had a change in surgical management and were excluded. The remaining 390 lesions constituted the ITT population. Here, resection margins were positive in 23 cases (11.8%) in the RSL group compared with 26 cases (13.3%) in the WGL group (P = 0.65). The per-protocol analysis revealed no difference in margin status (P = 0.62). There were no significant differences in the duration of the surgical procedure (P = 0.12), weight of the surgical specimen (P = 0.54) or the patients' pain perception (P = 0.28).RSL offers a major logistic advantage, as localization can be done several days before surgery without any increase in positive resection margins compared with WGL.

MicroRNAs have potential as diagnostic cancer biomarkers. The aim of this study was (1) to define microRNA expression patterns in formalin-fixed parafin-embedded tissue from pancreatic ductal adenocarcinoma, ampullary adenocarcinoma, normal pancreas and chronic pancreatitis without using micro-dissection and (2) to discover new diagnostic microRNAs and combinations of microRNAs in cancer tissue. The expression of 664 microRNAs in tissue from 170 pancreatic adenocarcinomas and 107 ampullary adenocarcinomas were analyzed using a commercial microRNA assay. Results were compared with chronic pancreatitis, normal pancreas and duodenal adenocarcinoma. In all, 43 microRNAs had higher and 41 microRNAs reduced expression in pancreatic cancer compared with normal pancreas. In all, 32 microRNAs were differently expressed in pancreatic adenocarcinoma compared with chronic pancreatitis (17 higher; 15 reduced). Several of these microRNAs have not before been related to diagnosis of pancreatic cancer (eg, miR-492, miR-614, miR-622). MiR-614, miR-492, miR-622, miR-135b and miR-196 were most differently expressed. MicroRNA profiles of pancreatic and ampullary adenocarcinomas were correlated (0.990). MicroRNA expression profiles for pancreatic cancer described in the literature were consistent with our findings, and the microRNA profile for pancreatic adenocarcinoma (miR-196b-miR-217) was validated. We identified a more significant expression profile, the difference between miR-411 and miR-198 (P=2.06 × 10(-54)) and a diagnostic LASSO classifier using 19 microRNAs (sensitivity 98.5%; positive predictive value 97.8%; accuracy 97.0%). We also identified microRNA profiles to subclassify ampullary adenocarcinomas into pancreatobiliary or intestinal type. In conclusion, we found that combinations of two microRNAs could roughly separate neoplastic from non-neoplastic samples. A diagnostic 19 microRNA classifier was constructed which without micro-dissection could discriminate pancreatic and ampullary adenocarcinomas from chronic pancreatitis and normal pancreas with high sensitivity and accuracy. Ongoing prospective studies will evaluate if these microRNA profiles are useful on fine-needle biopsies for early diagnosis of pancreatic cancer.

The aim of the present study was to identify a panel of microRNAs (miRNAs) that can predict overall survival (OS) in non micro-dissected cancer tissues from patients operated for pancreatic cancer (PC).MiRNAs were purified from formalin-fixed paraffin embedded (FFPE) cancer tissue from 225 patients operated for PC. Only a few of those patients received adjuvant chemotherapy. Expressions of miRNAs were determined with the TaqMan MicroRNA Array v2.0. Two statistical methods, univariate selection and the Lasso (Least Absolute Shrinkage and Selection Operator) method, were applied in conjunction with the Cox proportional hazard model to relate miRNAs to OS.High expression of miR-212 and miR-675 and low expression of miR-148a, miR-187, and let-7g predicted short OS independent of age, gender, calendar year of operation, KRAS mutation status, tumor stage, American Society of Anesthesiologists (ASA) score, localization (not miR-148a), and differentiation of tumor. A prognostic index (PI) based on these five miRNAs was calculated for each patient. The median survival was 1.09 years (Confidence Interval [CI] 0.98-1.43) for PImedian PI compared to 2.23 years (CI 1.84-4.36) for PImedian. MiR-212, miR-675, miR-187, miR-205, miR-944, miR-431, miR-194, miR-148a, and miR-769-5p showed the strongest prediction ability by the Lasso method. Thus miR-212, miR-675, miR-187, and miR-148a were predictors for OS in both statistical methods.The combination of five miRNAs expression in non micro-dissected FFPE PC tissue can identify patients with short OS after radical surgery. The results are independent of chemotherapy treatment. Patients with a prognostic indexmedian had a very short median OS of only 1 year.

Aims: Bladder pain syndrome/interstitial cystitis (BPS/IC), diagnosed according to the new 2008 criteria of the European Society for the Study of Interstitial Cystitis (ESSIC), may lead to detrusor fibrosis. In some inflammatory diseases, fibrosis is related to YKL-40. The aims were to examine YKL-40 antigenic expression in bladder tissue and levels in serum and urine in BPS/IC and to evaluate whether YKL-40 could be a non-invasive, prognostic biomarker for bladder fibrogenesis and treatment intensity. Methods and results: Immunohistochemistry, immunoelectron microscopy and enzyme-linked immunosorbent assay (ELISA) analyses in 45 patients showed YKL-40 expression in detrusor mast cell granules and submucosal macrophages, and elevated YKL-40 levels in serum and urine compared to healthy individuals (median 72 versus 7 μg/l, P < 0.001). Clinicopathological parameters showed associations of detrusor fibrosis with YKL-40-positive cells (P = 0.001), mast cells (P = 0.014) and urine YKL-40 (P = 0.009). Bladder capacity correlated inversely with YKL-40-positive cells (P < 0.001) and mast cells (P = 0.029). Treatment intensity was not associated with YKL-40. Conclusion: Serum and urine levels of YKL-40 may be used as non-invasive biomarkers in BPS/IC for the evaluation of bladder fibrogenesis.

Udgivelsesdato: 2010-May-8 The prognosis of ipsilateral supraclavicular lymph node recurrence after early breast cancer appears to be worse than for other loco-regional recurrences, but better than for distant metastases. The purpose of the present study was to investigate the relationship between different types of salvage treatment and primary patient characteristics, treatment response, and survival after supraclavicular recurrence (SR) in a large patient population. From the Danish Breast Cancer Cooperative Group treatment database 1977-2003, 305 patients were identified with SR without distant disease as site of first recurrence. Salvage treatment types as well as other factors were related to response and survival. The median follow-up time for progression after SR was 25 months. Complete remission was 76% among patients receiving excision surgery, 67% with combined loco-regional and systemic therapy, and 48% with systemic therapy alone. Median progression-free survival (PFS) and overall survival was 18 and 29 months, respectively. The 5-year PFS probability was 15%. In univariate analysis, combination salvage therapy, negative nodal status and low malignancy grade were related to longer PFS. In multivariate analysis, salvage therapy and malignancy grade remained independent factors for survival. In conclusion, the prognosis of SR is generally poor. However, it appears to be a curable condition. An independent marker of improved outcome is local and systemic combination salvage treatment, which can be considered.

To quantify the border versus centre enhancement of malignant breast tumours on dynamic magnetic resonance mammography.Fifty-two women diagnosed with primary breast cancer underwent dynamic magnetic resonance mammography (Omniscan 0.2 mmol/kg bodyweight) on a midfield scanner (0.6 T), prior to surgery. The following five variables were recorded from the border and centre regions of the tumours: Early Enhancement, Time to Peak, Wash-in rate, Wash-out rate and Area under Curve. Information on histology type, oestrogen and progesterone receptor status was collected. Statistical analysis was performed in SAS 9.1 as paired samples t-tests.Fifty of 52 malignant tumours displayed a faster Early Enhancement in the border region compared to the centre (p0.0001). Significant differences between the border and centre values were found for Time to Peak, Wash-in rate, Wash-out rate and Area under Curve. Hormone receptor positive tumours displayed an over-all highly significant difference between border and centre enhancement, whereas no significant differences for any of the five variables were recorded in neither oestrogen nor progesterone hormone receptor negative tumours.The border/centre enhancement difference in malignant breast tumours is easily visualized on midfield dynamic magnetic resonance mammography. The dynamic behaviour is significantly correlated to histological features and receptor status of the tumours.

Udgivelsesdato: 2007-Dec-22 YKL-40 is a new biomarker in serum with a prognostic value in several localized and metastatic malignancies. The current knowledge regarding the biological functions of YKL-40 in cancer links YKL-40 to increased aggressiveness of the tumor. Utilizing tissue microarrays, YKL-40 protein expression in tumor tissue was assessed by immunohistochemistry in a cohort of 630 high-risk breast cancer patients with a median estimated potential follow-up time of 10 and 13 years for disease-free (DFS) and overall survival (OS), respectively. YKL-40 protein expression was found in malignant tumor cells and in inflammatory cells. High expression was associated with positive estrogen and progesterone receptor status and high tumor differentiation. Contrary to studies on serum YKL-40 as a prognostic biomarker, a high YKL-40 expression in tumor cells was not significantly associated with DSF and OS in univariate and multivariate analyses

Elevated serum levels of the protein YKL-40 are associated with a poor prognosis in patients with solid and hematologic malignancies including breast cancer. The aim of this study was to develop a valid reproducible immunohistochemical method to visualize YKL-40 expression in normal breast tissue as well as in benign and malignant breast lesions. The presence of YKL-40 in breast tissue was verified by in situ hybridization and protein extraction procedures. An immunohistochemical method was developed and 4 different antibodies directed against YKL-40 were tested. Ten patients with normal breast tissue and benign breast lesions and 53 patients with localized breast carcinomas were analyzed immunohistochemically. The presence of YKL-40 in normal epithelial cells as well as in malignant tumor cells of the breast was established; however, a difference in staining intensity and staining pattern was observed. In normal breast tissue, a weak YKL-40 immunoreactivity was found in the cytoplasm of the epithelial cells with an additional strong dotlike staining between the nucleus and the gland lumen. In malignant lesions, 81% of the in situ carcinomas and 64% of the invasive carcinomas showed strong diffuse cytoplasmic YKL-40 immunoreactivity. No nuclear and membrane staining was found. A subpopulation of cells of macrophage morphology in normal breast tissue and in malignant lesions showed strong YKL-40 immunoreactivity.

YKL-40 is produced by cancer cells and tumour-associated macrophages. YKL-40 may play a role in cancer cell proliferation, differentiation, survival, invasiveness, metastasis, in angiogenesis and the inflammation and remodelling of the extracellular matrix surrounding the tumour. Serum YKL-40 is a biomarker of prognosis, confirmed in 13 different types of cancer including > 2500 patients. Highest serum YKL-40 is found in patients with metastatic cancer with the shortest recurrence-free interval and shortest overall survival. Serum YKL-40 provides independent information compared with clinical characteristics and biomarkers, such as HER2, carcinoembryonic antigen, CA-125, prostate-specific antigen and lactate dehydrogenase. The authors hypothesise that inhibition of YKL-40 by monoclonal antibodies either directly or towards its receptor may be as efficient a cancer therapeutic as the monoclonal antibodies against HER2, HER1, vascular endothelial growth factor and CD20. Drugs inhibiting YKL-40 should be explored as new cancer therapeutics.

OBJECTIVES The frequencies and prognostic role of KRAS and BRAF mutations in patients operated on for pancreatic ductal adenocarcinomas (PDACs) and ampullary adenocarcinomas (A-ACs) are scantily studied. METHODS KRAS and BRAF mutations were analyzed in formalin-fixed, paraffin-embedded tumor samples from primarily chemotherapy-naive patients operated on with radical intentions for PDAC (n = 170) and A-AC (n = 107). RESULTS Eighty percent of PDAC patients had KRAS mutations (codon 12 mutations: 74%) and 67% with A-AC (codon 12 mutations: 54%). BRAF mutations were less common, 16% in PDAC and 12% in A-AC, and no V600E mutations were found. Fourteen percent with PDAC and 7% with A-AC had mutations in both KRAS and BRAF. Multivariate analysis, including KRAS status, stage, and American Society of Anesthesiologists physical status classification system score, demonstrated that KRAS mutations in patients with A-AC were associated with short recurrence-free survival (RFS) (hazard ratio, 2.45; 95% confidence interval, 1.19-5.06; P = 0.015) and overall survival (OS) (1.93, 95% 1.12-3.31; P = 0.018). KRAS mutations in patients with PDAC were not associated with RFS and OS. BRAF mutations were not associated with RFS and OS. CONCLUSIONS KRAS mutations frequencies were high in PDAC and A-AC. KRAS mutations were associated with poor prognosis in patients with A-AC, but not in patients with PDAC.

Cancer in the gallbladder is rare, and each year about 160 cases are registered in Denmark. Most malignant tumours in the gallbladder are adenocarcinomas. We present a rare case of signet ring cell carcinoma of the gallbladder. Signet ring cell carcinomas are particularly aggressive. The tumours are often disseminated at the time of diagnosis, and it may be difficult to determine the primary origin.

Bladder pain syndrome/interstitial cystitis (BPS/IC), diagnosed according to the new 2008 criteria of the European Society for the Study of Interstitial Cystitis (ESSIC), may lead to detrusor fibrosis. In some inflammatory diseases, fibrosis is related to YKL-40. The aims were to examine YKL-40 antigenic expression in bladder tissue and levels in serum and urine in BPS/IC and to evaluate whether YKL-40 could be a non-invasive, prognostic biomarker for bladder fibrogenesis and treatment intensity.Immunohistochemistry, immunoelectron microscopy and enzyme-linked immunosorbent assay (ELISA) analyses in 45 patients showed YKL-40 expression in detrusor mast cell granules and submucosal macrophages, and elevated YKL-40 levels in serum and urine compared to healthy individuals (median 72 versus 7 μg/l, P0.001). Clinicopathological parameters showed associations of detrusor fibrosis with YKL-40-positive cells (P = 0.001), mast cells (P = 0.014) and urine YKL-40 (P = 0.009). Bladder capacity correlated inversely with YKL-40-positive cells (P0.001) and mast cells (P = 0.029). Treatment intensity was not associated with YKL-40.Serum and urine levels of YKL-40 may be used as non-invasive biomarkers in BPS/IC for the evaluation of bladder fibrogenesis.

YKL-40, a member of 'mammalian chitinase-like proteins', is secreted by macrophages, neutrophils, chondrocytes, endothelial-, vascular smooth muscle-, and cancer cells. High serum YKL-40 is a biomarker of poor prognosis in patients with cancer, inflammation and increased tissue remodelling. High YKL-40 protein expression assessed by immunohistochemistry is found in breast carcinomas associated with short disease-free survival and in glioblastomas with increased resistance to radiotherapy and decreased overall survival. In this chapter we describe the methods for the detection of (1) YKL-40 protein expression in human tissues (using immunohistochemistry) and cell cultures (using immunocytochemistry); (2) YKL-40 mRNA expression in human tissues (using in situ hybridization and Polymerase Chain Reaction); and (3) YKL-40 protein concentrations in serum or plasma (using Enzyme-Linked ImmunoSorbent Assay).

YKL-40, a member of 'mammalian chitinase-like proteins', is secreted by macrophages, neutrophils, chondrocytes, endothelial-, vascular smooth muscle-, and cancer cells. High serum YKL-40 is a biomarker of poor prognosis in patients with cancer, inflammation and increased tissue remodelling. High YKL-40 protein expression assessed by immunohistochemistry is found in breast carcinomas associated with short disease-free survival and in glioblastomas with increased resistance to radiotherapy and decreased overall survival. In this chapter we describe the methods for the detection of (1) YKL-40 protein expression in human tissues (using immunohistochemistry) and cell cultures (using immunocytochemistry); (2) YKL-40 mRNA expression in human tissues (using in situ hybridization and Polymerase Chain Reaction); and (3) YKL-40 protein concentrations in serum or plasma (using Enzyme-Linked ImmunoSorbent Assay).

YKL-40 is a glycoprotein secreted by macrophages, neutrophils and malignant tumor cells. Elevated serum levels of YKL-40 are associated with poor prognosis in several malignancies. In this study, we examined the prognostic value of serum YKL-40 before treatment and during follow-up in patients with squamous cell carcinoma of the head and neck (HNSCC). YKL-40 was determined by ELISA retrospectively in serum from 173 patients with primary HNSCC before treatment and up to 2 years after treatment. Median follow-up time was 7.9 years. YKL-40 protein expression in tumor biopsies was assessed by immunohistochemistry in 50 patients. Pretreatment serum YKL-40 was elevated in 53%. Patients with high serum YKL-40 had shorter survival than patients with normal serum YKL-40 (33 vs. 84 months; p = 0.008). Multivariate Cox analysis including pretreatment serum YKL-40, age, sex, primary tumor site, TNM classification and treatment demonstrated that TNM classification (HR = 2.61, p = 0.02) and serum YKL-40 (log-transformed continuous variable: HR = 1.55, p < 0.0001) were independent prognostic variables of overall survival (OS). Multivariate Cox analysis demonstrated that TNM classification (HR = 5.77, p = 0.001) and serum YKL-40 (dichotomous variable: HR = 2.75, p = 0.01) were independent predictors of recurrence-free survival. During follow-up after radiotherapy, a high serum YKL-40 (log-transformed continuous variable) in patients with TNM Stage III and IV disease predicted poorer OS within 6 months (HR = 1.95, p < 0.0001). Immunohistochemical analysis showed YKL-40 expression in the malignant tumor cells. In conclusion, serum YKL-40 was demonstrated to be an independent prognostic biomarker of recurrence-free and overall survival in patients with HNSCC.

YKL-40, a member of the “mammalian chitinase–like proteins,” is expressed and secreted by several types of solid tumors. The exact function of YKL-40 in cancer diseases is unknown and is an important objective of future studies. YKL-40 exhibits growth factor activity for cells involved in tissue remodeling processes. YKL-40 may have a role in cancer cell proliferation, survival, and invasiveness, in the inflammatory process around the tumor, angiogenesis, and remodeling of the extracellular matrix. YKL-40 is neither organ- nor tumor-specific. However, the present retrospective clinical studies of patients with eight different types of primary or advanced solid tumors suggest that serum concentration of YKL-40 may be a new biomarker in cancer patients used as a “prognosticator.” Elevated serum YKL-40 is found in a subgroup of patients with different types of solid tumors, including several types of adenocarcinomas, small cell lung carcinoma, glioblastoma, and melanoma. The highest serum YKL-40 is detected in patients with advanced cancer and with the poorest prognosis. In many cases, serum YKL-40 provides independent information of survival. Serum YKL-40 cannot be used as a single screening test for cancer. The use of serum YKL-40 has not received Food and Drug Administration approval for use as a biomarker for cancer or any other disease. Large multicenter retrospective and prospective studies of patients with different types of cancer are required to determine: (a) if serum YKL-40 is a useful prognostic cancer biomarker, (b) if serum YKL-40 can be of value in monitoring patients with cancer in order to provide information about metastases before these are detected by routine methods, and (c) if serum YKL-40 can be useful for screening of cancer together with a panel of other cancer biomarkers and imaging techniques. (Cancer Epidemiol Biomarkers Prev 2006;15(2):194–202)

4061 Background: The aim was to identify a panel of microRNAs (miRs) that can predict overall survival (OS) in non-microdissected tissues from patients operated for pancreatic cancer (PC). Methods:...

153 Background: The aim was: 1) to define the global microRNA (miRNA) expression pattern in pancreatic cancer (PC) and compare it with normal pancreas (NP) and chronic pancreatitis (CP); 2) to validate reported diagnostic miR profiles in PC; 3) to discover new diagnostic miRs in pancreas samples without micro-dissection of cancer cells. Methods: MiR expression patterns in FFPE tissue blocks from patients operated for pancreatic ductal adenocarcinoma (PDAC, n=170) and ampullary adenocarcinomas (A-AC, n=107) were determined using TaqMan Human miR microarrray (Applied Biosystem; 754 miRs were determined) and compared to profiles of CP (n=23) and NP (n=28). Raw Ct-values were normalized using quantile-normalisation. Results: 83miRs were differentially expressed in PDAC and NP (42 higher expression; 41 reduced expression in PDAC). 32 miRs were differentially expressed in PDAC and CP. The most differentially expressed miRs were miR-614, miR-492, miR-622, miR-135b* and miR-196. The miR signatures of PDAC and A-AC were highly correlated (0.99; only 5 miRs were significantly differentially expressed). An earlier reported diagnostic miR profile for PDAC was validated (mirR196b - miR217), and 3 other significant profiles were identified (Table). A more complex Lasso classifier using 19 miRs could separate PDAC from NP and CP (accuracy = 98%). Conclusions: We identified systematic differences in patterns of miR expressions between tumor tissue including both cancer cells and surrounding desmoplasia obtained from patients with PDAC and A-AC compared to tissue from patients with CP and NP. We validated the diagnostic miR expression profile (miR-196b, miR-217) described by Szafranska et al. (Clin Chem 2008;54:1716-24). Furthermore, we identified 5 miRs which were better to discriminate PDAC and A-AC from CP and NP. Prospective studies are needed to evaluate if this panel of miRs is useful for early diagnosis of patients with PDAC. [Table: see text] No significant financial relationships to disclose.

4043 Background: The results of cytostatic treatment in pancreatic ductal adenocarcinomas (PDAC) are still disappointing. Standard treatment is still gemcitabine, monotherapy or in combinations. St...

e15618 Background: Prognostic biomarkers in patients with pancreatic cancer might direct patients to optimal treatment. We studied the prognostic value of KRAS mutations in patients operated for pancreatic and other periampullary cancers. Methods: Retrospective study of 328 patients who were operated with a Whipples procedure at a single HBP-center during the period August 1976 to May 2008. For the present pilot study cancer tissue blocks were collected from 58 patients (31 men, 27 women, median age 64, range 33–81 years, ASA 1–3). All had localized pancreatic (n=26), ampullary (n=26) or duodenal cancer (n=6) and were operated during the period June 2001 to May 2008. 32 patients died (3 within the first month after operation and were excluded). Median follow-up time was 41 months (range 7–90). KRAS mutations in exon 1 and 2 were determined by LightScanner analysis (Idaho Technology). Results: KRAS mutations were found in 32 (55%) of the patients; 16 (62%) patients with pancreatic cancer, 13 (50%) with ampullary cancer and in 3 (50%) with duodenal cancer. KRAS status was neither associated with type of cancer (p=0.68), TNM stage (T stage p=0.64, N stage p=0.31). The median survival time for all patients was 22 months (KRAS mutation: 21, 95% CI: 10–40; Wildtype: not reached yet). Univariate Cox analysis showed that T stage (TNM) (1: HR=0.09, 95% CI: 0.02–0.48 p=0.004; 2: HR=0.18, 0.06- 0.51, p=0.0013; 3: HR=0.26, 0.09–0.71, p=0.0089) was a prognostic factor of OS. KRAS status (HR=0.70, 0.33–1.49, p=0.36) and N stage (TNM) (HR=0.53, 0.25–1.11, p=0.09) were not significant factors of OS, but there is a strong tendency for both factors. Conclusions: KRAS mutations are frequent in patients with localized pancreatic, ampullary and duodenal cancers. Preliminary analysis of 58 patients out of our cohort of 328 patients indicate that KRAS mutations may be a marker of overall survival and time to progression. No significant financial relationships to disclose.

21004 Background: YKL-40 is secreted by tumor cells, macrophages and neutrophils. The exact function of YKL-40 in cancer is unknown. It may be a growth, differentiation or survival factor and play a role in angiogenesis. High serum levels of YKL-40 are associated with a poor prognosis in a variety of solid and hematological malignancies. The aim of this study was to examine if serum YKL-40 is a prognostic biomarker in patients with cervical cancer. Methods: YKL-40 was determined by ELISA (Quidel, Santa Clara, CA) in pretreatment serum samples from 116 patients with cervical cancer (FIGO stage Ia (N=5), Ib (N=55), II (N=27), III (N=25) and IV (N=4); median age 50, range 27–87 years). The median follow-up time was 3.2 years (range 2.1–4.4 years). 18 patients had a recurrence and 28 patients died. Results: Serum YKL-40 was increased (pth percentile of serum YKL-40 in healthy women in 35% of the patients. Patients with high serum YKL-40 had significantly shorter survival than patients with normal serum YKL- 40 (HR=3.1, 95% CI: 1.5–6.5, p=0.002, logrank test). Univariate Cox analysis of serum YKL-40 (log transformed and treated as a continuous covariate) showed significant association with overall survival (HR=2.3, 95% CI: 1.7–3.2, p No significant financial relationships to disclose.

5551 Background: High serum YKL-40 is associated with poor prognosis in breast-, colorectal-, ovarian-, prostate-, small cell lung carcinoma and malignant melanoma. YKL-40 is secreted by cancer cells, macrophages and neutrophils. Its function in cancer is unknown. It may be a growth factor, play a role in angiogenesis or protect against apoptosis. The aim was to examine serum YKL-40 in patients with squamous cell carcinoma of the head and neck (SCCHN). Methods: YKL-40 was determined by ELISA (Quidel, Santa Clara, CA) in serum samples from 138 patients with SCCHN (median age 60, range 44–92 years) before surgery and/or fractionated radiotherapy (total dose of 60–68 Gy, 2-Gy fractions). 42 patients had stage 1, 28 stage 2, 22 stage 3, and 46 stage 4 disease. The median follow-up time was 4.0 years (range 15 days–8.5 years). 91 patients had died. Results: Serum YKL-40 was increased (p < 0.001) in patients with SCCHN (median 120 μg/l, range 25–1848 μg/l) compared to healthy controls (43 μg/l, 20–184 μg/l, n = 245). Serum YKL-40 was elevated in 52% of the patients. Patients with stage 1 disease had lower, but non-significant, serum YKL-40 compared to patients with stage 2–4 disease (median 113 μg/l, range 25–1000 μg/l vs. 139 μg/l, 29–1848 μg/l, p = 0.06). Patients with high serum YKL-40 had significantly shorter survival than patients with normal serum YKL-40 (33 months vs. 74 months, p = 0.01 logrank test). Univariate analysis of serum YKL-40 (log transformed and treated as a continuous covariate) showed significant association with overall survival after start of therapy (HR = 1.4, 95% CI: 1.2–1.7, p = 0.0004). Multivariate Cox analysis including age, stage and serum YKL-40 (log transformed and treated as a continuous variable) showed that stage (stage 3–4 vs. stage 1–2, HR = 3.0, 95% CI: 1.9–4.6, p < 0.0001) and serum YKL-40 (HR = 1.5, 95% CI: 1.2–1.8, p = 0.0003) were independent prognostic variables of overall survival. Conclusions: Serum YKL-40 is a prognostic biomarker of overall survival in SCCHN patients. No significant financial relationships to disclose.

18019 Background: Elevated serum levels of the protein YKL-40 are associated with a poor prognosis in several solid tumors including melanoma (H. Schmidt, JCO 2006, Cancer 2006 in press). YKL-40 is secreted by cancer cells, macrophages and neutrophils. The function of YKL-40 is only poorly understood. It may be a growth or differentiation factor, play a role in angiogenesis or protect against apoptosis. The aim was to describe the tissue expression of YKL-40 in naevocellular naevi, carcinomas and melanomas of the skin by immunohistochemistry. Methods: Paraffin-embedded tissue sections from 40 patients with different naevocellular naevi, carcinomas and melanomas of the skin were developed with EnVision+ System-HRP K4007 (DakoCytomation) using a monoclonal antibody against YKL-40. The intensity of YKL-40 staining was scored using a semi-quantitative method. Results: YKL-40 protein expression appeared as a cytoplasmic, granular staining with no staining of membranes, nuclei or extracellular matrix. In normal skin the basal epithelial layer stained weakly positive. Melanocytes and melanophages stained negative. Macrophages and leucocytes stained positive. Hair follicles, sebaceous glands and sweat glands stained positive. Three basal cell carcinomas all stained positive. In 10 squamous cell carcinomas, the epidermal components stained positive and the invasive tumor islands were negative. In 3 keratoacanthomas, the epithelial components as well as the infiltrative tumor islands stained positive. In naevi (2 junctional, 5 compound and 1 intradermal) the epithelial components stained positive, the intradermal components stained negative. In melanomas the tumor cells stained positive in 9 of 10 specimens. Both the epidermal and the invasive components of melanomas stained positive. Three melanoma metastases all stained positive. Conclusions: YKL-40 is expressed in normal skin and in benign and malignant lesions of the skin. Generally, YKL-40 expression is increased in cells of high activity, although the invasive tumor islands of squamous cell carcinomas stain negative. In melanomas, the tumor cells may be a source of the elevated YKL-40 serum levels observed in these patients. No significant financial relationships to disclose.

5047 Background: YKL-40 is secreted by cancer cells, macrophages and neutrophils. The exact function of YKL-40 is unknown. It may be a growth or differentiation factor, play a role in angiogenesis or protect against apoptosis. High serum YKL-40 is associated with poor prognosis in breast-, colorectal-, ovarian-, prostate-, small cell lung cancer and malignant melanoma. The aim was to examine serum YKL-40 in patients with cervical cancer and cervical intraepithelial neoplasia (CIN). Methods: YKL-40 was determined by ELISA (Quidel, Santa Clara, CA) in pretreatment serum samples from 116 patients with cervical cancer (FIGO stage Ia (N = 5), Ib (N = 55), II (N = 27), III (N = 25) and IV (N = 4); median age 50, range 27–87 years) and 152 patients with cervical CIN (34 years, 20–80 years). The controls included 63 healthy women undergoing sterilization (36 years, 25–59 years) and a group of 134 healthy women (51 years, 18–79 years). Results: Serum YKL-40 was increased (p < 0.001) in the patients with cervical cancer (median 76 μg/l, range 20–2310) compared to patients with CIN (45 μg/l, 20–288), women undergoing sterilization (37 μg/l, 20–125) and healthy women (43 μg/l, 20–172). Patients with CIN had slightly elevated serum YKL-40 levels. Serum YKL-40 increased with increasing stage (Ia: median 36 μg/l, range 26–137; Ib: 56 μg/l, 20–984; II: 92 μg/l, 28–2310; III: 163 μg/l, 40–1474; and IV: 224 μg/l, 137–391; p < 0.001 Kruskal-Wallis test). All stage IV patients, 72% stage III, 52% stage II, 24% stage Ib, 40% stage Ia and 13% of patients with CIN had elevated serum YKL-40 compared to healthy controls (95th percentile). Patients with squamous cell carcinoma had higher serum YKL-40 (median 92 μg/l, range 27–2310, N = 86) compared to patients with adenosquamous (67 μg/l, 20–93, N = 9, p = 0.027) and adenocarcinoma (43 μg/l, 20–1472, N = 20, p < 0.001). Conclusion: Median serum YKL-40 is elevated in patients with stage Ib - IV cervical cancer. The serum level of YKL-40 seems to be correlated to the stage. No significant financial relationships to disclose.

9665 Background: YKL-40, a heparinbinding glycoprotein, is a growth factor for connective tissue cells and stimulates migration of endothelial cells. High serum YKL-40 levels are associated with a ...