BACKGROUND:Abrogation of growth factor-dependent signaling represents an effective therapeutic strategy for patients with colorectal cancer (CRC). Here we evaluated the effectiveness of targeting the epidermal growth factor (EGF) receptors HER-2 and HER-3 in the three cell lines LS513, LS1034 and SW837.METHODS:Treatment with HER-2-specific antibodies trastuzumab and pertuzumab resulted in a mild reduction of cellular viability. In contrast, the antibody-drug conjugate T-DM1 mediated a strong and dose-dependent decrease of viability and Akt phosphorylation.RESULTS:The most striking effects were observed with the dual tyrosine kinase inhibitor lapatinib, and the Pan-ErbB inhibitor afatinib. Selectively, the effect of EGF receptor inhibition was augmented by a combination with 5-fluorouracil and oxaliplatin. Finally, high expression of HER-3 was detected in 121 of 172 locally advanced rectal cancers (70.3%). In conclusion, inhibition of EGF receptors effectively blocks downstream signaling and significantly impairs viability of CRC cells. However, the effectiveness of receptor inhibition highly depends on the inhibitors' mode of action, as targeting HER-2 alone is not sufficient.CONCLUSION:Since HER-2 and HER-3 are expressed in a relevant number of patients, targeting both receptors may represent a promising therapeutic strategy for CRC.

Background The small bowel is prone to ischemic injury during transport before transplantation, an injury that endangers the recipient patient. The small-bowel mucosal microcirculation in particular appears to be highly sensitive to injury. Current preservation solutions such as histidine-tryptophan-ketoglutarate (HTK) solution provide some protection to the graft. However, these were developed decades ago and do not address several critical processes, such as hypoxia-induced membrane pores and free radical-mediated hypothermic injury. Methods To protect the graft from cold ischemic injury, we implemented a modified HTK solution here, including glycine, alanine, and iron chelators in a heterotopic, syngeneic small-bowel transplantation model of the rat. The effects of the modified solution and its major components were compared against the conventional HTK solution using intravital microscopy in the early reperfusion period. Results The amino acid glycine, added to HTK solution, slightly improved mucosal perfusion. Both, the modified base solution (without iron chelators) and iron chelators increased functional capillary density of the mucosa during the early reperfusion period. The complete modified solution (with glycine, alanine, and iron chelators) significantly increased the perfusion index, functional capillary density of the mucosa, and red blood cell velocity in the grafts after reperfusion in comparison with the grafts preserved with HTK. Conclusions The modified preservation solution improved the microcirculation of the transplants and needs detailed evaluation in further models of small-bowel transplantation.

Detection of HER2/neu receptor overexpression and/or amplification is a prerequisite for efficient anti-HER2 treatment of breast and gastric carcinomas. Immunohistochemistry (IHC) of the HER2 protein is the most common screening test, thus precise and reproducible IHC-scoring is of utmost importance. Interobserver variance still is a problem; in particular in gastric carcinomas the reliable differentiation of IHC scores 2+ and 1+ is challenging.

The cellular sarcoma gene (SRC) is a proto-oncogene encoding for a tyrosine kinase.SRC expression was determined in locally advanced rectal adenocarcinoma tissue from pretreatment biopsies and resection specimens.The expression level was correlated with clinicopathological parameters to evaluate the predictive and prognostic capacity.For this monocentric analysis 186 patients with locally advanced rectal cancer (median: 63.7 years; 130 men (69.9%), 56 women (30.1%)) were included.Patients with a carcinoma of the upper third of the rectum were treated with primary tumor resection (n=27; 14.5%).All other patients received a preoperative chemoradiotherapy (CRT) with 50.4 Gy and concomitant 5-fluorouracil (5-FU) or 5-FU+oxaliplatin followed by postoperative chemotherapy with 5-FU or 5-FU+oxaliplatin.SRC expression was determined with immunohistochemical staining from pretreatment biopsies (n=152) and residual tumor tissue from the resection specimens (n=163).The results were correlated with clinicopathological parameters and long-term follow-up.The expression of SRC was determined in pretherapeutic biopsies (mean H-Score: 229) and resection specimens (mean H-Score: 254).High SRC expression in pretherapeutic tumor samples significantly correlated with a negative postoperative nodal status (p=0.005).Furthermore an increased protein expression in residual tumor tissue was associated with fewer distant metastases (p=0.04).The overexpression of SRC in pretreatment tumor biopsies showed also a trend for a longer cancer-specific survival (CSS; p=0.05) and fewer local relapses (p=0.06)during long-term follow-up.High SRC expression in rectal cancer seems to be associated with a better long-term outcome.This finding could help in the future to stratify patients for a recurrence risk adapted postoperative treatment.

Liver metastasis development in breast cancer patients is common and confers a poor prognosis. So far, the prognostic significance of surgical resection and clinical relevance of biomarker analysis in metastatic tissue have barely been investigated. We previously demonstrated an impact of WNT signaling in breast cancer brain metastasis. This study aimed to investigate the value of established prognostic markers and WNT signaling components in liver metastases. Overall N = 34 breast cancer liver metastases (with matched primaries in 19/34 cases) were included in this retrospective study. Primaries and metastatic samples were analyzed for their expression of the estrogen (ER) and progesterone receptor, HER-2, Ki67, and various WNT signaling-components by immunohistochemistry. Furthermore, β-catenin-dependent and -independent WNT scores were generated and analyzed for their prognostic value. Additionally, the influence of the alternative WNT receptor ROR on signaling and invasiveness was analyzed in vitro. ER positivity (HR 0.09, 95 % CI 0.01–0.56) and high Ki67 (HR 3.68, 95 % CI 1.12–12.06) in the primaries had prognostic impact. However, only Ki67 remained prognostic in the metastatic tissue (HR 2.46, 95 % CI 1.11–5.44). Additionally, the β-catenin-independent WNT score correlated with reduced overall survival only in the metastasized situation (HR 2.19, 95 % CI 1.02–4.69, p = 0.0391). This is in line with the in vitro results of the alternative WNT receptors ROR1 and ROR2, which foster invasion. In breast cancer, the value of prognostic markers established in primary tumors cannot directly be translated to metastases. Our results revealed β-catenin-independent WNT signaling to be associated with poor prognosis in patients with breast cancer liver metastasis.

AIMS:Acetone compression (AC) is an elution compression technique for the comprehensive pathological examination of fatty tissue. Here AC is combined with digital morphometry to evaluate the impact of preoperative (neoadjuvant) chemoradiotherapy (neoCRT) on lymph node (LN) numbers and morphology in locally advanced rectal cancer. AC is compared with complete embedding of the mesorectal fat (whole mesorectal embedding (WME)) to exclude artificial alterations and to the standard technique, manual dissectioning (MD).METHODS:320 rectal cancer specimens were subjected to LN morphometry. Neoadjuvant CRT was applied in 204 specimens. LNs were prepared either with AC (n=138), WME (n=51) or MD (n=131). 8523 LNs were assessed including 530 nodes with metastases.RESULTS:LN prepared by AC and WME showed similar morphologies. AC revealed reduced LN sizes in neoCRT specimens compared with primary resection (2.2; 2.4 mm, p=0.049) while the LN number was comparable (27; 30/specimen). AC yielded 28 LN/specimen on average, MD yielded 22 LN (p<0.001). In neoCRT specimens, MD yielded less LN compared with primary resection (19; 25). MD detected less small LN (<2 mm; MD: 25%; AC: 56%) while 24 of the 135 LN metastases found by AC were ≤2 mm in diameter.CONCLUSIONS:AC does not alter LN morphology and is especially suited to retrieve small LN after neoadjuvant CRT of rectal cancer. Neoadjuvant multimodality treatment caused reduced LN sizes while the LN numbers were not affected. When compared with MD, AC proved more reliable in the retrieval of LN from rectal cancer specimens after neoCRT.

Objective In this study, we evaluate the frequency of HER-2 and HER-3 expression in liver metastases from patients with colorectal cancer (CRLM). We analyzed the potential of HER-2 and HER-3 as therapeutic targets and evaluated their prognostic value. Patients and Methods Overall 208 patients with CRLM were enrolled. HER-2 and HER-3 expression were determined in metastatic tissue of diagnostic punch biopsies (n = 29) or resection specimens (n = 179). The results of immunohistochemistry (IHC) scoring and In-situ-hybridization (ISH)-amplification were correlated with clinical parameters and for the 179 resected patients with cancer-specific (CSS) and overall survival (OS). The mean follow-up time was 56.7 months. Results Positivity of HER-2 status (IHC score 2+/ISH+ and IHC 3+) was found in 8.2% of CRLM. High expression of HER-3 (IHC score 2+ and IHC 3+) was detected in 75.0% of liver metastases. CSS after liver surgery was determined and was independent from the HER-2 status (p = 0.963); however HER-3 was prognostic with a favorable course for patients showing an overexpression of HER-3 (p = 0.037). Conclusions HER-2 overexpression occurs in only 8% of patients with CRLM but with 75% of cases HER-3 is frequently overexpressed in CRLM. Therefore, HER-2 and particularly HER-3 could serve as novel targets to be addressed within multimodal treatment approaches.

567 Background: Even though treatment options for coloretal cancer liver metastases have improved with the implementation of multimodal strategies and new agents, progression of distant metastases is still limiting the prognosis of affected patients. In this context we investigated the expression of HER2 and HER3 in patients with metastatic colorectal cancer and primary tumors. Methods: In this study 226 consecutive patients with hepatic metastases of colorectal cancer were included. HER2 and HER3 expression were determined in the tissue from resected metastases and—if available—in primary tumors. Immunohistochemical scoring (IHC 0 to IHC 3) and S-ISH-amplification-detection (for HER2) were used to determine the HER2 and HER3 status. Results: A positive HER2 status was found in 8.4% of all hepatic metastases; an overexpression of HER-3 in 74,8% of all cases. There was a high congruence of the expression pattern of HER2 and HER3 between hepatic metastases and available primary tumors (>90%). Conclusions: HER2 amplification and HER3 overexpression is detectable in a significant proportion of hepatic metastases of colorectal cancer. These results suggest that innovative new targeted treatment agents might be possible opportunities for the specific therapy of patients with HER2/HER3 positive metastatic colorectal cancer and should be further assessed within prospective clinical trials.

Purpose: We have previously shown that the addition of cetuximab to chemoradiation therapy failed to improve complete response rates (pCR) in rectal cancer. Here we report the long-term results of the cetuximab added to preoperative radiation therapy with capecitabine and oxaliplatin (CET-CAPOX-RT) phase 1/2 study that evaluated preoperative chemoradiation with cetuximab, capecitabine, and oxaliplatin in patients with rectal cancer.Methods and Materials: The median follow-up was 63 months (range, 5-73 months). Sixty patients were enrolled; 3 patients were excluded due to protocol violation, and 4 died before surgery. Total mesorectal excision was performed in 53 patients, in 85% (n=45) with curative intention (M0-status). Secondary end points including overall survival (OS) disease-free survival (DFS) and cancer-specific survival (CSS) were calculated. The prognostic value of KRAS mutation status was also assessed.Results: Histopathological examination confirmed ypUICC stages 0 (n=4; pCR), I (n=17), II (n=10), III (n=14), and IV (n=8). For patients who underwent surgery (n=53), OS at 1, 3, and 5 years was 88.7%, 83%, and 75.5%, respectively, whereas CSS rates were 94.1%, 88.1%, and 78.1%, respectively. In the 45 patients who were treated with curative intent (M0), the OS rates at 1, 3, and 5 years were 91.1%, 88.9%, and 86.7%, respectively; whereas CSS rates were 97.6%, 95.2%, and 90.3%, respectively; and DFS rates were 90.7%, 88.3%, and 88.3%, respectively. We did not find any locoregional failure in patients with M0-status (n=45). Chronic toxicity was rare. KRAS mutations, as detected in 33.3%, showed no correlation with the clinicopathological parameters nor significance for either OS (P=.112), CSS (P=.264), or DFS (P=.565).Conclusions: Taken together, chemoradiation therapy combined with cetuximab is safe, feasible, and offers excellent survival rates. KRAS mutation status was not a predictive factor. Importantly, lack of improvement in pCR rate did not translate to poor survival in our clinical trial. (C) 2013 Elsevier Inc.

Leukocytoclastic vasculitis is a multicausal systemic inflammatory disease of the small vessels, histologically characterized by inflammation and deposition of both nuclear debris and fibrin in dermal postcapillary venules. The clinical picture typically involves palpable purpura of the lower legs and may be associated with general symptoms such as fatigue, arthralgia and fever. Involvement of the internal organs, most notably the kidneys, the central nervous system or the eyes, is possible and determines the prognosis. Oxaliplatin-induced leukocytoclastic vasculitis is a very rare event that limits treatment options in affected patients. We report 2 patients who developed the condition under chemotherapy for advanced rectal and metastatic colon carcinoma, respectively; a termination of the therapy was therefore necessary. While current therapies for colorectal cancer include the combination of multimodal treatment with new and targeted agents, rare and unusual side effects elicited by established agents also need to be taken into account for the clinical management.

In patients with advanced rectal cancer (cUICC II and III) multimodality therapy resulted in better long-term local tumor control. Ongoing clinical trials are focusing on therapy intensification to improve disease-free (DFS) and cancer-specific survival (CSS), the integration of biomarkers for prediction of individual recurrence risk, and the identification of new targets. In this context, we investigated HER-2, a member of the epidermal growth factor receptor family, whose expression pattern and role was unclear in rectal cancer. A total of 264 patients (192 male, 72 female; median age 64 y) received standardized multidisciplinary treatment according to protocols of phase II/III trials of the German Rectal Cancer Study Group. HER-2 status was determined in pretherapeutic biopsies and resection specimens using immunohistochemistry scoring and detection of silver in situ hybridization amplification. Tumors with an immunohistochemistry score of 3 or silver in situ hybridization ratios of ≥2.0 were classified HER-2 positive; these results were correlated with clinicopathologic parameters [eg, resection (R) status, nodal status ((y)pN)], DFS, and CSS. Positive HER-2 status was found in 12.4% of biopsies and in 26.7% of resected specimens. With a median follow-up of 46.5 months, patients with HER-2 positivity showed in trend a better DFS (P=0.1) and a benefit in CSS (P=0.03). The 5-year survival rate was 96.0% (HER-2 positive) versus 80.0% (HER-2 negative). In univariate and multivariate analyses, HER-2 was an independent predictor for CSS (0.02) along with the (y)pN status (P<0.00001) and R status (P=0.011). HER-2 amplification is detectable in a relevant proportion (26.7%) of rectal cancer patients. For the development of innovative new therapies, HER-2 may represent a promising target and should be further assessed within prospective clinical trials.

INTRODUCTION:The phase III SATURN study demonstrated that first-line maintenance erlotinib extended progression-free survival (PFS) and overall survival (OS) versus placebo in patients with advanced non-small cell lung cancer (NSCLC). Analysis of epidermal growth factor receptor (EGFR) expression by immunohistochemistry (IHC) found no significant interaction between EGFR IHC status and PFS (p = 0.63) or OS (p = 0.52). The FLEX study of first-line cetuximab plus chemotherapy demonstrated that EGFR IHC expression was predictive of improved OS with cetuximab when assessed by H-score with a magnification rule. This novel method was used to reassess samples from SATURN.METHODS:The H-score method assigned a score of 0-300 to each patient, based on the percentage of cells stained at different intensities viewed at various magnifications. The discriminatory threshold was set at 200, per the FLEX study, and existing samples were re-read and classed as low (H-score < 200) or high (≥200) EGFR expression. PFS and OS were re-analyzed based on these new classifications.RESULTS:In the overall and EGFR wild-type populations, erlotinib provided a consistent survival benefit versus placebo. Hazard ratios (HRs) in the overall population were similar between EGFR IHC-positive and -negative patients for median PFS (HR 0.68 [95% confidence interval (CI) 0.53-0.86] and 0.76 [95% CI 0.62-0.93], respectively) and OS (HR 0.80 [95% CI 0.62-1.05] and 0.80 [95% CI 0.64-1.01] for IHC-positive and IHC-negative, respectively). In the EGFR wild-type population, HRs were again similar between EGFR IHC-positive and -negative subpopulations for PFS (HR 0.69 [95% CI 0.51-0.95] and 0.84 [95% CI 0.63-1.12], respectively) and OS (HR 0.78 [95% CI 0.55-1.10] and 0.76 [95% CI 0.55-1.05], respectively).CONCLUSIONS:These data suggest that EGFR IHC does not have value as a marker to predict erlotinib benefit in the first-line maintenance setting for advanced NSCLC.

e14162 Background: Preoperative application of cetuximab and radiochemotherapy combined with 5-FU+oxaliplatin (CET-CAPOX-RT-phase-I/II-study) failed to increase the complete remission rate (pCR, primary endpoint) in rectal cancers. Here, we report longterm survival in respect of biomarkers. Methods: In 45 of 53 patients (85%) total mesorectal excision (TME) was performed with curative intention. Disease-/progression-free survival (DFS/PFS) and overall cancer specific survival (CSS) were correlated with clinicopathological findings (e.g. tumor regression grading (TRG), KRAS-, HER-2-, Ki67-, EGFR-, IGF1R-, Src-, PTEN-, Survivin-, XIAP- and thymidylatesynthase(TS)-status). Results: After triple RCT, ypUICC stages were 0 (4x), I (17x), II (10x), III (14x) and IV (7x); RCT-induced T-level- (49%) and UICC-downcategorizing (53%) correlated with major (38% TRG 3-4) versus minor response (62.3% TRG 0-2) (p=0.005 and p=0.02, respectively). During follow-up (63 months) 7/53 patients with M1-status at surgery died. In 45 patients with M0-status no locoregional failure but in 5 patients metastatic recurrences were detected. DFS at 1, 3 and 5 years was 90.7% (CI: 82.5-99.8), 88.3% (CI: 79.2-98.5) and 88.3% (CI: 79.2-98.5); the CSS was 97.6% (CI: 93.1-100), 95.2% (CI: 89-100), and 90.3% (CI: 82-99.8), respectively. Patients with ypN0-status demonstrated better DFS (p=0.027), PFS (p=0.024) and CSS (p=0.016). KRAS mutations (33%) had no prognostic influence but a positive HER-2-status (18%) seemed to be associated with better CSS (p=0.11). High Ki-67-expression (p=0.09) as well as the loss of RCT-induced Survivin-downregulation (p=0.05) correlated with reduced CSS. No prognostic influence was found for EGFR-, IGF1R-, Src-, PTEN-, XIAP- and TS-expression. Conclusions: The disappointing pCR rate of the CET-CAPOX-RT-phase-I/II-study did not translate to poor longterm survival.

Introduction: The pathogenesis of nasal polyposis and nasal hyperplasia is still unknown. The localization of caspases in nasal polyps and nasal hyperplasia of patients with and without allergic rhinitis was studied. Methods: Sections of human nasal polyps (n = 5) and hyperplastic nasal turbinates (5 with, 5 without allergy) were stained for active caspase-3 and caspase-8. Double immunofluorescence was used to evaluate colocalization of the caspases with Ki-M1P and tryptase. TUNEL was performed. Results: Active caspase-3 and caspase-8 were seen in nearly all nasal polyps and hyperplastic nasal turbinates. Active caspase-3 was predominantly localized in stromal cells, identified as mast cells. Caspase-8 was localized in mast cells with the pattern similar to active caspase-3 and additionally found in epithelial cells at the nasal and polyp surface and in epithelial cells of glands. Conclusion: Our results suggest that mast cell apoptosis may be involved in the pathological mechanisms which characterize and sustain chronic inflammatory disorders of the nasal mucosa with and without allergy.

493 Background: With the implementation of multimodal treatment strategies including neoadjuvant radiochemotherapy (RCT), the prognosis of locally advanced rectal cancer has been improved over the last two decades. Most actual clinical trials aim to further develop therapy by intensification of agents administered. We examined Her2/neu in rectal cancer patients to evaluate its expression as a potential drug target as well as its capacity as predictive and prognostic biomarker. Methods: Two-hundred-seventy-seven patients (205 male, 72 female; median age 63.5 years) with locally advanced rectal cancer (cUICC-II/III) were included in this study. Preoperative RCT (50.4 Gy and concomitant either 5-FU or 5-FU and oxaliplatin) was administered in 236 patients followed by surgical resection with total mesorectal excision (TME). Another 41 patients received postoperative RCT. All patients were treated within phase-II/-III-trials of the German Rectal Cancer Study Group or analogous to these standardized protocols. Her2-/neu expression from pre-treatment biopsies and corresponding resection specimens were assessed by immunohistochemical staining and SISH-analysis. Results: A positive Her-2/neu expression was found in 12.3% of all pre-treatment tumor biopsy samples and in 28.7% of the resection specimens. The correlation of the pre-treatment Her-2/neu status did not show a significant correlation with the grade of tumor regression. However, patients with a higher Her-2/neu protein expression as measured in the resection specimen showed a significant survival benefit (p=0.0277) compared with patients having low intratumoral Her-2/neu expression during the follow-up (median 43 months). The 10-year survival rate was 73.2% (Her-2/neu positive) versus 60.1% (Her-2/neu negative). Conclusions: The proportion of patients with a positive Her-2/neu status is relevant in rectal cancer. Her-2/neu might potentially serve as a promising drug target in rectal cancer and should be further assessed within prospective clinical trials. Furthermore the Her-2/neu status seems to be a valuable prognostic marker within the multimodal treatment of advanced rectal cancer.