Clinical significance of HER2 and EGFR expression in colorectal cancer patients with ovarian metastasis

Brain metastases (BM) from colorectal cancer (CRC) are a rare but increasing event. Surgical resection of oligometastatic disease, including BM, may produce a survival benefit in selected patients. Previous studies described the HER-2 expression patterns in CRC patients, but its prognostic role still remains controversial. Information on the HER-2 expression in BM from CRC is currently lacking. Among the over 500 patients treated at our Department of Neurosurgery in the last 13 years (1999–2012), we identified a cohort of 50 consecutive CRC patients resected for BM. Clinical data were retrospectively reviewed using electronic hospital charts and surgical notes. Formalin-fixed, paraffin-embedded tissue samples were retrieved and histologically reviewed. HER-2 status was assessed on 4-μm sections by HerceptTest™, and scored by two pathologists according to gastric cancer HER-2 status guidelines. In score 2+ cases HER-2 gene copy number was analyzed by FISH, performed using the PathVysion HER-2 DNA Probe Kit. Median age at time of BM resection was 65 years (35–82); most patients were males (60%) with a good performance status. The majority of the BM were single (74%) and sited in the supratentorial area (64%); 2–4 lesions were diagnosed in 9 patients (18%), and >4 in 3 patients (6%). The rate of HER-2 positivity (defined as IHC score 3+ or IHC score 2+ and FISH gene amplification) was 8.1% for the primary CRC tumors and 12% for their corresponding BM. The concordance rate between primary tumors and matched BM was 89%. Median overall survival after neurosurgery was 6.5 months for HER-2 IHC score 0 vs. 4.6 months for HER-2 IHC score 1+/2+/3+; the difference was statistically significant (p = 0.01, Log-rank test). HER-2 positivity of our case cohort was low but comparable to literature. Concordance rate of HER-2 expression between BM and corresponding primary tumors is high and similar to those reported for breast and gastric cancers. Our data suggest a potential negative prognostic value of HER-2 expression in brain lesions from CRC.

Simple SummaryTreatment with anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (mAbs)—cetuximab and panitumumab—produced clinical benefits in a subset of patients with metastatic colorectal cancer (mCRC). Here, the authors investigated the relative expression and predictive value of HER family members in 144 patients with CRC. They found high levels of expression of HER2 in patients treated with cetuximab; these were associated with shorter progression free survival (PFS). The results provide support for the emergence of HER2 as a therapeutic target in patients with mCRC.The overexpressed HER2 is an important target for treatment with monoclonal antibody (mAb) trastuzumab, only in patients with breast and gastric cancers, and is an emerging therapeutic biomarker in metastatic colorectal cancer (mCRC) treated with anti-epidermal growth factor receptor (EGFR) mAbs cetuximab and panitumumab. In this study, we investigated the relative expression and predictive value of all human epidermal growth factor receptor (HER) family members in 144 cetuximab-treated patients with wild type RAS mCRC. The relative expression of EGFR and HER2 have also been examined in 21-paired primary tumours and their metastatic sites by immunohistochemistry. Of the 144 cases examined, 25%, 97%, 79%, 48%, and 10% were positive for EGFR, HER2, HER3, and HER4 and all four HER family members, respectively. The expression of EGFR was an indicator of poorer overall survival and the membranous expression of HER2 and HER3 3+ intensity was associated with a shorter progression free survival (PFS). In contrast, the cytoplasmic expression of HER2 was associated with better PFS. In 48% and 71% of the cases, there were discordance in the expression of EGFR or one or more HER family members in paired primary and related metastatic tumours, respectively. Our results implicate the importance of a large prospective investigation of the expression level and predictive value of not only the therapeutic target (i.e., EGFR protein) but also HER2 and other HER family members as therapeutic targets, or for response to therapy with anti-EGFR mAbs and other forms of HER inhibitors, in both the primary tumours and metastatic sites in mCRC.

The aim of this study was to explore the lncRNAs expression in colorectal cancer (CRC) patients with type 2 diabetes (T2DM) and evaluate the diagnostic value of lncRNAs expression in CRC patients with T2DM. The present study was conducted on two cohorts with CRC patients. The tissues levels of lncRNAs were measured by real-time PCR analysis. The results showed that H19 and MALAT1 expression were higher in CRC tissues than in normal colorectal mucosa (p = 1.59 × 10−6 and p = 6.95 × 10−9, respectively), whereas lincRNA-p21 showed lower expression in CRC tissues (p = 1.10 × 10−4). Logistic regression analysis results indicated that the expression of H19 was significantly lower in CRC patients with T2DM compared with CRC patients without T2DM (p = .032). H19 expression in CRC group without T2DM was significantly associated with hypertension (p = .040). Additionally, the area under the receiver operating characteristic curve of H19 was 0.672 of the group CRC with T2DM, which suggests that H19 could be a useful biomarker and predictive targets for diagnosis of T2DM in CRC patients.

Members of the epidermal growth factor receptor, EGFR, family are interesting as targets for radionuclide therapy using targeting agents labeled with α- or β-emitting radionuclides, especially when EGFR-positive colorectal carcinomas, CRC, are resistant to EGFR inhibiting agents like cetuximab and various tyrosine kinase inhibitors. The expression of EGFR, HER2 and HER3 was therefore analyzed in CRC samples from primary tumors, corresponding lymph node metastases and, in a few cases, liver metastases. The expression of HER2 and EGFR was scored from immunohistochemical preparations using the HercepTest criteria 0, 1+, 2+ or 3+ for cellular membrane staining while HER3 expression was scored as no, weak or strong cytoplasm staining. Material from 60 patients was analyzed. The number of EGFR 2+ or 3+ positive primary tumors was 16 out of 56 (29%) and for lymph node metastases 8 out of 56 (14%) whereas only one out of nine (11%) liver metastases were positive. Thus, there was lower EGFR positivity in the metastases. Only one among 53 patients was strongly HER2 positive and this in both the primary tumor and the metastasis. Eight out of 49 primary tumors (16%) were strongly HER3 positive and the corresponding numbers for lymph node metastases were 9 out of 49 (18%) and for liver metastases 2 out of 9 (22%). The observed number of strongly EGFR positive cases was somewhat low but EGFR might be, for the cases with high EGFR expression in metastases, a target for radionuclide therapy. HER2 seems not to be of such interest due to rare expression, neither HER3 due to mainly expression in the cytoplasm. The requirements for successful EGFR targeted radionuclide therapy are discussed, as well as patient inclusion criteria related to radionuclide therapy.

Previous studies indicated that approximately 3.4% of female colorectal cancer (CRC) patients are at increased risk of developing ovarian metastases (OM). It has been suggested that young women more frequently develop this form of metastatic disease. This study evaluated, in 6 Dutch hospitals, the proportion of young women with CRC who developed OM. In a cohort of 200 young (age ≤ 55) women with CRC, the proportion of patients diagnosed with synchronous or metachronous OM was calculated. This study revealed that 5% (n = 10) of young female CRC patients developed ovarian metastases resulting in a 5-year overall survival rate of approximately 40%. Furthermore, six patients had concurrent peritoneal metastases, five patients had bilateral ovarian metastases, and five patients had synchronous metastases, while the median time of the occurrence of metachronous metastases (n = 5) was 19months. This retrospective multicenter cohort study indicates that 5% of young women with CRC either present with or develop OM. This result appears to be clinically relevant and demonstrates the need for improved surveillance for young women diagnosed with CRC.

Despite recent clinical trials, the sensitivity and resistance of metastatic gastric cancer to anti-HER2 and anti-EGFR therapy are still unclear. To clarify the HER2 and EGFR expression status in the metastatic sites, we immunohistochemically compared HER2 and EGFR expression between primary and metastatic tumors from 52 gastric cancer patients with liver metastases and 85 patients with peritoneal metastases. The HER2 positivity rate of primary and metastatic tumors in patients with liver metastases, especially with intestinal-type histology (70.6 and 80.0 %, respectively), was significantly higher than in primary and metastatic tumors (22.4 and 16.4 %, respectively) in patients with peritoneal metastases. HER2 positivity of the primary tumor and liver metastases showed good concordance (87.5 %) in patients with liver metastases. In contrast, the EGFR positivity rate of metastatic tumors (70.1 %) in patients with peritoneal metastases was significantly higher than that of metastatic tumors (37.5 %) in patients with liver metastases. HER2 and EGFR expression tended to be mutually exclusive, and HER2/EGFR double-positive cases were rare in patients with liver or peritoneal metastases. In four such patients with HER2/EGFR double-positive primary tumors, the HER2- and EGFR-positive areas were separate, and corresponding liver metastasis was only positive for HER2 and peritoneal metastasis only positive for EGFR. These results indicate that HER2 and EGFR are preferentially expressed in the liver and peritoneal metastases, respectively, which would be potential targets for anti-HER2 and anti-EGFR molecular therapy in metastatic gastric cancer patients.

Objectives: In the 9th edition of the Japanese Classification of Colorectal, Appendiceal, and Anal Carcinoma (JCCRC), ovarian metastasis is classified as distant metastasis. We assessed the significance of resection of ovarian metastases and the validity of this 9th edition of JCCRC for ovarian metastases from colorectal cancer (CRC). Methods: We retrospectively analyzed the clinicopathological factors and overall survival of 17 patients with ovarian metastases from CRC who underwent resection and 110 female CRC patients with Stage IV (M1a) disease. Results: The patients with only ovarian metastases who underwent resection had a longer median survival time than patients with both ovarian and peritoneal metastases who underwent resection (45.4 months vs. 9.3 months, P = 0.029). The 5-year overall survival of the patients with only ovarian metastases who underwent R0 resection was as long as that of the female Stage IV (M1a) CRC patients after R0 resection (50% vs. 48%, P = 0.334). Conclusions: We found that, after resection, patients with only ovarian metastases had significantly better prognoses than patients with ovarian and peritoneal metastases. R0 resection of ovarian metastasis indicated as good prognosis as R0 resection of metastasis to one distant organ without ovaries. So the 9th edition of JCCRC, which classifies ovarian metastasis from CRC as distant metastasis, is appropriate.

e16023 Background: Krukenberg tumors of gastric origin have a poor prognosis. Their clinicopathologic characteristics are rarely reported and they lack optimal clinical management. There is an urgent need to explore new strategies to improve patient survival and quality of life. Methods: The clinicopathological characteristics, treatments and survival data were collected and analyzed from 130 patients with Krukenberg tumors of gastric cancer. Results: The median age of the patients was 41 years. A total of 63.1% of patients had synchronous ovarian metastasis, 70.8% had bilateral ovarian metastasis, 68.5% had peritoneum metastasis, and 98.4% had pathologically poorly differentiated adenocarcinoma. The positive rate of HER-2 was 1.8%. Only one patient showed dMMR. The follow-up rate was 90.8%. The median OS from the diagnosis of ovarian metastasis was 13.0 months. Univariate analysis showed that the significant survival factors included menstrual status, size of the gastric lesion and ovarian metastasis, number of lymph node metastases, interval time to ovarian metastasis, resection of gastric foci, peritoneal metastasis, oophorectomy, chemotherapy after ovarian metastasis, 2-drug regimen chemotherapy, albumin, serum CA-125 level, platelet count, D-dimer, fibrinogen, pretreatment high FAR, PLR and SII. Further multivariable analysis found that albumin, peritoneal metastasis and oophorectomy were independent prognostic factors. Conclusions: Patients of gastric cancer with Krukenberg tumors had some unique clinical characteristics. Active improvement of hypoalbuminemia, treatment of hypercoagulability, management of peritoneal metastasis, timely oophorectomy and chemotherapy can improve the patients’ prognosis. This suggests that we should focus on the nutritional status and coagulation function of patients with Krukenberg tumors in our clinical work.

The expression of EGFR, HER2 and HER3 receptors were analyzed in immunohistochemical preparations from primary esophageal tumours and corresponding lymph node metastases. The goal was to evaluate whether any of these receptors are suitable as targets for radionuclide based imaging and therapy. The receptor expressions were evaluated in parallel samples, primary tumour and metastasis, from each patient (n=51). The majority of the cases were esophageal squamous cell carcinomas, ESCC (n=40). The HercepTest scoring was used for the analysis of both HER2 and EGFR expression (0, 1+, 2+ or 3+). HER3 was only evaluated as negative, weak or strong staining. EGFR overexpression (2+/3+) was found in 67.5% (27/40) of both the ESCC primary tumours and the corresponding lymph node metastases. There were only a few changes in these EGFR-scores: two cases from 2+/3+ to 0/1+ when the primary tumours were compared to the corresponding metastases and 2 changes the other way around. HER2 overexpression (2+/3+) was found in only 3 of the primary ESCC tumours and 2 of the lymph node metastases. EGFR and HER2 stainings were found mainly in the cell membranes. The HER3 staining (weak or strong) was mainly cytoplasmic and granular and was observed in about half (20/39) of the cases, for both the ESCC primary tumours and the corresponding lymph node metastases. It was concluded that ESCC lymph node metastases generally have a strong expression of EGFR in their cell membranes and to the same extent as in the primary tumours. The stability in EGFR expression is encouraging for efforts to develop radionuclide based EGFR imaging agents. It is also possible that EGFR targeting agents (e.g. Iressa, Tarceva, Erbitux or radiolabelled antibodies) can be applied for therapy of ESCC.

e14697 Background: 5-30 % of all ovarian tumors are metastatic and only 3-14 % of them derive from gastrointestinal tract. The aim of this study is to determine the rate of colorectal cancer patients with ovarian metastases and also to identify the features in the management of these patients. Methods: 972 colorectal cancer patients admitted to our clinic between 01/2001 and 12/2011 were included in the study. Among these patients, only 9 had ovarian metastases. Age, menopausal status, initial symptom, operation status, localisation of colorectal tumor, stage at diagnosis, tumor grade, histopathological type, colorectal tumor-ovarian metastases interval, having synchronous or metachronous metastases, the place of metastases and concurrent metastatic disease were evaluated. SPSS 16 is used. Results: Mean age of patients was 45 years (range between 21-72 years). 66.7 % had premenopausal state. 55 % of them had right colon tumor. 5 patients had stage IV disease. 5 patients had synchronous metastases (55 %). Colorectal cancer-ovarian metastases interval was 6-49 months in the patients with metachronous metastases. Among patients; 5 had right and 1 had left ovarian metastases and 3 had metastases to both ovaries. 2 of 3 bilateral ovarian metastases were derived from right-sided tumors (66.6 %). 7 patients also had metastases to other different parts and most of those had peritoneal involvement (85.7 %). PFS was between 2 and 27 months. At the time of ovarian metastases, 7 patients had high CA 125 levels and 3 had high CEA levels. All patients with high levels of CA 125 during diagnosis continued to have high levels of CA 125 with ovarian metastases. Conclusions: Premenopausal patients seem to have higher risk of ovarian metastases. This study support that examining CA 125 levels in colorectal cancer patients who have abnormal findings in the gynecological examination preoperatively may help not to miss synchronous ovarian metastases.The finding of ovarian metastases should make consideration that the disease is disseminated. Ovaries should be examined preoperatively and it should be kept in mind that CA 125 levels may be a valuable marker in this setting.

The cytoplasmic ribonuclease DICER1 is one of the key enzymes in microRNA (miRNA) processing, essential for the production of mature miRNA. The effect of DICER1 expression in tumor cells on the prognosis of patients with several cancers has been examined with controversial results in various cancer types. In particular, the clinical significance of DICER1 expression in colorectal cancer (CRC) patients has yet to be determined. The aim of this study was to evaluate the correlation between the DICER1 mRNA levels and the clinicopathological characteristics and prognostic significance in CRC patients. Tumor and normal adjacent tumor tissues from 260 patients with CRC (Dukes' stage A: 40 cases, Dukes' B: 68 cases, Dukes' C: 88 cases and Dukes' D: 64 cases) were examined. The DICER1 mRNA levels were measured using the TaqMan real-time reverse transcription polymerase chain reaction (RT-PCR) method. The expression levels of DICER1 mRNAs showed a significant decrease in CRC tissues as compared to normal ones (P=0.039). A statistically significant association was observed between DICER1 mRNA expression and tumor size, depth of invasion, lymph node metastasis, lymphatic invasion and Dukes' stage. In Kaplan-Meier survival curve analysis, overall survival (OS) and disease-free survival (DFS) rates of patients with a low DICER1 mRNA expression were significantly worse compared to patients with a high DICER1 mRNA expression (OS P<0.001; DFS P<0.001). In the Cox multivariate analysis, DICER1 mRNA expression in CRC tissues was identified as an independent prognostic factor for OS [hazard ratio (HR), 0.30; 95% confidence interval (CI), 0.13-0.64; P=0.001] and DFS (HR, 0.23; 95% CI, 0.10-0.48; P=0.001). This study demonstrated that a reduced DICER1 mRNA expression is associated with poor prognosis in CRC patients.

4527^ Background: HER2 overexpression may be a prognostic factor for poor outcomes in pts with high-risk UC. Publications report a wide variability of HER2 expression in UC, with ≥2+ HER2 expression by immunohistochemistry (IHC) reported in &lt;10% to &gt;50% of cases. DN24-02 is an investigational autologous immunotherapy targeting HER2, based on the same manufacturing platform used for sipuleucel-T. NeuACT (N10-1; NCT01353222) is designed to evaluate whether DN24-02 can prolong survival when given as adjuvant therapy following surgical resection in pts with high risk HER2-expressing UC (Bajorin, et al. ASCO 2012). Here we report preliminary data for HER2 expression on primary tumor and positive lymph node samples, and covariate analyses. Methods: Trial eligibility criteria include surgical resection of a primary UC, with either ≥pT2 or pN+ staging, and HER2 expression ≥1+ IHC. Surgical specimens are screened for HER2 expression by central pathology laboratory review and HER2 positivity is scored using the Dako HercepTest system. Results: As of December 2012, tumor specimens from 114 pts have been screened. Of these pts, 84 (74%; 95% CI: 65–82%) had a HER2 expression score ≥1+ in the primary tumor, with 36 (32%) having a 2+ score and 5 (4%) having a 3+ score. Thirty-eight pts also had HER2 expression levels evaluated in lymph node samples. Of these 38 pts, 35 (92%; 95% CI: 79–98%) had a HER2 expression score ≥1+ in the lymph nodes, with 17 (45%) having a 2+ score and 4 (11%) having a 3+ score. Gender, primary tumor site, nodal stage and prior neoadjuvant chemotherapy were not significantly correlated with HER2 expression (p≥0.10). Conclusions: To date, high frequencies (≥74%) of HER2 expression ≥1+ in primary tumor and lymph node samples of UC pts have been observed. No baseline variables, including prior neoadjuvant chemotherapy, appear to affect the rate of HER2 expression. Although preliminary, these data are consistent with prior studies that have noted a higher incidence of HER2 expression in UC lymph node tumor vs. primary tumor and suggest that HER2 protein expression is common in high-risk UC. Clinical trial information: NCT01353222.

Purpose. The aim of this study was to compare the overexpression of specific biomarkers in primary advanced and recurrent epithelial ovarian cancers.Methods. Biomarker expression by epithelial ovarian cancer specimens from primary and metastatic sites was examined by immunohistochemistry and flow cytometry. Biomarker expression by subpopulations of tissues consisting of matched pairs of synchronous and metachronous lesions was also studied.Results. A total of 3173 epithelial ovarian cancer specimens were retrieved from women with FIGO Stage III/IV disease. These included lesions from 1036 primary and 2137 metastatic sites. The percentages of biomarker expression for primary and metastatic lesions, respectively, were MDR1, 12 and 10%; p53, 55 and 60%; HER2, 12 and 11%; EGF-R, 26 and 33%; increased microvessel counts (CD31), 21 and 36%. Approximately 73% of both primary and metastatic specimens were aneuploid, and approximately 57% of both sets had an S-phase fraction >7%. Only EGF-R and CD31 expression were found to be significantly different between the primary and metastatic tumors (P < 0.05). Of the paired synchronous cases (n = 48) evaluated, 88% of aneuploid primary lesions were associated with aneuploid metastases. Similarly, the distributions for MDR1, HER2, and p53 expression did not vary significantly between primary and metastatic sites. Pairings of metachronous cases (n = 66) revealed that nearly 80% of primary aneuploid tumors (n = 39) retained their aneuploid status at the time of relapse. Furthermore, there were no significant changes in MDR1, p53, or HER2 expression at relapse.Conclusions. With the exception of EGF-R and CD31, clonal divergence of the biomarkers evaluated in this study probably does not play a significant role in imparting clinical heterogeneity during the advanced and recurrent stages of epithelial ovarian cancer. These particular genes likely undergo alterations early in the tumorigenesis process before metastases have become established.

The macrophage-associated molecule CD163 has been reported as a prognostic biomarker in different cancer types, but its role in colorectal cancer (CRC) is unclear. We studied CD163 in the tumor microenvironment and circulation of patients with CRC in relation to clinicopathological parameters. An enzyme-linked immunosorbent assay (ELISA) was used to measure the serum sCD163 levels and multiparameter flow cytometry was used to study the peripheral blood monocytes and their CD163 expression in CRC patients (N = 78) and healthy donors (N = 50). The distribution of tumor-associated macrophages (TAMs) was studied in primary colorectal tumors with multiplex immunofluorescence. We showed that CRC patients with above-median sCD163 level had a shorter overall survival (OS, p = 0.035) as well as disease-free survival (DFS, p = 0.005). The above-median sCD163 remained significantly associated with a shorter DFS in the multivariate analysis (p = 0.049). Moreover, a shorter OS was observed in CRC patients with an above-median total monocyte percentage (p = 0.007). The number and phenotype of the stromal and intraepithelial TAMs in colorectal tumors were not associated with clinical outcome. In conclusion, sCD163 and monocytes in the circulation may be potential prognostic biomarkers in CRC patients, whereas TAMs in the tumor showed no association with clinical outcome. Thus, our results emphasize the importance of the innate systemic immune response in CRC disease progression.

Human epidermal growth factor receptor 2, HER-2, is overexpressed in various tumours, e.g. breast- and bladder tumours. The aim of this study was to predict the potential use of HER-2 receptors as targets in systemic treatment of disseminated bladder tumours. HER-2 expression was assessed in bladder carcinoma metastases and the corresponding primary tumours, and subsequently compared with the EGFR expression. HER-2 and EGFR expression was analysed by immunohistochemistry in formalin-fixed, paraffin-embedded tissues from 21 patients with metastatic bladder carcinoma. HER-2 was overexpressed in 81% of the primary tumours and in 67% of the metastases. All HER-2-positive metastases were from HER-2-positive primary tumours. The results for EG FR were 71% of both primary and metastases-positive tumours. In 90% of the primary tumours and 86% of the metastases, at least one of the receptors was overexpressed. These results suggest that HER-2 targeted therapy can be considered as an alternative or a complement to other modalities in the treatment of metastatic urinary bladder carcinoma.