Formalin-fixed Paraffin-embedded Primary Tumor Tissue Research Articles

Protein expression and localization of ABC transporters in pancreatic adenocarcinoma: Prognostic role of ABCC8

BackgroundATP-binding cassette (ABC) transporters translocate various substances across cellular membranes. Their deregulation may cause cancer drug resistance or perturbations in the supply of building blocks for cancer cells and modify patients’ prognosis. This study investigated protein expression and cellular localization of the previously suggested putative prognostic biomarkers – ABCB2/TAP1, ABCC7/CFTR, ABCC8/SUR1, and ABCD4 in patients with pancreatic ductal adenocarcinoma (PDAC). MethodsProtein expression and localization were assessed by immunohistochemistry in formalin-fixed paraffin-embedded primary tumor tissue blocks of 61 PDAC patients and associated with clinical data and the survival of patients. ResultsNo CFTR protein expression was observed in PDAC, while TAP1 and ABCC8 were expressed predominantly in the cytoplasm of tumor cells. Most samples (81 %) had detectable both membranous and cytoplasmic ABCD4 staining and 42 % had ABCD4 expressed in the apical orientation. Negative membranous ABCD4 staining was significantly more frequent in advanced stage III or IV tumors (p = 0.022). Small or medium counts of individual ABCC8-positive cells in the stroma surrounding tumor tubules were also more often found in stage III or IV (p = 0.044). Patients with moderate or strong ABCC8 cytoplasmic staining intensity in tumor cells had a 3.5-fold higher risk of disease progression than those with weak staining (p = 0.002). ConclusionsThe study shows for the first time that the cytoplasmic ABCC8 protein expression has prognostic value in PDAC.

PL01.4. A LATE BREAKING ABSTRACT: A LONG PEPTIDE VACCINE TARGETING THE CLONAL DRIVER MUTATION H3K27M IN ADULT PATIENTS WITH DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND Clonal and mutually exclusive substitution of lysine 27 to methionine in histone H3-3A (H3K27M) defines an aggressive subtype of diffuse glioma. These diffuse midline gliomas (DMG) are aggressive, incurable primary brain tumors that occur predominantly in a midline location in children and young adults. Surgical treatment options remain limited, response to chemoradiation is poor and palliative radiotherapy remains the only intervention with proven benefit, resulting in a median overall survival between 10 and 15 months after initial diagnosis. Previous studies have shown that a H3K27M-specific long peptide vaccine (H3K27M-vac) induces mutation-specific immune responses capable of controlling H3K27M+ tumors in a major histocompatibility complex (MHC)-humanized mouse model. MATERIAL AND METHODS Here we describe the first-in-human treatment with H3K27M-vac of eight adult patients with recurrent H3K27M+ DMG. Induction of mutation-specific immune responses was determined in peripheral blood using Interferon-γ ELISpot assays. Class II HLA-restricted peripheral H3K27M-specific T cells were expanded in vitro to characterize and track unique H3K27M-reactive T cell responses in blood and cerebrospinal fluid (CSF). RESULTS Repeat vaccinations with H3K27M-vac were safe and induced mutation-specific immune responses in five of eight patients after a median of two vaccinations. Median progression free survival after start of vaccination was 6.2 months and median overall survival was 12.8 months. In vitro restimulation of peripheral CD4+ and CD8+ T cells with H3 mutant peptide or wild type peptide revealed that H3K27M specific immune responses are CD4-mediated and can be suppressed by blocking antibodies against MHC II, but not MHC I. Furthermore, proximity ligation assay of formalin-fixed paraffin-embedded primary tumor tissue revealed that H3K27M neoepitope co-localizes with HLA class II-DR. One patient showed radiographic pseudoprogression (PsPD) according to iRANO criteria within six weeks after first detection of mutation-specific peripheral immune response. Following PsPD, three out of the top ten vaccine-induced, K27M-expanded CD4+ TCR clonotypes from peripheral blood were detectable in the CSF of this patient who went into sustained complete remission for > 31 months. CONCLUSION H3K27M-vac is safe and induces H3K27M-specific CD4 T cell responses in patients with H3K27M+ DMG. An ongoing multicenter, phase 1 clinical trial for adult patients with newly diagnosed H3K27M+ DMG will assess the safety and immunogenicity of H3K27M-vac in combination with atezolizumab in standard-of-care radiotherapy (NCT04808245).

Single-nucleotide polymorphisms and the effectiveness of taxane-based chemotherapy in premenopausal breast cancer: a population-based cohort study in Denmark

PurposeTaxane-based chemotherapy is the primary treatment for premenopausal breast cancer. Although being inconsistent, research suggests that variant alleles alter pharmacokinetics through reduced function of OATP transporters (limiting hepatic uptake), CYP-450 enzymes (hampering drug metabolism), and ABC transporters (decreasing clearance). Reduced function of DNA repair enzymes may hamper effectiveness through dose-limiting toxicities. We investigated whether single-nucleotide polymorphisms (SNPs) were associated with breast cancer recurrence or mortality in premenopausal women diagnosed with breast cancer.MethodsWe conducted a population-based cohort study of premenopausal women diagnosed with non-distant metastatic breast cancer in Denmark during 2007‒2011, when guidelines recommended adjuvant combination chemotherapy (taxanes, anthracyclines, and cyclophosphamide). Using archived formalin-fixed paraffin-embedded primary tumor tissue, we genotyped 26 SNPs using TaqMan assays. Danish health registries provided data on breast cancer recurrence (through September 25, 2017) and death (through December 31, 2019). We fit Cox regression models to calculate crude hazard ratios (HRs) and 95% confidence intervals (CIs) for recurrence and mortality across genotypes.ResultsAmong 2,262 women, 249 experienced recurrence (cumulative incidence: 13%) and 259 died (cumulative incidence: 16%) during follow-up (median 7.0 and 10.1 years, respectively). Mortality was increased in variant carriers of GSTP1 rs1138272 (HR: 1.30, 95% CI 0.95–1.78) and CYP3A rs10273424 (HR: 1.33, 95% CI 0.98–1.81). SLCO1B1 rs2306283 (encoding OATP1B1) variant carriers had decreased recurrence (HR: 0.82, 95% CI 0.64–1.07) and mortality (HR: 0.77, 95% CI 0.60–0.98).ConclusionDocetaxel effectiveness was influenced by SNPs in GSTP1, CYP3A, and SLCO1B1 in premenopausal women with non-distant metastatic breast cancer, likely related to altered docetaxel pharmacokinetics. These SNPs may help determine individual benefit from taxane-based chemotherapy.

Abstract P3-13-06: Single nucleotide polymorphisms and mortality after docetaxel-based chemotherapy in premenopausal breast cancer: A population-based cohort study in Denmark

Abstract Background Taxane-based chemotherapy is first line treatment in various cancers, including premenopausal breast cancer, but the inter-individual effectiveness is unpredictable. Differences in expression and activity of docetaxel-metabolizing enzymes and transporters (DMETs) may modify docetaxel effectiveness. Although reported data are inconsistent, some findings suggests that variant alleles that reduce the function of 1) SLC-transporters reduce drug influx into hepatocytes; 2) CYP-450 enzymes hamper drug metabolism; and 3) ABC-transporters decrease docetaxel clearance. The net effect is hypothesized to be increased docetaxel exposure and effectiveness. In contrast, GSTP1 variants have been associated with poorer docetaxel effectiveness. We investigated whether single nucleotide polymorphisms (SNPs) in drug metabolizing enzymes and transporters were associated with mortality in premenopausal breast cancer patients. Materials and methods Using the Danish Breast Cancer Group (DBCG) clinical database, we identified data on premenopausal women aged 18–55 years, diagnosed with non–metastatic breast cancer during 2007–2011. All women were recommended epirubicin, cyclophosphamide and docetaxel-based adjuvant chemotherapy, and tamoxifen if the disease was estrogen receptor (ER) positive. From DBCG and other Danish administrative and medical registries we retrieve data on death, emigration, and tumor characteristics. We collected archived formalin-fixed paraffin-embedded primary tumor tissue from Danish pathology departments. We genotyped 17 candidate SNPs using TaqMan SNP genotyping assays and, for each SNP, categorized the women as having two normal alleles (wildtype) or at least one variant allele. We followed the women from six months after breast cancer diagnosis until death, emigration or 30th June 2019, whichever came first. Comparing variant carriers with wildtype, we computed cumulative incidence proportions (CIPs) and used Poisson regression models to calculate unadjusted incidence rate ratios (IRRs) and associated 95% confidence intervals (CIs) of all–cause mortality. We stratified by ER status to evaluate interaction. Analyses were repeated for breast cancer specific mortality. Results Our cohort included 2,262 women. During follow–up (median 9.6 years, interquartile range: 8.4–11.0), 250 women died (CIP: 14%); 219 due to breast cancer (CIP: 11%). Genotyping was successful for ≥95% of the study cohort, with exception of two SNPs, which were excluded from analyses. We detected decreased mortality in variant carriers of SLCO1B1 rs2306283 (IRR: 0.75, 95% CI 0.58–0.97), and ABCB1 rs1128503 (IRR: 0.81, 95% CI: 0.63–1.05), ABCB1 rs2032582 (IRR: 0.80, 95% CI: 0.62–1.04) and ABCC2 rs12762549 (IRR: 0.80, 95% CI: 0.62–1.04). In contrast, mortality was increased in carriers of GSTP1 rs1138272 (IRR: 1.28, 95% CI: 0.93–1.76) and, unexpectedly, in carriers of CYP3A rs10273424 (IRR: 1.36, 95% CI: 0.99–1.86) variant carriers. Sensitivity analyses yielded similar findings. ER status did not modify the associations. Conclusions In this study, mortality may be associated with SNPs in ABCB1, ABCC2, SLCO1B1, CYP3A and GSTP1 in premenopausal women with non-metastatic breast cancer receiving docetaxel. The mechanisms underlying our findings remains unclear but may be related to docetaxel pharmacokinetics. Citation Format: Cathrine F Hjorth, Per Damkier, Tore B Stage, Søren Feddersen, Bent Ejlertsen, Timothy L Lash, Stephen Hamilton-Dutoit, Thomas P Ahern, Mikael Rørth, Henrik T Sørensen, Deirdre Cronin-Fenton. Single nucleotide polymorphisms and mortality after docetaxel-based chemotherapy in premenopausal breast cancer: A population-based cohort study in Denmark [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-13-06.

Evaluation of tumor-infiltrating lymphocytes and association with prognosis in BRCA-mutated breast cancer

Background: Patients with a BRCA1 or BRCA2 mutation (BRCA-mutated breast cancer) are frequently diagnosed with low differentiated and highly proliferating breast cancer characterized by high amounts of tumor-infiltrating lymphocytes (Tils). Stromal Tils (sTils) are highly prognostic in sporadic triple-negative and HER2 positive breast cancer however, their prognostic importance in BRCA-mutated breast cancers is unknown.Material and methods: Formalin-fixed paraffin-embedded primary tumor tissue from 411 patients with a germline BRCA1 or BRCA2 mutation and diagnosed with early breast cancer was included. The percentage of sTils was quantified on full HE sections according to guidelines proposed by the Immuno-Oncology Biomarker in Breast Cancer Working Group. Distribution of sTils and associates with patient and tumor characteristics were assessed according to categorical sTils groups defined as low (<10%), intermediate (10–59%) and high (≥60%). Prognostic associations of sTils were evaluated as a continuous variable in univariate and multivariate models. Only follow-up time beyond date of BRCA mutation test was included.Results: A large proportion had high sTils (27% in the full cohort, 36% in BRCA1-mutated, and 44% in ER negative breast cancers). Higher sTils were associated with BRCA1, ER negative breast cancer, high histological grade and medullary histology. In combined analysis for BRCA1 and BRCA2-mutated breast cancers, increasing sTils in 10% intervals were significantly associated with OS (HR 0.92, 95% CI 0.84–1.00, p = .05). For each 10% increment of sTils in BRCA1 breast cancers, a 10% reduction of mortality (adjusted HR 0.90 95% CI 0.81–0.99, p = .03) and a 13% reduction in risk of DFS-event (HR 0.87 95% CI 0.76–1.00, p = .05) was observed even after adjustment for ER status. No significant association with survival was of observed in the BRCA2 subgroup. Test for interaction of sTils and BRCA status was not statistically significant (p = .3).Conclusions: Breast cancer patients with a germline BRCA mutation had higher sTils than previously reported in sporadic breast cancers, and sTils were associated with favorable survival among BRCA carriers.

Analytic validity of DecisionDx-Melanoma, a gene expression profile test for determining metastatic risk in melanoma patients

BackgroundThe DecisionDx-Melanoma test provides prognostic information for patients with cutaneous melanoma (CM). Using formalin-fixed paraffin-embedded primary tumor tissue, the RT-PCR-based test classifies patients into a low- (Class 1) or high-risk (Class 2) category for recurrence based on expression of 31 genes. The current study was designed to assess the analytical validity of this test.MethodsInter-assay, inter-instrument, and inter-operator studies were performed to evaluate reliability of the 31-gene expression test results, sample stability and reagent stability. From March 2013 through June 2016, the gene expression test was performed on 8244 CM tumors. De-identified data from Pathology Reports were used to assess technical success.ResultsRobust sample and reagent stability was observed. Inter-assay concordance on 168 specimens run on 2 consecutive days was 99% and matched probability scores were significantly correlated (R2 = 0.96). Inter-instrument concordance was 95%, and probability scores had a correlation R2 of 0.99 (p < 0.001). From 8244 CM specimens submitted since 2013, 85% (7023) fulfilled pre-specified tumor content parameters. In these samples with sufficient tumor requirements, the technical success of the test was 98%.ConclusionDecisionDx-Melanoma is a robust gene expression profile test that demonstrates strong reproducibility between experiments and has high technical reliability on clinical samples.

Prognostic significance of RACGAP1 mRNA expression in high-risk early breast cancer: a study in primary tumors of breast cancer patients participating in a randomized Hellenic Cooperative Oncology Group trial

RACGAP1 is a Rac GTPase-activating protein involved in cell growth regulation, cell transformation and metastasis. The aim of the present study was to explore the prognostic and/or predictive significance of RACGAP1 mRNA expression on disease-free survival (DFS) and overall survival (OS) in high-risk early breast cancer patients and compare it to that of Ki67 protein expression and to the Nottingham prognostic index (NPI). A total of 595 high-risk breast cancer patients were treated in a two-arm trial evaluating postoperative dose-dense sequential chemotherapy with epirubicin followed by CMF with or without paclitaxel. RNA was extracted from 314 formalin-fixed paraffin-embedded primary tumor tissue samples followed by one-step quantitative RT-PCR for assessing RACGAP1 mRNA expression. High RACGAP1 mRNA expression (above the median) was associated with poor DFS (log-rank, p = 0.002) and OS (p < 0.001). High histological grade, as well as high Ki67 protein expression, was more frequent in the high-expression group of RACGAP1. Results of the Cox multivariate regression analysis revealed that high RACGAP1 mRNA expression independently predicted poor overall survival (Wald's p = 0.008). High Ki67 protein expression was also an adverse prognostic factor for death (p = 0.016), while high NPI score values were not. High RACGAP1 mRNA expression, as assessed by qRT-PCR, was found to be of adverse prognostic significance in high-risk early breast cancer patients treated with dose-dense sequential chemotherapy. The utility of RACGAP1 mRNA expression in patient selection for treatment with aggressive chemotherapy regimens should be further explored and validated in larger cohorts.

832P - C-MYC as a New Predictive Biomarker for Sunitinib in Metastatic Renal Clear Cell Carcinoma

ABSTRACT Background Sunitinib is a tyrosin kinase inhibitor with proven efficacy in renal clear cell carcinoma (RCC). However we still lack properly validated molecular predictors of response. Two subtypes of sporadic VHL-deficient RCC were proposed based on HIF1α and HIF2α expression and c-Myc activation. This molecular subclassification could provide a framework for current targeted therapies. This study aims to explore the predictive value of this molecular signature. Methods An observational prospective study involving 10 Spanish Hospitals was designed to collect formalin-fixed paraffin-embedded primary tumor tissue samples. We enrolled consecutively attending adults who had a centralized pathologically confirmed diagnosis of RCC with a clear-cell histology and advanced disease. Eligible patients were treatment naive and scheduled for sunitinib following routine clinical practice. The protocol was approved by the medical ethics committee of all participating institutions, and signed informed consent was obtained for all patients. Selected biomarkers were analyzed by immunohistochemistry following established protocols. Results From March 2009 to February 2011, 80 patients were included. At the time of the analysis, tumor samples were available for 64 patients and 58 were evaluable for c-Myc expression. Only 33% (19/58) showed c-Myc immunostainig (any score: 1–3) and 67% (39/58) were negative for c-Myc expression (scored as 0). Both groups were well balanced and non significant differences in risk prognostic factors were found. Regarding Sunitinib efficacy, significant differences in PFS were found between both molecular subgroups. A median PFS of 5.4 months was found in patients with c-Myc positive primary tumors vs. 11.4 months in patients with c-Myc negative primary tumors (HR = 2.54, 95%CI (1.28, 5.07), p = 0.0062). Correlation of c-Myc expression with other related biomarkers is underway. Conclusions Although, this is a small sample and validation is still needed, these results suggest a promising value for c-Myc immunostaining as a novel tool to identify those patients with RCC with more probabilities of obtaining benefit with Sunitinib through an accessible and reproducible technique. This study was supported by an Independent Investigator Research grant from Pfizer Inc. Disclosure P. Maroto: Compensation of consultant/Advisory role at Pfizer. E. Esteban: Compensation of consultant/Advisory role at Pfizer. All other authors have declared no conflicts of interest.

Improved Outcome of High-Risk Early HER2 Positive Breast Cancer With High CXCL13-CXCR5 Messenger RNA Expression

BackgroundChemokines are important in cell migration and are thought to play a key role in metastasis. We explored the prognostic significance of C-X-C ligand-motif (CXCL) 12, CXCL13, and receptor (CXCR) 5 on disease-free survival (DFS) and overall survival (OS) in early breast cancer. MethodsA total of 595 patients at high risk for early breast cancer were treated in a 2-arm trial (HE10/97) with dose-dense sequential epirubicin followed by cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) with or without paclitaxel. RNA was extracted from 321 formalin-fixed paraffin-embedded primary tumor tissue samples and quantitative reverse-transcriptase polymerase chain reaction was used to assess messenger RNA (mRNA) expression of CXCL12, CXCL13, and CXCR5; estrogen receptor; progesterone receptor (PgR); microtubule-associated protein tau and human epidermal growth factor receptor 2 (HER2). ResultsCXCL13 and CXCR5 were found to be negatively associated with estrogen receptor and microtubule-associated protein tau mRNA expression and with dense lymphocytic infiltration, and were positively associated with nuclear grade. Only CXCL13 was positively associated with HER2. Multivariate analysis revealed an association between high CXCL13 mRNA expression and improved DFS (hazard ratio [HR] 0.48 [95% CI, 0.25-0.90]; Wald, P = .023) but not OS; whereas high CXCL12 expression was significantly associated with increased OS (HR 0.53 [95% CI, 0.33-0.85]; Wald, P = .009). In the HER2 mRNA overexpressing subgroup, high CXCL13 mRNA expression was associated with improved DFS (P < .001), whereas high CXCR5 was associated with increased DFS and OS (P = .004 and P = .049, respectively). ConclusionsThe CXCL13-CXCR5 axis is associated with classic determinants of poor prognosis, such as high grade, hormone receptor negativity, and axillary node involvement. Interestingly, this chemokine axis seems to be strongly associated with improved outcome in patients with HER2+ disease.

Association of matrix metalloproteinase-1 (MMP-1) and matrix metalloproteinase-2 (MMP-2) expression with KRAS/BRAF mutational status and survival outcomes in metastatic colorectal cancer (CRC) patients.

e14088 Background: MMP-1 and MMP-2 proteins are felt to be involved in tumor growth, invasion and metastasis and may be regulated by the KRAS pathway. Methods: A tissue microarray was constructed from archival formalin-fixed paraffin-embedded primary tumor tissue samples of 96 patients who had undergone treatment for metastatic CRC. MMP-1 and MMP-2 expression was measured semiquantitatively by immunohistochemistry and graded 0 to 2. Mutation analysis was done for KRAS in codons 12 and 13, and patients without KRAS mutations were tested for BRAF V600E mutation. Chi-squared test, Fisher’s exact test and Cox-proportionate hazard ratios were used for statistical analysis. Results: 96 patients had samples available for analysis (median age at diagnosis 60, 57% male, 46% stage IV at diagnosis, 33/93 (35%) KRAS mutated, and 7/51 (14%) BRAF mutated. At the time of the analysis, all but 2 patients received first line therapy for metastatic disease. First line therapy was 5-Fluorouracil monotherapy among 7 pts (8%), irinotecan based in 50 pts (52%), and oxaliplatin based therapy in 35 pts (37%). 21 pts (22%) received anti-EGFR therapy. No significant association was identified between MMP-1 and KRAS (p=0.88) or BRAF (p=0.54), or between MMP-2 and KRAS (p=0.08) or BRAF (p=0.41). Neither overall survival nor survival from diagnosis of metastatic disease were affected by MMP-1 or MMP-2 expression. Conclusions: MMP-1 and MMP-2 expression is not associated with KRAS or BRAF mutational status and is not a prognostic factor in this cohort of CRC patients.

Abstract 235: Beyond KRAS mutation status: Influence of KRAS copy number alteration and microRNAs in response to cetuximab in metastatic colorectal cancer patients

Abstract Introduction: KRAS mutation is a negative predictive factor for treatment with anti-epidermal growth factor receptor (EGFR) antibodies in metastatic colorectal cancer (mCRC). Novel predictive markers are required to further improve the selection of patients for this treatment. We assessed the influence of modification of KRAS by gene copy number aberration (CNA) and microRNAs (miRNAs) in correlation to clinical outcome in mCRC patients treated with cetuximab in combination with chemotherapy and bevacizumab. Patients and methods: Formalin-fixed paraffin-embedded primary tumor tissue was used of 34 mCRC patients from a phase III trial (CAIRO2), who were selected based upon their good (n=17) and poor (n=17) progression-free survival (PFS) upon treatment with cetuximab in combination with capecitabine, oxaliplatin, and bevacizumab. Gene CNA at the KRAS locus was assessed using a high resolution genome-wide array CGH and the expression levels of 17 miRNAs targeting KRAS were determined by real-time PCR. Results: Copy number loss of the KRAS locus was observed in the tumor of 5 patients who were all good responders including patients with a KRAS mutation. Copy number gains in two wild type-KRAS tumors were associated with a poor PFS. Increased expression of several miRNAs (presented at the meeting) targeting KRAS correlated with PFS of patients treated with cetuximab, bevacizumab and chemotherapy. Conclusion: Our results indicate that the assessment of KRAS CNA and miRNAs targeting KRAS further optimize the selection of mCRC eligible for anti-EGFR therapy, in addition to KRAS mutation status. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 235. doi:10.1158/1538-7445.AM2011-235

Evaluation of the association of matrix metalloproteinase-1 (MMP-1) and matrix metalloproteinase-2 (MMP-2) expression with KRAS and BRAF mutational status or survival outcomes in colorectal cancer (CRC) patients.

452 Background: MMP-1 and MMP-2 proteins are felt to be involved in tumor growth, invasion and metastasis and may be regulated by the KRAS pathway. Methods: A tissue microarray was constructed from archival formalin-fixed paraffin-embedded primary tumor tissue samples of 96 patients with metastatic CRC. MMP-1 and MMP-2 expression was measured semiquantitatively by immunohistochemistry and graded 0 to 2. Mutation analysis was done for KRAS in codons 12 and 13, and patients without KRAS mutations were tested for BRAF V600E mutation. Chi-squared test, Fisher's exact test and Cox-proportionate hazard ratios were used for statistical analysis. Results: 96 patients had samples available for analysis (median age at diagnosis 60, 57% male, 46% stage IV at diagnosis, 33/93 (35%) KRAS mutated, 7/51 (14%) BRAF mutated, 22% received anti-EGFR therapy). No significant association was identified between MMP-1 and KRAS (p=0.88) or BRAF (p=0.54), or between MMP-2 and KRAS (p=0.08) or BRAF (p=0.41). Neither overall survival nor survival from diagnosis of metastatic disease were affected by MMP-1 or MMP-2 expression. Conclusions: MMP-1 and MMP-2 expression is not associated with KRAS or BRAF mutational status and is not a prognostic factor in our cohort of CRC patients. No significant financial relationships to disclose.

Abstract 3731: Elevated mRNA expression of CXCL13 predicts improved outcome in patients with high-risk early breast cancer: A possible molecular predictor of treatment benefit in the context of a Hellenic Cooperative Oncology Group trial

Abstract Chemokines are important in cell migration and are therefore thought to play a key role in metastasis. Chemokine (C-X-C motif) ligand 13 (CXCL13) was recently found to be overexpressed in breast cancer and its overexpression is thought to be associated with poor outcome. The aim of the present study was to explore the prognostic significance of CXCL13 on disease-free survival (DFS) and overall survival (OS) in high-risk operable breast cancer enrolled in a randomized phase III trial. The association of CXCL13 with hormonal receptor and HER family expression at the m-RNA level, as well as with other clinicopathological characteristics of patients was also investigated. 595 high-risk breast cancer patients were treated in a two-arm trial (HE10/97) investigating postoperative dose-dense sequential chemotherapy with epirubicin (E) followed by CMF with or without paclitaxel (T). RNA was extracted from 312 formalin-fixed paraffin-embedded primary tumor tissue samples followed by kinetic one-step RT-PCR for assessment of mRNA expression of CXCL13, ER, PgR, and HER2. Values of CXCL13 and HER2 mRNA above the 75th percentile were considered high expression. CXCL13 expression was correlated with known clinicopathological parameters, such as HER2, ER and PgR by IHC, tumor grade, and nodal involvement. With a median follow up of 8 years the total number of events (disease relapses) was 109/312 (35%), and the total number of deaths 78/312 (25%). High CXCL13 mRNA expression was associated with improved DFS (log-rank, p=0.043). This remained unchanged when adjusted for treatment group (Wald, p=0.045). Furthermore, CXCL13 was found to be negatively correlated (p&amp;lt;0.001) with ER and PgR mRNA expression. Results of the Cox multivariate regression analysis revealed that, in the presence of treatment group, high CXCL13 mRNA expression was associated with a significantly decreased risk for relapse (HR=0.50, 95% CI: 0.31-0.82, p=0.006) and a significantly decreased risk for death (HR=0.56, 95% CI: 0.31-0.99, p=0.047). The number of ER/PgR IHC positive patients was significantly lower in the high expression group of CXCL13 compared to the low expression group (67.5% vs 81.5%, p=0.017). Finally, higher tumor grade was more likely to be present in the high expression group of CXCL13 than in the low expression group (69.2% vs 42.7%, p&amp;lt;0.001), as was HER2 mRNA overexpression (41.4% vs 26.5%, p=0.024). Furthermore, high expression of CXCL13 was associated with lower risk for relapse in HER2 mRNA overexpressing patients. High CXCL13 mRNA expression is associated with a significantly decreased risk for relapse and death in high-risk breast cancer patients treated with dose-dense sequential chemotherapy. This association appears to be more pronounced in HER2 overexpressing patients. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3731.

Predicting response to alkylating chemotherapy in breast cancer patients using array comparative genomic hybridization.

Abstract Abstract #6050 Background: Breast cancer cells lacking the DNA repair mechanism Homologous Recombination (HR) have recently been shown to be hypersensitive to bifunctional alkylating agents such as carboplatin and poly(ADP-ribose)polymerase(PARP)-inhibitors, a novel class of targeted agents. We have previously reported the construction of an array Comparative Genomic Hybridization (aCGH) classifier that detects the characteristic copy number aberrations of BRCA1-mutated tumors which are thought to be caused by HR deficiency. We hypothesized that metastatic breast cancer patients with tumors harboring such a BRCA1-profile may specifically benefit (longer progression-free survival (PFS)) from intensive treatment with bifunctional alkylating agents compared to patients without this tumor-profile.&amp;#x2028; Methods: We isolated DNA from formalin-fixed paraffin-embedded primary tumor tissue collected from 3 prior studies carried out in our institute, in which metastatic breast cancer patients received intensive alkylating chemotherapy with carboplatin, thiotepa and cyclophosphamide (CTC). DNA from 40 tumors was tested for the presence of the BRCA1-profile by aCGH. Furthermore, we screened for the majority of BRCA1 and BRCA2 mutations that frequently occur in Dutch BRCA1 and BRCA2 families.&amp;#x2028; Results: Sixteen patients had a tumor with a BRCA1-profile and had a better response to CTC-treatment, defined by achievement of a complete remission (p=0.01) and longer PFS (p=0.001, figure 1), with a hazard ratio for progression of 0.30 (p=0.002).&amp;#x2028; &amp;#x2028; All 6 patients who remain in continuous complete remission (56+ to 150+ months) had a tumor with a BRCA1 profile. Two BRCA1 and 2 BRCA2 mutations were found in 4 patients of whom 3 had a BRCA1-profile and a long PFS (&amp;gt; 30months), linking HR deficiency (BRCA1 and BRCA2 mutations) in breast cancer patients to good outcome on platinum-based chemotherapy.&amp;#x2028; Discussion: Using an aCGH classifier initially constructed to identify BRCA1-mutated tumors we were able to identify a group of metastatic breast cancer patients treated with platinum-based chemotherapy with a high complete remission-rate and long progression free survival. Moreover, this group contained all patients who were apparently cured by intensive alkylating chemotherapy. Our aCGH assay may represent an effective test for BRCA-ness and could be important to select patients with sporadic tumors for therapy targeted at cells deficient in Homologous Recombination. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6050.