Silvia Calabuig

Deletions Affecting Codons 557-558 of the c-KIT Gene Indicate a Poor Prognosis in Patients With Completely Resected Gastrointestinal Stromal Tumors: A Study by the Spanish Group for Sarcoma Research (GEIS)

PURPOSE To explore the prognostic value of mutations in c-KIT and PDGFR-alpha genes with respect to relapse-free survival (RFS) in patients with gastrointestinal stromal tumors (GIST). We have investigated the prognostic relevance of the type and position of the mutations, in addition to other clinicopathologic factors, in a large series of patients with GIST. METHODS For this study, 162 patients were selected according to the following criteria: completely resected tumors with negative margins attended between 1994 and 2001; no metastasis at diagnosis; tumor larger than 2 cm, c-KIT-positive immunostaining; and no other primary tumors. RESULTS The median follow-up was 42 months for patients free of recurrence. Mutations were detected in 96 tumors (60%): 82 cases involving c-KIT and 14 cases involving PDFGR-alpha. Univariate analysis demonstrated the following as poor prognostic factors for RFS: tumors larger than 10 cm (P < .0001); mitotic count higher than 10 mitoses per 50 high-power fields (P < .0001); high risk index (P < .0001); intestinal GIST location (P = .0041); high cellularity (P < .0001); tumor necrosis (P < .0001); deletions affecting exon 11 (P = .0007); and deletions affecting codons 557 to 558 (P < .0001). After the multivariate analysis, only the high risk index (relative risk [RR], 12.36), high cellularity (RR, 3.97), and deletions affecting codons 557 to 558 of c-KIT (RR, 2.57) corresponded to independent prognostic factors for RFS in GIST patients. CONCLUSION Deletions affecting codons 557 to 558 are relevant for the prognosis of RFS in GIST patients. This critical genetic alteration should be considered to be a new prognostic stratification variable for randomized trials exploring imatinib mesylate in the adjuvant setting in GIST patients.

Serum metabolomic profiling facilitates the non-invasive identification of metabolic biomarkers associated with the onset and progression of non-small cell lung cancer

Lung cancer (LC) is responsible for most cancer deaths. One of the main factors contributing to the lethality of this disease is the fact that a large proportion of patients are diagnosed at advanced stages when a clinical intervention is unlikely to succeed. In this study, we evaluated the potential of metabolomics by 1H-NMR to facilitate the identification of accurate and reliable biomarkers to support the early diagnosis and prognosis of non-small cell lung cancer (NSCLC). We found that the metabolic profile of NSCLC patients, compared with healthy individuals, is characterized by statistically significant changes in the concentration of 18 metabolites representing different amino acids, organic acids and alcohols, as well as different lipids and molecules involved in lipid metabolism. Furthermore, the analysis of the differences between the metabolic profiles of NSCLC patients at different stages of the disease revealed the existence of 17 metabolites involved in metabolic changes associated with disease progression. Our results underscore the potential of metabolomics profiling to uncover pathophysiological mechanisms that could be useful to objectively discriminate NSCLC patients from healthy individuals, as well as between different stages of the disease.

MRP1 Overexpression Determines Poor Prognosis in Prospectively Treated Patients with Localized High-Risk Soft Tissue Sarcoma of Limbs and Trunk Wall: An ISG/GEIS Study

Patients with localized high-risk soft tissue sarcomas (STS) of the limbs and trunk wall still have a considerable metastatic recurrence rate of more than 50%, in spite of adjuvant chemotherapy. This drug-ceiling effect of chemotherapy in sarcoma setting could be explained, at least partially, by multidrug resistance (MDR) mechanisms. The aim of this study was to ascertain whether mRNA and protein expression of ABCB1 (P-glycoprotein), ABCC1 (MRP1), and GSTA1 (glutathione S-transferase pi) was prognostic in localized high-risk STS. Immunohistochemistry and reverse transcriptase-PCR studies were performed from biopsies at the time of diagnosis. Patients of this series were prospectively enrolled into a phase III trial that compared three versus five cycles of epirubicin plus ifosfamide. The series of 102 patients found 41 events of recurrence and 37 of death with a median follow-up of 68 months. In univariate analysis, variables with a statistically significant relationship with relapse-free survival (RFS) were: MRP1 expression (5-year RFS rate of 23% in positive cases and 63% in negative cases, P = 0.029), histology (5-year RFS rate of 74% in undifferentiated pleomorphic sarcoma and 43% in synovial sarcoma, P = 0.028), and ABCC1 expression (5-year RFS rate of 33% in overexpression and 65% in downregulation, P = 0.012). Combined ABCC1/MRP1 was the only independent prognostic factor for both RFS (HR = 2.704, P = 0.005) and overall survival (HR = 2.208, P = 0.029). ABCC1/MRP1 expression shows robust prognostic relevance in patients with localized high-risk STS treated with anthracycline-based chemotherapy, which is the standard front line treatment in STS. This finding deserves attention as it points to a new targetable protein in STS. Mol Cancer Ther; 13(1); 249–59. ©2013 AACR.

CCL27–CCR10 and CXCL12–CXCR4 chemokine ligand-receptor mRNA expression ratio: new predictive factors of tumor progression in cutaneous malignant melanoma

CXCR4, CCR7 and CCR10 chemokine receptors are known to be involved in melanoma metastasis. Our goal was to compare the relative intratumoral mRNA expression of these receptors with that of their corresponding chemokine ligands, CXCL12, CCL19, CCL21, and CCL27 across the full spectrum of human melanoma progression: thin and thick primary melanomas, as well as “in transit”, lymph node, and distant metastases. Expression was quantified by real-time RT-PCR in 103 melanoma samples: 51 primary tumors and 52 metastases. Particular emphasis was focused on chemokine ligand-receptor expression ratios. Immunohistochemistry was performed to identify the cell types expressing these molecules. CXCL12–CXCR4 and CCL27–CCR10 ratios were higher in thin than in thick primary melanomas, and all four chemokine-receptor ratios were higher in primary tumors than in melanoma metastases. CCL27–CCR10 and CXCL12–CXCR4 expression ratios in primary tumors were inversely associated with the development of distant metastases, and improved the predictive value of tumor thickness for distant metastasis, which is important since chemokine ligand-receptor ratios are not affected by the endogenous gene employed for normalizing mRNA expression. Both receptor and ligand immunolabeling were detected in neoplastic cells suggesting autocrine mechanisms. Our results support the concept that low CCL27/CCR10 and CXCL12/CXCR4 intratumoral mRNA ratios are associated with melanoma progression, and in combination with Breslow thickness, are the best predictive factors for the development of distant metastases in primary cutaneous melanoma.

Análisis del gen de fusión COL1A1-PDGFB en un caso de dermatofibrosarcoma protuberans con componente de fibrosarcoma

Resumen El dermatofibrosarcoma protuberans (DFSP) es un tumor poco frecuente, de malignidad intermedia, con poca tendencia a desarrollar metastasis, pero con una alta frecuencia de recidiva local. Citogeneticamente, el DFSP se caracteriza por presentar la translocacion reciproca t(17;22) (q22;q13) que condiciona en la fusion del gen del colageno tipo Iα (COL1A1), en el cromosoma 17q, con el gen de la cadena β del factor de crecimiento derivado de las plaquetas (PDGFB), en el cromosoma 22q. La fusion de estos genes es variable, implicando a alguno de los 51 exones del gen COL1A1 con el exon 2 del gen PDGFB. Presentamos el caso de una mujer de 37 anos con una tumoracion en el brazo cuya histologia muestra una infiltracion neoplasica del tejido celular subcutaneo constituido por celulas fusiformes de nucleo elongado con un patron estoriforme y otras celulas mas pleomorficas con un patron en espina de pescado siendo compatible con DFSP con componente de fibrosarcoma. El estudio de biologia molecular con material incluido en parafina mediante transcripcion inversa y reaccion en cadena de la polimerasa (RT-PCR) y posterior secuenciacion muestra una nueva fusion del exon 19 del gen COL1A1 con el exon 2 de PDGFB, apoyando un diagnostico de DFSP. El estudio de los productos de fusion COL1A1-PDGFB es util en casos donde la histologia y la inmunohistoquimica son insuficientes para el diagnostico diferencial del DFSP con otros sarcomas y ademas, justifica la aplicacion de nuevas vias de tratamiento farmacologico con los inhibidores de la tirosincinasa.

Analysis of the immune microenvironment in resected non-small cell lung cancer: the prognostic value of different T lymphocyte markers

The prognosis of non-small cell lung cancer (NSCLC) remains poor and heterogeneous and new biomarkers are needed. As the immune system plays a pivotal role in cancer, the study of immune-related markers may provide valuable prognostic information of NSCLC. In 122 formalin-fixed, paraffin-embedded tumor tissue samples from early-stage NSCLC, tumor and tumor-near stromal areas were microdissected and gene expression levels of conventional and regulatory T cell markers were assessed by quantitative polymerase chain reaction. Also, the presence of infiltrating CD4+, CD8+, and FOXP3+ cells in tumor samples was assessed by immunohistochemistry. The relative proportion of conventional and regulatory T cells present in the tumor environment was assessed and found to be key to understand the importance that the immune system analysis has in the prognostics of NSCLC patients. The presence of CD8+ cells in the tumor compartment was associated with better outcome, whereas the presence of FOXP3+ cells was associated with worse overall survival. The negative prognostic value of combined biomarkers, indicating high levels of FOXP3 in the stroma and low levels of CD4 or CD8 in tumors, was observed at mRNA level and was validated by immunohistochemistry.In conclusion, the proportion of T helper and cytotoxic cells vs. regulatory T cells in different locations of the tumor microenvironment have opposite prognostic impacts in resected NSCLC.

Biphasic dermatofibrosarcoma protuberans with a labyrinthine plexiform high-grade fibrosarcomatous transformation

Several variants of dermatofibrosarcoma protuberans, a low‐grade superficial sarcoma, are well recognized. The most prognostically important is the fibrosarcomatous variant. We report a case of biphasic dermatofibrosarcoma protuberans in which the high‐grade component exhibited a previously undescribed plexiform pattern. A clinicopathological study complemented with immunohistochemical, ultrastructural, reverse transcription polymerase chain reaction and fluorescence in situ hybridization analyses of this unique case. Histopathologically, a conventional low‐grade dermatofibrosarcoma protuberans was admixed with intratumoral high‐grade areas showing a striking labyrinthine plexiform pattern characterized by a higher cellularity of larger and slightly atypical tumor cells. CD34 expression was present in both components, while Ki‐67 immunostaining was significantly higher in the plexiform high‐grade areas. Focal epithelial membrane antigen and claudin‐1 immunostaining was present at the interphase between high‐ and low‐grade areas. COL1A1‐PDGFB fusion transcripts, with breakpoints at exon 25 of COL1A1 and exon 2 of PDGFB, were present in both components, being more numerous, as the extra copies of both genes, in the high‐grade areas. A previously undescribed histopathologic pattern of high‐grade sarcomatous transformation of dermatofibrosarcoma protuberans is reported: a biphasic tumor with a labyrinthine plexiform high‐grade component.

1416PDINTEGRATING GENOTYPE IN RISK CLASSIFICATION FOR GIST RECURRENCE. A SPANISH GROUP FOR SARCOMA RESEARCH (GEIS) STUDY

ABSTRACT Aim: Relevant prognostic factors for relapse free survival (RFS) in GIST relies on clinical and pathologic variables as size, mitoses, location or tumor rupture. However, being GIST a solid tumor model for molecular research and targeted therapies, it seems legitimate to explore the integration of relevant molecular prognostic factors into the risk classification. Several authors have pointed out the detrimental prognostic role of deletion type mutations involving 557/558 (DEL-5758) codons of exon 11 in KIT gene. On the other hand, mutations of PDGFR (MUT-PDGFR) gene have been associated with lower risk of recurrence. We will analyze the influence of these genotype factors for each Miettinen risk category (MRC). Methods: Clinical data, therapeutic and follow-up procedures stemmed from the GIST Registry of GEIS. Main inclusion criteria were: localized GIST, adequate surgery, size>2 cm, complete genotype for KIT and PDGFR genes, no adjuvant imatinib and minimum follow-up of 3 years. RFS was measured by Kaplan-Meier method. For each MRC, RFS was estimated for those harboring DEL-5758 or MUT-PDGFR. Univariate and multivariate analysis were performed using log-rank test and Cox regression. Results: 429 patients were identified according to the inclusion criteria, 35 of them had insufficient data, thus 394 patients entered into the analysis. Median size was 7 cm and with a median follow-up of 84 months there were 137 recurrences (37%). DEL-5758 and MUT-PDGFR were present in 65 and 25 cases respectively. The 7-year RFS for low MRC were 93% in MUT-PDGFR, 76% in DEL-5758 and 90% in the remainder (p=0.35), for Intermediate MRC were 80%, 26% and 64% (p=0-009) respectively and for high MRC were 40%, 21% and 28% (p=0.79) respectively. On univariate analysis: mitoses, size, location (gastric vs non gastric) and genotype were found to be significantly correlated with RFS. Multivariate analyses revealed that size (HR 1.75; CI 1.04-2.90), location (HR 1.79; CI 1.24-2.60), mitoses (RR 3.4; CI 2.31-5.05) and DEL-5758 (RR 1.5; CI 1.02-2.32) were independent prognostic factors for RFS. Conclusions: Genotype significantly affects prognosis in localized GIST and therefore should be integrated into the risk classification especially in intermediate MCR. Disclosure: All authors have declared no conflicts of interest.

Abstract 4330: Immune checkpoint expression score is an independent prognostic biomarker in resectable non-small cell lung cancer

BACKGROUND Immune checkpoints blockade, which activate antitumor immunity, has demonstrated promising clinical results in NSCLC. In this study we have investigated the prognostic role of immune checkpoint expression markers and its correlation with immune-cells infiltration and clinico-pathological characteristics in a cohort of resectable NSCLC patients. MATERIAL AND METHODS RNA was isolated from fresh-frozen lung specimens (tumor and normal lung) (n = 178). RTqPCR was performed to analyze the expression of CTLA-4, PD-1 and PD-L1 by the use of hydrolysis probes. Relative gene expression was assessed by Pfaffl formula and normalized by the use of CDKN1B, GUS and ACTB as endogenous genes (selected by GeNorm algorithm). These data was used to develop a gene expression score. Furthermore, the presence of CD4+, CD8+ and FOXP3+ lymphocytes was also assessed in FFPE samples from 63 of these patients by immunohistochemistry (IHC). All statistical analysis were considered significant at p RESULTS Patient9s median age was 65 years [26-85], 86.5% were male and 43.8% were adenocarcinomas (ADC). Since CTLA-4 and PD-1 were moderately associated with prognosis based on COX regression analysis (|Z-score| CONCLUSIONS The immune checkpoint score based on the expression levels of CTLA-4 and PD-1 correlates with the presence of CD8+ infiltrating lymphocytes in the tumor microenvironment. This score provides relevant prognostic information for a better characterization of early-stage NSCLC patients with strikingly different outcomes who may be candidates for immune-based therapies. Supported by grants PS09-01149 and RD12/0036/0025 from ISCIII. Citation Format: Marta Uso, Eloisa Jantus-Lewintre, Rafael Sirera, Silvia Calabuig, Enrique Pastor, Jeronimo Forteza, Carlos Camps. Immune checkpoint expression score is an independent prognostic biomarker in resectable non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4330. doi:10.1158/1538-7445.AM2015-4330

227PPROGNOSIS OF PHOSPHORYLATED-INSULIN GROWTH FACTOR RECEPTOR (P-IGF-1R) AND METALLOPROTEINASE-3 (MMP3) EXPRESSION IN ADVANCED GASTROINTESTINAL STROMAL TUMORS (GIST) PATIENTS TREATED WITH IMATINIB. A GEIS STUDY

ABSTRACT Aim: Most GISTs have mutations in KIT or PDGFRA. Patients with advanced GIST with KIT exon 9, with PDGFRA mutation or WT for KIT and PDGFRA have a worse progression-free survival (PFS) compared to patients with KIT exon 11 mutated tumors. We evaluated expression of p-IGF-1R (Y1316) and MMP3 as predictors of PFS or overall survival (OS) Methods: Ninety-six advanced GIST patients included in GEIS-16 study with KIT and PDGFRA mutational information were examined for p-IGF-1R (Y1316) and MMP3 expression in a tissue micro-array. To study activation of the IGF-1R system, we have used an antibody (anti-pY1316) that specifically recognizes the phosphorylated (active) form of the IGF-1R. DNA was extracted from paraffin-embedded tissues and intronic PCR primers were used to amplify exons 9,11,13 and 17 of KIT, 12 and 18 of PDGFRA. Bidirecctional sequencing with specific primers was performed on a ABI3100 sequencer using the Big Dye Terminator v3.1 kit. Multivariate model was built using a stepwise automated variable selection approach with criterion to enter the variable in the model of p Results: MMP3 was overexpressed in 10% of cases and p-IGF-1R in only 2% of cases. 68% of patients had KIT mutations, 4% had PDGFRA mutations and 28% were WT for KIT and PDGFRA. At univariate analysis KIT exon 11/13 vs rest (WT/WT, KIT exon 9 mutations and PDGFRA mutations) had better PFS (p = 0.038; HR: 0.58; 95%CI (0.35-0.96). Less than 24 months disease free-interval (HR 24.2, 95% CI 10.5-55.8), poor performance status (PS) (HR 6.3, 95% CI 2.5-15.9), extension of disease; > 1 organ (HR 1.89; 95% CI 1.03-3.4) and positive expression of p-IGF-1R or MMP3 (HR 2.1; 95% CI 1.01-4,2) but not mutational analysis (HR 1.02; 95%CI 0.53-1.96) were the strongest prognostic factors for PFS in the multivariate analysis. For OS only PS, disease free-interval and number of metastatic sites remain significant. Conclusions: Our findings suggest that p-IGF-1R (Y1316) and MMP3 expression have major prognostic significance for PFS in advanced GIST treated with imatinib therapy. Disclosure: All authors have declared no conflicts of interest.