Pascale Mariani

Characterization of a Large Panel of Patient-Derived Tumor Xenografts Representing the Clinical Heterogeneity of Human Colorectal Cancer

Purpose: Patient-derived xenograft models are considered to represent the heterogeneity of human cancers and advanced preclinical models. Our consortium joins efforts to extensively develop and characterize a new collection of patient-derived colorectal cancer (CRC) models. Experimental Design: From the 85 unsupervised surgical colorectal samples collection, 54 tumors were successfully xenografted in immunodeficient mice and rats, representing 35 primary tumors, 5 peritoneal carcinoses and 14 metastases. Histologic and molecular characterization of patient tumors, first and late passages on mice includes the sequence of key genes involved in CRC (i.e., APC, KRAS, TP53), aCGH, and transcriptomic analysis. Results: This comprehensive characterization shows that our collection recapitulates the clinical situation about the histopathology and molecular diversity of CRC. Moreover, patient tumors and corresponding models are clustering together allowing comparison studies between clinical and preclinical data. Hence, we conducted pharmacologic monotherapy studies with standard of care for CRC (5-fluorouracil, oxaliplatin, irinotecan, and cetuximab). Through this extensive in vivo analysis, we have shown the loss of human stroma cells after engraftment, observed a metastatic phenotype in some models, and finally compared the molecular profile with the drug sensitivity of each tumor model. Through an experimental cetuximab phase II trial, we confirmed the key role of KRAS mutation in cetuximab resistance. Conclusions: This new collection could bring benefit to evaluate novel targeted therapeutic strategies and to better understand the basis for sensitivity or resistance of tumors from individual patients. Clin Cancer Res; 18(19); 5314–28. ©2012 AACR.

Genomic Profiling and Identification of High-Risk Uveal Melanoma by Array CGH Analysis of Primary Tumors and Liver Metastases

PURPOSE Incurable metastases develop in approximately 50% of patients with uveal melanoma (UM). The purpose of this study was to analyze genomic profiles in a large series of ocular tumors and liver metastases and design a genome-based classifier for metastatic risk assessment. METHODS A series of 86 UM tumors and 66 liver metastases were analyzed by using a BAC CGH (comparative genomic hybridization) microarray. A clustering was performed, and correlation with the metastatic status was sought among a subset of 71 patients with a minimum follow-up of 24 months. The status of chromosome 3 was further examined in the tumors, and metastases with disomy 3 were checked with an SNP microarray. A prognostic classifier was constructed using a log-linear model on minimal regions and leave-one-out cross-validation. RESULTS The clustering divides the groups of tumors with disomy 3 and monosomy 3 into two and three subgroups, respectively. Same subgroups are found in primary tumors and in metastases, but with different frequencies. Isolated monosomy 3 was present in 0% of metastatic ocular tumors and in 3% of metastases. The highest metastatic rate in ocular tumors was observed in a subgroup defined by the gain of 8q with a proximal breakpoint, and losses of 3, 8p, and 16q, also most represented in metastases. A prognostic classifier that included the status of these markers led to an 85.9% classification accuracy. CONCLUSIONS The analysis of the status of these specific chromosome regions by genome profiling on SNP microarrays should be a reliable tool for identifying high-risk patients in future adjuvant therapy protocols.

Surgical management of liver metastases from uveal melanoma: 16 years' experience at the Institut Curie

BACKGROUND Uveal melanoma is characterised by a high prevalence of liver metastases and a poor prognosis. AIM To review the evolving surgical management of this challenging condition at a single institution over a 16-year period. PATIENTS AND METHODS Between January 1991 and June 2007, among 3873 patients with uveal melanoma, 798 patients had liver metastases. We undertook a detailed retrospective review of their clinical records and surgical procedures. The data was evaluated with both uni- and multivariate statistical analysis for predictive survival indicators. RESULTS 255 patients underwent surgical resection. The median interval between ocular tumour diagnosis and liver surgery was 68 months (range 19-81). Liver surgery was either microscopically complete (R0; n = 76), microscopically incomplete (R1; n = 22) or macroscopically incomplete (R2; n = 157). The median overall postoperative survival was 14 months, but increased to 27 months when R0 resection was possible. With multivariate analysis, four variables were found to independently correlate with prolonged survival: an interval from primary tumour diagnosis to liver metastases >24 months, comprehensiveness of surgical resection (R0), number of metastases resected (< or = 4) and absence of miliary disease. CONCLUSIONS Surgical resection, when possible, is able to almost double the survival and appears at present the optimal way of improving the prognosis in metastatic uveal melanoma. Advances in medical treatments will be required to further improve survival.

Detection rate and prognostic value of circulating tumor cells and circulating tumor DNA in metastatic uveal melanoma

Circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) have been recently investigated in several cancer types, but their respective clinical significance remains to be determined. In our prospective study, we compared the detection rate and the prognostic value of these two circulating biomarkers in patients with metastatic uveal melanoma. GNAQ/GNA11 mutations were characterized in archived tumor tissue. Using a highly sensitive and mutation‐specific bidirectional pyrophosphorolysis‐activated polymerization (bi‐PAP) technique, GNAQ c.626A>T, GNAQ c.626A>C and GNA11 c.626A>T copy numbers were quantified in plasma from 12 mL of blood. CTCs were detected at the same time in 7.5 mL of blood by the CellSearch® technique. Patient characteristics and outcome were prospectively collected. CTCs (≥1) were detected in 12 of the 40 included patients (30%, range 1–20). Among the 26 patients with known detectable mutations, ctDNA was detected and quantified in 22 (84%, range 4–11,421 copies/mL). CTC count and ctDNA levels were associated with the presence of miliary hepatic metastasis (p = 0.004 and 0.03, respectively), with metastasis volume (p = 0.005 and 0.004) and with each other (p < 0.0001). CTC count and ctDNA levels were both strongly associated with progression‐free survival (p = 0.003 and 0.001) and overall survival (p = 0.0009 and <0.0001). In multivariate analyses, ctDNA appeared to be a better prognostic marker than CTC. In conclusion, ctDNA and CTC are correlated and both have poor prognostic significance. CTC detection can be performed in every patient but, in patients with detectable mutations, ctDNA was more frequently detected than CTC and has possibly more prognostic value.

French guidelines for enhanced recovery after elective colorectal surgery.

Enhanced recovery after surgery provides patients with optimal means to counteract or minimize the deleterious effects of surgery. This concept can be adapted to suit a specific surgical procedure (i.e., colorectal surgery) and comes in the form of a program or a clinical pathway covering the pre-, intra- and postoperative periods. The purpose of these Expert Panel Guidelines was firstly to assess the impact of each parameter typically included in the fast-track programs on six foreseeable consequences of colorectal surgery: surgical stress, postoperative ileus, fluid and electrolyte imbalances, decreased postoperative mobility, sleep disorders and postoperative complications; secondly, to validate the value of each parameter in terms of efficacy criteria for success of rapid rehabilitation programs. Two primary endpoints were selected to evaluate the impact of each parameter: the duration of hospital stay and rate of postoperative complications. For some of the parameters, the lack of information in the literature forced the experts to assess the parameter using different criteria (i.e., the duration of postoperative ileus or quality of analgesia); improvement in endpoints favored the implementation of a rapid rehabilitation program. After analysis of the literature, 19 parameters were identified as potentially impacting at least one of the foreseeable consequences of colorectal surgery. GRADE(®) methodology was applied to determine a level of evidence and the strength of recommendation regarding each parameter. After synthesis of the work of experts on the 19 parameters using GRADE(®) methodology, the organizing committee reached 35 formal recommendations. The recommendations were submitted and amended by a group of reviewers. After three rounds of Delphi quotes, strong agreement was obtained for 28 recommendations (80%) and weak agreement for seven recommendations. Consensus was reached among anesthesiologists and surgeons on a number of tactics that are insufficiently applied in current rehabilitation programs in colorectal surgery such as: pre-operative intake of carbohydrates; optimization of intra-operative volume control; resumption of oral feeding within 24 hours; gum chewing after surgery; getting the patient out of bed and walking on D1. The panel also clarified the value and place of such approaches as: patient information; pre-operative immunonutrition; laparoscopic surgery; antibiotic prophylaxis; prevention of hypothermia; systematic medication to prevent nausea and vomiting; morphine-sparing analgesia techniques; indications and techniques for bladder catheterization. The panel also confirmed the futility of other methods such as: bowel preparation for colon surgery; maintaining a nasogastric tube; surgical drainage for colorectal surgery.

Pyrophosphorolysis-activated polymerization detects circulating tumor DNA in metastatic uveal melanoma.

Purpose: To develop a molecular tool to detect circulating tumor–derived DNA (ctDNA) in the plasma from patients with uveal melanoma as a marker of tumor burden and monitor treatment efficacy. Experimental Design: A real-time PCR was developed on the basis of bidirectional pyrophosphorolysis-activated polymerization (bi-PAP) for the quantification of ctDNA using 3′blocked primer pairs specific for the 3 recurrent mutually exclusive mutations of Gα subunits GNAQ and GNA11. Results: Sensitivity and specificity of bi-PAP were assessed on serial dilutions of tumor DNA in normal DNA for the 3 recurrent mutations. Each assay could detect a single mutated molecule per reaction, whereas 104 copies of normal DNA were not detected. The ctDNA was readily detected in plasma of mice bearing uveal melanoma xenografts in amounts proportional to circulating human DNA. Finally, plasma was almost always found positive (20 of 21 tested patients) in a prospective analysis of patients with metastatic uveal melanoma. Conclusions: Bi-PAP assays detect and quantify ctDNA in patients with metastatic uveal melanoma. A prospective study is ongoing to assess the clinical usefulness of ctDNA level in uveal melanoma. Clin Cancer Res; 18(14); 3934–41. ©2012 AACR.

Preoperative staging of liver metastases from uveal melanoma by magnetic resonance imaging (MRI) and fluorodeoxyglucose-positron emission tomography (FDG-PET)

BACKGROUND Microscopically complete (R0) resection of metastases from uveal melanoma prolongs median overall survival compared to incomplete surgery. The aim of this study was to compare the sensitivity of dynamic-enhanced magnetic resonance imaging (MRI) with fluorodeoxyglucose-positron emission tomography (FDG-PET) in the preoperative diagnosis of liver metastases from uveal melanoma. PATIENTS AND METHODS Fifteen consecutive patients (mean age: 56 years) underwent FDG-PET and liver MRI. Extrahepatic metastatic disease was excluded by whole body computed tomography and bone scintigraphy. MRI and FDG-PET were performed with a mean of 19 days (range: 1-30) before surgery. Imaging findings were compared with surgical (including intraoperative ultrasonography) and histological findings on a lesion by lesion analysis. RESULTS R0 resection was performed in 12 patients. A total of 28 lesions were resected with 27 histologically proven metastases. Nine lesions were smaller than 5mm, 7 measured 5-10mm and 11 were larger than 10mm. Sensitivity and positive predictive value were 67% and 95% for MRI compared to 41% and 100% for FDG-PET. The difference between the two modalities was statistically significant (p=0.01; McNemar test). In remaining 3 patients, diffuse miliary disease (>10 capsular lesions) was discovered intraoperatively, and was suspected on preoperative MRI in 2 cases. Only one extrahepatic lesion identified by FDG-PET was falsely positive. CONCLUSIONS In this preliminary study, MRI was superior to FDG-PET for staging of liver metastases from uveal melanoma. Although miliary disease was suggested by MRI in some cases, preoperative confirmation remains imperfect.

Recommendations for bowel obstruction with peritoneal carcinomatosis.

This article reports on the clinical practice guidelines developed by a multidisciplinary group working on the indications and uses of the various available treatment options for relieving intestinal obstruction or its symptoms in patients with peritoneal carcinomatosis. These guidelines are based on a literature review and expert opinion. The recommended strategy involves a clinical and radiological evaluation, of which CT of the abdomen is a crucial component. The results, together with an analysis of the prognostic criteria, are used to determine whether surgery or stenting is the best option. In most patients, however, neither option is feasible, and the main emphasis, therefore, is on the role and administration of various symptomatic medications such as glucocorticoids, antiemetic agents, analgesics, and antisecretory agents (anticholinergic drugs, somatostatin analogues, and proton-pump inhibitors). Nasogastric tube feeding is no longer used routinely and should instead be discussed on a case-by-case basis. Recent studies have confirmed the efficacy of somatostatin analogues in relieving obstruction-related symptoms such as nausea, vomiting, and pain. However, the absence of a marketing license and the high cost of these drugs limit their use as the first-line treatment, except in highly selected patients (early recurrence). When these medications fail to alleviate the symptoms of obstruction, venting gastrostomy should be considered promptly. Rehydration is needed for virtually every patient. Parenteral nutrition and pain management should be adjusted according to the patient needs and guidelines.

Facteurs pronostiques du mélanome malin de l’uvée: Étude rétrospective sur 2 241 patients et apport recent de la recherche de la monosomie 3

Introduction Nous avons realise une etude retrospective des facteurs cliniques influencant le pronostic local et general chez des patients atteints de melanome choroidien. Nous avons effectue une analyse preliminaire de la valeur pronostique de la monosomie 3. Patients et methode Les patients adresses a l’Institut Curie pour un melanome de l’uvee ont un bilan initial complet, un traitement conservateur radiotherapique ou une enucleation et un suivi a long terme local et general. Depuis 5 ans, les tumeurs traitees par enucleation ont une etude du statut du chromosome 3 par FISH. Les donnees concernant le bilan initial, le traitement et le suivi sont enregistrees systematiquement de maniere prospective. Nous avons realise une etude retrospective avec analyse multivariee des facteurs cliniques influencant la recidive locale, la conservation oculaire, les metastases et la survie, et en particulier des effets de la monosomie 3. Resultats Deux mille deux cent quarante et un patients ont ete traites avec un recul median de 72 mois. 92,8 % des patients ont eu un traitement conservateur par curietherapie a l’iode 125 ou par protontherapie, et 7,2 % des patients ont eu une enucleation (n = 160). Parmi les patients enuclees, 120 ont eu une etude genetique de leur tumeur par FISH. La survie globale a ete de 76,3 %, et le taux de metastases, de 19,5 %. Les facteurs cliniques influencant la survie en analyse multivariee sont la taille et la localisation de la tumeur, l’âge, le sexe et le traitement initial. Les facteurs influencant le risque metastatique sont identiques, avec en plus, l’existence d’un decollement de retine initial et la recidive locale. La monosomie 3 influence de facon significative le risque metastatique. Discussion Cette etude retrouve les facteurs de risque habituels, mais differe legerement des precedentes series car la localisation a cheval sur l’equateur apparait plus pejorative que la localisation au niveau du corps ciliaire pour les metastases et la survie. Par ailleurs, le decollement de retine initial apparait comme un facteur de risque significatif pour la recidive locale et les metastases. Actuellement, la recherche de la monosomie 3 n’est possible que sur des pieces d’enucleation ; mais, il est probable qu’elle puisse etre applicable aux biopsies a l’aiguille. Cette recherche pourrait etre proposee en meme temps que le traitement conservateur. Conclusion Cette etude confirme les resultats precedents sur les facteurs pronostiques du melanome uveal et la valeur pronostique de la monosomie 3. L’identification de plus en plus precise d’un groupe de patients a haut risque devrait permettre prochainement de proposer des therapeutiques adjuvantes a ces patients et d’adapter le suivi a long terme.INTRODUCTION We conducted a retrospective study on the clinical factors influencing the local and general prognosis of patients treated for uveal melanoma with a preliminary analysis of the prognostic value of monosomy 3. PATIENTS and method: The patients sent to Curie Institute for uveal melanoma have a complete initial clinical evaluation, conservative management by radiotherapy or enucleation, and local and general long-term follow-up. Over the last 5 years, the status of chromosome 3 has been assessed by FISH in the tumors of enucleated patients. Findings concerning the initial workup, treatment, and follow-up are recorded prospectively. We conducted a retrospective study with multivariate analysis of the clinical factors influencing local recurrence, ocular conservation metastasis, and survival and studied the effect of monosomy 3. RESULTS A total of 2241 patients were registered with a median follow-up of 72 months. Of these patients, 92.8% had conservative management with iodine 125 brachytherapy or proton beam therapy and 7.2% of the patients had enucleation (n=160). Tumors from 120 patients were studied for the status of chromosome 3 by FISH. The overall survival rate was 76.3% and the metastatic rate was 19.5%. The clinical factors influencing survival were the size and location of the tumor, age of the patient, gender, and initial treatment. The factors influencing the metastatic risk were the same plus retinal detachment and local recurrence. Monosomy 3 was a significant risk factor for metastatic disease. DISCUSSION This study found the usual risk factors with the difference that location on the equator seems to be of worse prognosis than ciliary body involvement for survival and metastasis. In addition, the initial retinal detachment appears to be a risk factor for local recurrence and metastasis. At present, the evaluation of chromosome 3 is available for enucleated tumors but it could probably be done on needle biopsy performed during conservative management as well. CONCLUSION This study confirms previous results on the prognostic factors of uveal melanoma and on the value of monosomy 3. The increasingly precise identification of a group of high-risk patients should allow us to propose adjuvant therapy and to adapt follow-up.

Disseminated and circulating tumor cells in gastrointestinal oncology

Circulating (CTCs) and disseminated tumor cells (DTCs) are two different steps in the metastatic process. Several recent techniques have allowed detection of these cells in patients, and have generated many results using different isolation techniques in small cohorts. Herein, we review the detection results and their clinical consequence in esophageal, gastric, pancreatic, colorectal, and liver carcinomas, and discuss their possible applications as new biomarkers.