Hany Soliman

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.

Non-invasive molecular detection of bladder cancer recurrence

Transitional cell carcinoma (TCC) is the most common bladder tumor and ≈90% of bladder TCC are superficial at initial diagnosis. High recurrence rate and possible progression to muscle invasive disease that is eventually indicated for radical cystectomy are established features of these tumors. Therefore, reliable predictors of tumor recurrence are of critical importance for management of superficial bladder TCC. Successful molecular diagnosis of bladder cancer by detecting genetic lesions: loss of heterozygosity (LOH) or microsatellite instability (MSI) in cells exfoliated in urine has been reported by several groups including ours. The aim of our study was to evaluate the predictive potential of microsatellite analysis of cells exfoliated in urine in the detection of superficial bladder TCC recurrence. We studied 47 Caucasian patients with confirmed superficial bladder TCC (37 pTa, 10 pT1) at initial diagnosis. Blood samples were obtained once from every patient whereas urine samples were collected before each cystoscopy (initial and follow‐up). Matched DNAs from blood and urine were subjected to microsatellite analysis in a blinded fashion. The follow‐up period ranged 12–48 months after tumor resection. Microsatellite analysis correctly identified 94% (44/47) of primary tumors and 92% (12/13) of tumor recurrences. Interestingly enough, 75% (9/12) of tumor recurrences were molecularly detected 1–9 months before cystoscopic evidence of recurrent disease. This study demonstrated clearly that not only urine microsatellite analysis reliably detected superficial bladder tumors, but also was a reliable test for detecting and predicting tumor recurrence in Caucasian patients. These results warrant multicenter randomized trials.

Immunohistochemical and molecular analyses of HER2 status in breast cancers are highly concordant and complementary approaches

Background:Immunohistochemistry (IHC) and fluorescent in situ hybridisation (FISH) are currently the most commonly used methods to assess HER2 status. PCR-based assays allow quantitative determination of HER2 amplification (Q-PCR) or overexpression (Q-RT–PCR), but are not routinely used. We evaluated the relevance of Q-RT–PCR for HER2 status determination.Methods:We compared IHC and Q-RT–PCR in 466 breast tumours. In discordant or equivocal cases, five additional methods (IHC with two other antibodies, FISH, silver in situ hybridisation (SISH) and Q-PCR) were combined to determine HER2 status. Two cases with HER2 intra-tumour heterogeneity were further explored by allelic profiles analysis and HUMARA clonality determination after microdissection.Results:We observed 97.3% concordance between Q-RT–PCR and non-equivocal IHC. Twelve out of 466 cases (3%) revealed discordances between the two methods. The power of Q-RT–PCR to predict HER2 status (defined by seven methods) was similar to that of IHC. Although rare, some discordances between techniques might be due to HER2 intra-tumour heterogeneity and we report two examples, one tumour containing two distinct clones, another tumour consisting of HER2 amplified and non-amplified subclones.Conclusion:Q-RT–PCR and IHC are highly concordant methods for HER2 status assessment, and Q-RT–PCR allows a highly reliable quantitative assessment and could be a useful adjunct to IHC.

Association Between a High Number of Isolated Lymph Nodes in T1 to T4 N0M0 Colorectal Cancer and the Microsatellite Instability Phenotype

HYPOTHESIS Stage I or II colorectal carcinomas with microsatellite instability (MSI) are characterized by more isolated lymph nodes in the resected specimen than their counterparts with microsatellite stability (MSS). DESIGN Prospective study. SETTING Academic research. PATIENTS Using a pentaplex polymerase chain reaction assay, MSI status was determined prospectively for 135 operative patients. MAIN OUTCOME MEASURES Mismatch repair defects were investigated by immunohistochemistry on tumors demonstrating MSI. RESULTS Among 82 stage I or II colorectal carcinomas, 11 had MSI, and 71 had MSS, with a mean (SD) number of 23.6 (3.1) and 13.7 (1.0) negative lymph nodes, respectively (P = .001). The mean number of lymph nodes for all resected stage I or II colorectal carcinomas analyzed at our hospital was 15. The prevalence of MSI among tumors with more than 15 lymph nodes in the specimen was 25% (9 of 36), and 82% (9 of 11) of MSI tumors belonged to this group. CONCLUSIONS A high number of isolated lymph nodes in stage I or II colorectal carcinomas was associated with the MSI phenotype. Good prognosis that is usually associated with tumors having a high number of uninvolved lymph nodes might reflect the high prevalence of MSI among these tumors. The number of examined lymph nodes as a quality criterion should be used with caution. For stage I or stage II colorectal carcinomas, restricting MSI phenotyping to tumors with more than the mean number of lymph nodes identifies almost all MSI tumors.

A meta-analysis of the relationship between FGFR3 and TP53 mutations in bladder cancer.

TP53 and FGFR3 mutations are the most common mutations in bladder cancers. FGFR3 mutations are most frequent in low-grade low-stage tumours, whereas TP53 mutations are most frequent in high-grade high-stage tumours. Several studies have reported FGFR3 and TP53 mutations to be mutually exclusive events, whereas others have reported them to be independent. We carried out a meta-analysis of published findings for FGFR3 and TP53 mutations in bladder cancer (535 tumours, 6 publications) and additional unpublished data for 382 tumours. TP53 and FGFR3 mutations were not independent events for all tumours considered together (OR = 0.25 [0.18–0.37], p = 0.0001) or for pT1 tumours alone (OR = 0.47 [0.28–0.79], p = 0.0009). However, if the analysis was restricted to pTa tumours or to muscle-invasive tumours alone, FGFR3 and TP53 mutations were independent events (OR = 0.56 [0.23–1.36] (p = 0.12) and OR = 0.99 [0.37–2.7] (p = 0.35), respectively). After stratification of the tumours by stage and grade, no dependence was detected in the five tumour groups considered (pTaG1 and pTaG2 together, pTaG3, pT1G2, pT1G3, pT2-4). These differences in findings can be attributed to the putative existence of two different pathways of tumour progression in bladder cancer: the CIS pathway, in which FGFR3 mutations are rare, and the Ta pathway, in which FGFR3 mutations are frequent. TP53 mutations occur at the earliest stage of the CIS pathway, whereas they occur would much later in the Ta pathway, at the T1G3 or muscle-invasive stage.

Determination of Angptl4 mRNA as a Diagnostic Marker of Primary and Metastatic Clear Cell Renal-Cell Carcinoma

Background We have previously shown that angiopoietin-like 4 (angptl4) mRNA, a hypoxia-inducible gene, is highly expressed in clear cell renal-cell carcinoma (ccRCC), the most common subtype of RCC for which no specific marker is available. We here investigated whether angptl4 mRNA 1) could be a useful diagnostic and/or prognostic marker of ccRCC in a large and comprehensive retrospective series, 2) induction is dependent on the VHL status of tumors. Methodology/Principal Findings Using in situ hybridization, we report that angptl4 mRNA is expressed in 100% of both sporadic (n = 102) and inherited (n = 6) primary ccRCCs, without any statistical association with nuclear grade (p = 0.39), tumor size (p = 0.09), stage grouping (p = 0.17), progression-free survival (p = 0.94), and overall survival (p = 0.80). Angptl4 mRNA was also expressed in 26 (87%) of 30 secondary ccRCCs but neither in any other secondary RCCs (n = 7). In contrast, angptl4 mRNA was neither expressed in 94% non-ccRCC renal tumors (papillary RCCs (n = 46), chromophobe RCCs (n = 28), and oncocytomas (n = 9)), nor in non-renal clear cell carcinomas (n = 39). Angptl4 expression was also examined in tumors associated (n = 23) or not associated (n = 66) with VHL disease. 40 (98%) hemangioblastomas expressed angptl4 whereas all pheochromocytomas (n = 23) and pancreatic tumors (n = 25) were angptl4-negative, whatever their VHL status. Conclusions/Significance Angptl4 mRNA expression was highly associated with ccRCC (p = 1.5 10−49, Chi square test) allowing to define its expression as a diagnosis marker for primary ccRCC. Moreover, angptl4 mRNA allows to discriminate the renal origin of metastases of clear-cell carcinomas arising from various organs. Finally, inactivation of VHL gene is neither necessary nor sufficient for angptl4 mRNA induction.

Spectrum of molecular alterations in colorectal, upper urinary tract, endocervical, and renal carcinomas arising in a patient with hereditary non-polyposis colorectal cancer

Hereditary nonpolyposis colon cancer (HNPCC) syndrome is the most frequent hereditary cancer syndrome predisposing to cancers of various locations, especially colon, endometrium, stomach, and upper urinary tract. Carcinomas of the kidney parenchyma are not considered as an HNPCC-related tumor. HNPCC tumors are characterized by microsatellite instability (MSI) due to a defect in mismatch repair (MMR) and carry somatic frameshift mutations in mononucleotide repeats within the coding regions of key genes. We report the first case of a papillary carcinoma of the kidney in an HNPCC patient who developed carcinomas of the upper urinary tract, endocervix, and colon. Whereas the HNPCC-related tumors demonstrated MSI phenotype, loss of MSH2 protein expression, and frameshift mutations in several of the 13 target genes analyzed, the kidney cancer displayed MSS phenotype, normal MMR protein expression, and no frameshift mutation in target genes. Our observations do not support the possibility that papillary carcinomas are part of HNPCC syndrome.

Identification of a three-gene expression signature of early recurrence in non-muscle-invasive urothelial cell carcinoma of the bladder

OBJECTIVES To identify the expression profile of early recurring non-muscle-invasive bladder cancer (NMI-BC). MATERIALS AND METHODS We selected primary NMI-BC according to the following criteria: complete resection, primary occurrence of urothelial cell carcinoma, stage cTa or T1, low grade, no carcinoma in situ. All patients underwent a transurethral resection of the bladder and were followed according to the Guidelines of European Association of Urology. Expression of 110 target genes was studied by using real time quantitative RT-PCR. The correlation between the absolute expression and early recurrence was analyzed by using a Cox regression model. The ability of a gene to distinguish the tumors with early recurrence from those with late recurrence was determined by using a ROC-AUC test. A hierarchical classification was established by using the software of GeneANOVA and tested by a χ(2) test. RESULTS Forty-seven tumors were studied: 25 with early recurrence vs. 22 with late or null recurrence. These 2 groups of tumors have a significantly different expression profile in 3 genes among the 110-gene expression profile: peroxysome proliferator-activated receptor-γ (PPARG) (P = 0.031), stathmin-1 (STMN1) (P = 0.041), Caveolin-2 (CAV2) (P = 0.004). The combination of the expression of these 3 genes was significantly predictive for early recurrence leading to a correct hierarchical classification of the NMI-BC regarding the time-to-recurrence (P < 0.001). CONCLUSIONS This study identifies a concise 3-gene panel that is associated with time to recurrence. This 3-gene signature has to be confirmed in a prospective study and in larger cohort of patients. However, our preliminary results are promising and gene expression profiling seems to be an interesting approach in cTa-T1 tumors for recurrence prediction.

Prognostic Value of Loss of Heterozygosity at Chromosome 9p in Non–muscle-invasive Bladder Cancer

OBJECTIVE To investigate the prognostic value of loss of heterozygosity (LOH) at chromosome 9p in patients with non-muscle-invasive bladder cancer (NMI-BC). METHODS Between 2000 and 2006, we included in the study 84 patients with NMI-BC. LOH analyses were performed on tumor tissue using 3 microsatellite markers at chromosome 9p. Associations of LOH with recurrence and progression of the tumors were evaluated. RESULTS Frequency of LOH at 9p was 11.1%, 29.0%, and 31.6% in pTaG1, pTaG2, and pT1G3 tumors, respectively. Recurrence occurred in 27 patients. None of the markers was able to predict recurrence. Progression occurred in 9.5% of the cases, with progression to muscle-invasive bladder cancer (MI-BC) in 4.8% of the cases. LOH at IFN-alpha was significantly associated with progression to MI-BC (P = .006). In the case of LOH at IFN-alpha, 2-year progression-free survival and progression-free survival to MI-BC were both 59.3%, compared with 97.1% and 98.6%, respectively, in case of conservation of LOH in multivariable analysis, LOH at IFN-alpha remained statistically associated with progression and progression to MI-BC. LOH at IFN-alpha was a significant and independent predicting factor of progression and progression to MI-BC, with P = .05 and 0.01 (HR 4.8 for progression; HR 24.2 for muscle invasion). CONCLUSIONS Our study suggests that LOH at IFN-alpha is an independent prognostic factor for progression to MI-BC. LOH analysis of bladder tumors may help in the management of NMI-BC. Specifically, it could be of use in selecting patients for early aggressive treatment and/or in planning close follow-up schedule.

Changes in allelic imbalances in locally advanced breast cancers after chemotherapy

In advanced breast cancers, TP53 mutation is highly predictive of complete response to high-dose epirubicin/cyclophosphamide chemotherapy. In these tumours with an altered control of genomic stability, accumulation of chemotherapy-induced genetic alterations may contribute to cell death and account for complete response. To explore the effects of chemotherapy on stability of the tumour genome, allelic profiles were obtained from microdissected tumour samples before and after chemotherapy in 29 unresponsive breast cancers (9 with TP53 mutation). Ninety-four per cent allelic profiles remained unchanged after treatment. Interestingly, 11 profiles (6%) showed important changes after treatment; allelic imbalances significantly increased (four cases) or decreased (seven cases) after chemotherapy in three distinct experiments, two of which using laser microdissected tumour cells. These genetic changes were not linked to the TP53 status, but one tumour showed complete disappearance of TP53-mutated cells in the residual tumour after treatment. Altogether, these observations carry important implications for the clonal evolution of breast cancers treated with DNA-damaging agents, as they point both to the importance of tumour heterogeneity and chemotherapy-driven selection of subclones.