Real-time molecular optical micro-imaging of EGFR mutations using a fluorescent erlotinib based tracer.

BACKGROUND: EGFR mutations are routinely explored in lung adenocarcinoma by sequencing tumoral DNA. The aim of this study was to evaluate a fluorescent-labelled erlotinib based theranostic agent for the molecular imaging of mutated EGFR tumours in vitro and ex vivo using a mice xenograft model and fibred confocal fluorescence microscopy (FCFM). METHODS: The fluorescent tracer was synthesized in our laboratory by addition of fluorescein to an erlotinib molecule. Three human adenocarcinoma cell lines with mutated EGFR (HCC827, H1975 and H1650) and one with wild-type EGFR (A549) were xenografted on 35 Nude mice. MTT viability assay was performed after exposure to our tracer. In vitro imaging was performed at 1 µM tracer solution, and ex vivo imaging was performed on fresh tumours excised from mice and exposed to a 1 µM tracer solution in PBS for 1 h. Real-time molecular imaging was performed using FCFM and median fluorescence intensity (MFI) was recorded for each experiment. RESULTS: MTT viability assay confirmed that addition of fluorescein to erlotinib did not suppress the cytotoxic of erlotinib on tumoral cells. In vitro FCFM imaging showed that our tracer was able to distinguish cell lines with mutated EGFR from those lines with wild-type EGFR (p < 0.001). Ex vivo FCFM imaging of xenografts with mutated EGFR had a significantly higher MFI than wild-type (p < 0.001). At a cut-off value of 354 Arbitrary Units, MFI of our tracer had a sensitivity of 100% and a specificity of 96.3% for identifying mutated EGFR tumours. CONCLUSION: Real time molecular imaging using fluorescent erlotinib is able to identify ex vivo tumours with EGFR mutations.

Clinical Relevance of Gene Copy Number Variation in Metastatic Clear Cell Renal Cell Carcinoma.

BACKGROUND: Gene copy number variations (CNVs) have been reported to be frequent in renal cell carcinoma (RCC), with potential prognostic value for some. However, their clinical utility, especially to guide treatment of metastatic disease remains to be established. Our objectives were to assess CNVs on a panel of selected genes and determine their clinical relevance in patients who underwent treatment of metastatic RCC. PATIENTS AND METHODS: The genetic assessment was performed on frozen tissue samples of clear cell metastatic RCC using quantitative multiplex polymerase chain reaction of short fluorescent fragment method to detect CNVs on a panel of 14 genes of interest. The comparison of the electropherogram obtained from both tumor and normal renal adjacent tissue allowed for CNV identification. The clinical, biologic, and survival characteristics were assessed for their associations with the most frequent CNVs. RESULTS: Fifty patients with clear cell metastatic RCC were included. The CNV rate was 21.4%. The loss of CDKN2A and PLG was associated with a higher tumor stage (P < .05). The loss of PLG and ALDOB was associated with a higher Fuhrman grade (P < .05). The loss of ALDOB was also associated with a worse Heng prognostic score (95% vs. 66%; P = .029) and lower 24-month survival rate (18% vs. 58%; P = .012). The loss of both ALDOB and PLG was frequent (32%) and was associated with a higher tumor stage and grade (P < .05). CONCLUSION: As expected, we showed that several CNVs were associated with clinical relevance, especially those located on CDKN2A, PLG, and ALDOB, in a homogeneous cohort of patients with clear cell metastatic RCC.

Copy number variations in DCC/18q and ERBB2/17q are associated with disease-free survival in microsatellite stable colon cancer.

We conducted a prospective study to assess the prognostic impact of selected copy number variations (CNVs) in Stage II-III microsatellite stable (MSS) colon cancer. A total of 401 patients were included from 01/2004 to 01/2009. The CNVs in 8 selected target genes, DCC/18q, EGFR/7p, TP53/17p, BLK/8p, MYC/8q, APC/5q, ERBB2/17q and STK6/20q, were detected using a quantitative multiplex polymerase chain reaction of short fluorescent fragment (QMPSF) method. The primary end-point was the impact of the CNVs on the 4-year disease-free survival (DFS). The recurrence rate at 4 years was 20.9%, corresponding to 14% Stage II patients versus 31% Stage III patients (p < 0.0001). The 4-year DFS was significantly decreased in patients with a loss at DCC/18q (p = 0.012) and a gain at ERBB2/17q (p = 0.041). The multivariate analysis demonstrated that Stage III, a loss at DCC/18q and a gain at ERBB2/17q were independent factors associated with DFS. A combination of DCC/18q and ERBB2/17q was also associated with relapse, with the hazard ratio increasing from 1 to 2.4 (95% confidence interval (CI), 1.5-4.1) and 3.1 (95% CI, 1.2-8.4) in the presence of 0, 1 or 2 alterations, respectively (p = 0.0013). CNVs in DCC/18q and ERBB2/17q are significantly associated with DFS in Stage II-III MSS colon cancer.

Photodynamic therapy using methylene blue in lung adenocarcinoma xenograft and hamster cheek pouch induced squamous cell carcinoma.

BACKGROUND: Photodynamic therapy (PDT) is used to treat early proximal bronchial cancer during a flexible bronchoscopy. The technique relies on the excitation of a photosensitizer by an appropriate wavelength, which is delivered into the bronchus in close contact with the tumor. OBJECTIVE: To assess methylene blue (MB) as a PDT agent for the treatment of respiratory tract cancer in animal models. METHODS: MB-induced PDT was performed on 7 subcutaneous NCI-H460 lung adenocarcinoma xenografts in nude mice and 9 induced squamous cell cancer in the hamster cheek pouch model. In mice, PDT was carried out on right-sided tumors after intratumoral injection of methylene blue 1% (w/v) and illumination at 630nm at 200J/cm (Diomed PDT 630), with the left tumor used as control (illumination alone or MB alone). The tumoral volume was assessed before and 15 days after PDT. RESULTS: Fourteen xenografts were treated in mice, including seven treated with MB-PDT, producing a 52% mean tumor volume regression (1568mm(3)vs. 544mm(3)) compared to seven control cases in which tumor volume increased (p=0.007; Mann-Whitney test). Nine cheek pouch induced carcinomas were treated in the hamster group, with a mean volume decrease of 85.8% (from 44.8% to 100%) (initial mean volume=210mm(3)vs. post PDT mean volume=97mm(3)). Histology analysis showed 4/9 complete responses. CONCLUSION: Intratumoral MB appears efficient as PDT agent for cancer treatment in animal models. Further studies are needed to assess the safety and efficacy of MB-associated PDT for the treatment of lung cancer in humans.

Integrative Analyses of Colorectal Cancer Show Immunoscore Is a Stronger Predictor of Patient Survival Than Microsatellite Instability.

Microsatellite instability in colorectal cancer predicts favorable outcomes. However, the mechanistic relationship between microsatellite instability, tumor-infiltrating immune cells, Immunoscore, and their impact on patient survival remains to be elucidated. We found significant differences in mutational patterns, chromosomal instability, and gene expression that correlated with patient microsatellite instability status. A prominent immune gene expression was observed in microsatellite-instable (MSI) tumors, as well as in a subgroup of microsatellite-stable (MSS) tumors. MSI tumors had increased frameshift mutations, showed genetic evidence of immunoediting, had higher densities of Th1, effector-memory T cells, in situ proliferating T cells, and inhibitory PD1-PDL1 cells, had high Immunoscores, and were infiltrated with mutation-specific cytotoxic T cells. Multivariate analysis revealed that Immunoscore was superior to microsatellite instability in predicting patients' disease-specific recurrence and survival. These findings indicate that assessment of the immune status via Immunoscore provides a potent indicator of tumor recurrence beyond microsatellite-instability staging that could be an important guide for immunotherapy strategies.

Clinical value of chip-based digital-PCR platform for the detection of circulating DNA in metastatic colorectal cancer.

BACKGROUND: The detection of circulating DNA is considered a promising strategy in cancer patients. Digital PCR has emerged as a sensitive method able to quantify both circulating free and tumour DNA. AIM: The aim of this study was to prospectively evaluate the clinical value of a chip-based digital PCR for the detection of circulating DNA. METHODS: Digital PCR was used in 34 metastatic colorectal cancer patients to detect and quantify circulating free and tumour DNA based on K-ras mutational status. Clinical outcomes were analyzed according to circulating DNA measurements. RESULTS: Digital PCR yielded a detection rate of 69% for circulating tumour DNA. The median concentrations of circulating free and tumour DNA were 20 and 6.8 ng/mL, respectively, with significant correlation between both biomarkers (p<0.001). Median overall survival was 4.8 months in patients with high circulating free DNA (>75% quartile) versus not reached in patients with a low level (<25% quartile) (p=0.029). Moreover, median overall survival was significantly decreased in patients with detectable circulating tumour DNA compared to those without (respectively 11.8 months versus not reached, p=0.04). CONCLUSIONS: Chip-based digital PCR is a simple and non-invasive method allowing the efficient detection of circulating DNA. Our results highlight that levels of these circulating markers may have a potential prognostic value.

Correlation between Density of CD8+ T-cell Infiltrate in Microsatellite Unstable Colorectal Cancers and Frameshift Mutations: A Rationale for Personalized Immunotherapy.

Colorectal cancers with microsatellite instability (MSI) represent 15% of all colorectal cancers, including Lynch syndrome as the most frequent hereditary form of this disease. Notably, MSI colorectal cancers have a higher density of tumor-infiltrating lymphocytes (TIL) than other colorectal cancers. This feature is thought to reflect the accumulation of frameshift mutations in sequences that are repeated within gene coding regions, thereby leading to the synthesis of neoantigens recognized by CD8(+) T cells. However, there has yet to be a clear link established between CD8(+) TIL density and frameshift mutations in colorectal cancer. In this study, we examined this link in 103 MSI colorectal cancers from two independent cohorts where frameshift mutations in 19 genes were analyzed and CD3(+), CD8(+), and FOXP3(+) TIL densities were quantitated. We found that CD8(+) TIL density correlated positively with the total number of frameshift mutations. TIL densities increased when frameshift mutations were present within the ASTE1, HNF1A, or TCF7L2 genes, increasing even further when at least one of these frameshift mutations was present in all tumor cells. Through in vitro assays using engineered antigen-presenting cells, we were able to stimulate peripheral cytotoxic T cells obtained from colorectal cancer patients with peptides derived from frameshift mutations found in their tumors. Taken together, our results highlight the importance of a CD8(+) T cell immune response against MSI colorectal cancer-specific neoantigens, establishing a preclinical rationale to target them as a personalized cellular immunotherapy strategy, an especially appealing goal for patients with Lynch syndrome.

Safety and performance analysis of acriflavine and methylene blue for in vivo imaging of precancerous lesions using fibered confocal fluorescence microscopy (FCFM): an experimental study.

BACKGROUND: Fibered confocal fluorescence microscopy (FCFM) allows in vivo investigation of pulmonary microstructures. However, the bronchial epithelium can only be imaged using exogenous fluorophores. The objective of this study is to compare methylene blue (MB) and acriflavine genotoxicity and to assess FCFM performance for in vivo imaging of precancerous lesions. METHODS: Genotoxicity was assessed using the comet assay on both cultured human lymphocytes and NCI-H460 cells, which had been exposed to MB or acriflavine before being illuminated at 660 or 488 nm, respectively. FCFM was performed on precancerous lesions in the hamster cheek pouch model, following topical application of the fluorophores. FCFM data were analyzed according to histology. RESULTS: No genotoxicity was found using 0.01% (w/v) MB after illumination at 660 nm for 2 and 15 min (5 mW). Acriflavine exposure (0.025%) led to DNA damages, increasing from 2 to 15 min of light exposure at 448 nm in both lymphocytes (83.4 to 88%, p = 0.021) and NCI H460 cell populations (79.9 to 84.6%, p = 0.045). In total, 11 invasive carcinoma, 24 reserve cell hyperplasia, and 17 dysplasia lesions were imaged using FCFM in vivo. With both fluorophores, the cellular density increased from hyperplasia to high-grade dysplasia (p < 0.05). With MB, the cellular diameter significantly decreased (48.9 to 13.9 µm) from hyperplasia to carcinoma (p < 0.05). In this model, a cut-off diameter of 30 µm enabled the diagnosis of high-grade lesions with a sensitivity of 94.7% and a specificity of 97%. CONCLUSION: Methylene blue can be used safely to image precancerous lesions in vivo. This study does not support the use of acriflavine in humans.

A multiplex technology platform for the rapid analysis of clinically actionable genetic alterations and validation for BRAF p.V600E detection in 1549 cytologic and histologic specimens.

CONTEXT: Current clinicopathologic assessment of malignant neoplastic diseases entails the analysis of specific genetic alterations that provide diagnostic, prognostic, or therapy-determining information. OBJECTIVE: To develop and validate a robust molecular method to detect clinically relevant mutations in various tissue types and anatomic pathology specimens. DESIGN: Genes of interest were amplified by multiplex polymerase chain reaction and sequence variants identified by liquid bead array cytometry. The BRAF assay was fully characterized by using plasmids and genomic DNA extracted from cell lines, metastatic colorectal cancer formalin-fixed, paraffin-embedded (FFPE) tissues, and thyroid nodule fine-needle aspirates. RESULTS: Qualitative multiplex assays for 22 different mutations in the BRAF, HRAS, KRAS, NRAS, or EGFR genes were established. The high signal-to-noise ratio of the technology enabled reproducible detection of BRAF c.1799T>A (p.V600E) at 0.5% mutant allele in 20 ng of genomic DNA. Precision studies with multiple operators and instruments showed very high repeatability and reproducibility with 100% (98.7%-100%) qualitative agreement among 292 individual measures in 38 runs. Evaluation of 1549 representative pathologic specimens in 2 laboratories relative to independent reference methods resulted in 99.0% (97.6%-99.6%) agreement for colorectal FFPE tissues (n = 416) and 98.9% (98.2%-99.4%) for thyroid fine-needle aspiration specimens (n = 1133) with an overall diagnostic odds ratio of 10 856 (2451-48 078). CONCLUSIONS: The multiplex assay system is a sensitive and reliable method to detect BRAF c.1799T>A mutation in colorectal and thyroid lesions. This optimized technology platform is suitable for the rapid analysis of clinically actionable genetic alterations in cytologic and histologic specimens.

The MDM2 285G-309G haplotype is associated with an earlier age of tumour onset in patients with Li-Fraumeni syndrome.

In the Li-Fraumeni syndrome (LFS) resulting from germline TP53 mutations, the MDM2 SNP309G allele has been shown to be associated with an earlier age of tumour onset, however the significance of this association is controversial. The 285C variation, also located in the MDM2 promoter, has been shown to reduce the strength of Sp1 binding to MDM2 promoter, antagonizing the effect of the 309G variation. In this study, we investigated the interaction of the MDM2 SNP285 and 309 in a large series of 195 LFS patients. Although we observed a lower mean age of tumour onset in patients with MDM2 SNP309 T/G or G/G genotype (23.1 years) than in patients with T/T genotype (27.3 years), the difference was not statistically significant. In contrast, patients with the 285-309 G-G haplotype develop tumours 5 years earlier than patients harbouring other haplotypes (p = 0.044). This result shows that the MDM2 285-309 G-G is a higher risk haplotype in patients with germline TP53 mutations. This study confirms that the MDM2 309G variation is deleterious when its effect is not neutralized by the 285C variation and illustrates the interfering effects of SNPs located within a gene acting as modifier factor in a Mendelian disease.

Genetic variations of the A13/A14 repeat located within the EGFR 3' untranslated region have no oncogenic effect in patients with colorectal cancer.

BACKGROUND: The EGFR 3' untranslated region (UTR) harbors a polyadenine repeat which is polymorphic (A13/A14) and undergoes somatic deletions in microsatellite instability (MSI) colorectal cancer (CRC). These mutations could be oncogenic in colorectal tissue since they were shown to result into increased EGFR mRNA stability in CRC cell lines. METHODS: First, we determined in a case control study including 429 CRC patients corresponding to different groups selected or not on age of tumor onset and/or familial history and/or MSI, whether or not, the germline EGFR A13/A14 polymorphism constitutes a genetic risk factor for CRC; second, we investigated the frequency of somatic mutations of this repeat in 179 CRC and their impact on EGFR expression. RESULTS: No statistically significant difference in allelic frequencies of the EGFR polyA repeat polymorphism was observed between CRC patients and controls. Somatic mutations affecting the EGFR 3'UTR polyA tract were detected in 47/80 (58.8%) MSI CRC versus 0/99 microsatellite stable (MSS) tumors. Comparative analysis in 21 CRC samples of EGFR expression, between tumor and non malignant tissues, using two independent methods showed that somatic mutations of the EGFR polyA repeat did not result into an EGFR mRNA increase. CONCLUSION: Germline and somatic genetic variations occurring within the EGFR 3' UTR polyA tract have no impact on CRC genetic risk and EGFR expression, respectively. Genotyping of the EGFR polyA tract has no clinical utility to identify patients with a high risk for CRC or patients who could benefit from anti-EGFR antibodies.

Drastic effect of germline TP53 missense mutations in Li-Fraumeni patients.

In contrast to other tumor suppressor genes, the majority of TP53 alterations are missense mutations. We have previously reported that in the Li-Fraumeni syndrome (LFS), germline TP53 missense mutations are associated with an earlier age of tumor onset. In a larger series, we observed that mean age of tumor onset in patients harboring dominant negative missense mutations and clearly null mutations was 22.6 and 37.5 years, respectively. To assess the impact of heterozygous germline TP53 mutations in the genetic context of the patients, we developed a new functional assay of the p53 pathway on the basis of induction of DNA damage in Epstein-Barr-virus-immortalized lymphocytes, followed by comparative gene-expression profiling. In wild-type lymphocytes, we identified a core of 173 genes whose expression was induced more than twofold, of which 46 were known p53 target genes. In LFS lymphocytes with canonical missense mutations, the number of induced genes and the level of known p53 target genes induction were strongly reduced as compared with controls and LFS lymphocytes with null mutations. These results show that certain germline missense TP53 mutations, such as those with dominant negative effect, dramatically alter the response to DNA damage. This probably explains why TP53 alterations are predominantly missense mutations.

Metastatic colorectal cancer KRAS genotyping in routine practice: results and pitfalls.

KRAS genotyping is mandatory before anti-epidermal growth factor receptor monoclonal antibody therapy in metastatic colorectal cancer, which is the second leading cause of cancer-related death in the United States and in Europe. Thus, large-scale KRAS mutation screening is needed for efficient patient management and in the future metastatic colorectal cancer genotyping might also include the detection of the BRAF V600E mutation, which is a very strong negative prognostic factor in colorectal cancer. We report our experience of routine KRAS/BRAF mutation screening practice performed on 1130 formalin-fixed paraffin-embedded tumor samples from 992 colorectal cancer patients. DNA was extracted from macrodissected tumor areas highlighted by a pathologist, KRAS codons 12/13 and BRAF V600E mutations were assessed in a single SNaPshot® multiplex assay and each mutation was confirmed by an independent analysis. KRAS and BRAF mutations were, respectively, present in 41.8 and 6.5% of the tumor samples. If KRAS and BRAF mutations were mutually exclusive, four samples presented two concomitant KRAS mutations. Genotyping of paired primary tumors and metastases from 44 patients indicated that 5 patients (11.4%) presented discordant KRAS mutational status. KRAS genotype heterogeneity was also observed within primary tumor sites in seven cases. Non-reproducible KRAS artefactual mutations were detected in 53 samples (4.7%). We found that the prominent mechanism leading to these artefactual mutations was the fragmentation of DNA occurring during tissue processing. Routine KRAS genotyping performed on formalin-fixed paraffin-embedded tissues requires, therefore, the development of quality control scheme for molecular pathology, especially because of DNA damages induced by formalin fixation. The tumor heterogeneity observed in some patients indicates that it should be more appropriate to perform KRAS genotyping on metastases if sample is available.

HLA-A*0201-restricted CEA-derived peptide CAP1 is not a suitable target for T-cell-based immunotherapy.

Carcinoembryonic antigen (CEA) is a potential target for antigen-specific immunotherapy, as it is frequently overexpressed in human carcinomas. Moreover, an epitope derived from CEA, designated CAP1 (YLSGANLNL), has been proposed as naturally processed and presented by tumors in the human leukocyte antigen (HLA)-A*0201 context. Our aim was to fully characterize and assess the clinical relevance of the HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) response against CEA. Stable and potent artificial antigen presenting cells (AAPCs) were used to evaluate T-cell response against CEA. These cells efficiently activate CTLs against tumor-derived epitopes after transduction with the antigenic peptides or full-length proteins. We found that AAPCs genetically modified to express CAP1, the agonist peptide CAP1-6D, or the whole CEA protein were not able to activate CAP1-specific CTLs from HLA-A*0201+ healthy donors or patients with colorectal carcinoma, even after multiple stimulations. In addition, we showed that a CAP1-specific T-cell clone, obtained after multiple stimulations of T cells of a HLA-A*0201+ healthy donor in vitro with autologous antigen presenting cells, recognized CEA(-) HLA-A*0201+ tumors transduced with a minigene encoding CAP1 but failed to react against HLA-A*0201+ tumor cells expressing CEA. Finally, AAPCs expressing the whole CEA protein did not induce any specific CTL response against CEA+ HLA-A*0201+ tumor cells highlighting the potential difficulty of mounting an efficacious T-cell response against this autoantigen. Altogether, our data indicate that CAP1 is not efficiently processed and presented by CEA+ tumor cells, and therefore, is not an appropriate target for T-cell-based immunotherapy.

Tumor-infiltrating lymphocytes in colorectal cancers with microsatellite instability are correlated with the number and spectrum of frameshift mutations.

Colorectal cancers with microsatellite instability are characterized by an important density of tumor-infiltrating lymphocytes and a good prognosis. Microsatellite instability results from the inactivation of the DNA mismatch repair system and induces secondary somatic frameshift mutations within target genes harboring repeat sequences in their coding frame. By disrupting the open reading frame, frameshift mutations can result in the appearance of potentially immunogenic neopeptides. To determine the frameshift mutations inducing a T-cell response during the development of a tumor with microsatellite instability, we studied in 61 colorectal cancer patients with microsatellite instability, using a fluorescent multiplex PCR comparative analysis, the relative frequency of frameshift mutations within 19 target genes and analyzed the correlation of these frameshift mutations with the density of CD3+ tumor-infiltrating lymphocytes. The four most frequently mutated genes were ACVR2 (92%), TAF1B (84%), ASTE1/HT001 (80%) and TGFBR2 (77%). The vast majority (95%) of the tumors exhibited at least three frameshift mutations, and the number of frameshift mutations was associated with tumor progression (TNM stage, wall invasion and tumor diameter). Tumor-infiltrating lymphocyte density was associated with the overall number of frameshift mutations and with the presence of frameshift mutations within two target genes, namely ASTE1/HT001 and PTEN. These results strongly argue for the clinical relevance of immunotherapy of colorectal cancers with microsatellite instability.

Gradual reduction of BUBR1 protein levels results in premature sister-chromatid separation then in aneuploidy.

Biallelic and heterozygous mutations of the BUB1B gene have been reported in mosaic variegated aneuploidy (MVA), a rare disorder characterized by constitutional mosaic aneuploidies associated to severe intrauterine growth retardation, microcephaly and, in most cases, to premature chromatid separation (PCS), highlighting the key role of human BUBR1 in chromosome segregation. To study the consequences of gradual reduction of the BUBR1 protein levels, inhibition of BUB1B expression in model cells was induced using short hairpin RNAs (shRNAs). We obtained stable shRNA-transduced HeLa cells displaying a gradient of residual BUBR1 protein (8.5, 10, 14, 58, and 77%), mimicking the situation of patients' cells harboring one or two BUB1B mutations. Induction of PCS was detected in all transduced cells and its level was correlated to the decrease of BUBR1. Aneuploidy was clearly detected in cells with residual BUBR1 below 50%. Our data demonstrate that the function of the human BUBR1 protein in the spindle checkpoint is remarkably dosage-dependent and that the biological consequences of BUB1B expression reduction on premature chromatid separation and aneuploidy depend on the residual amount of BUBR1. This provides a biological explanation for the mode of inheritance of PCS, which is dominant, and of MVA, which can be recessive in some families and result from the combination of a null allele associated to a common hypomorphic allele in others.

Molecular predictive factors for progression of high-grade preinvasive bronchial lesions.

RATIONALE: The outcome of precancerous bronchial lesions is not well known, and their management is subject to controversy. Many molecular alterations are present in preinvasive lesions, but none has been assessed to predict the evolution of the lesions. OBJECTIVES: To analyze the outcome of high-grade precancerous lesions according to their molecular profile. METHODS: Twenty-three severe dysplasia and 31 carcinoma in situ (CIS) lesions in 37 patients were monitored using repeated autofluorescence bronchoscopy over a 12-year period. Microdissection and polymerase chain reaction analysis were performed on paraffin tissue sections to assess loss of heterozygosity (LOH) and microsatellite instability on chromosome 3p, 5q, and 9p. Histology and molecular status at baseline were compared between 7 lesions that became invasive, 11 that relapsed after treatment, 17 that were eradicated with local treatment, and 19 that spontaneously regressed. MEASUREMENTS AND MAIN RESULTS: Ninety-four percent of lesions that progressed or relapsed were CIS at baseline, whereas 79% of spontaneously regressing lesions were severe dysplasia (P < 0.0001). 3p and 9p LOH was more frequent in CIS than in severe dysplasia (P = 0.03). In the whole group of lesions as well as in the CIS group, 3p LOH was strongly associated with progression (P < 0.0001 and P = 0.02, respectively). Microsatellite instability was not associated with the outcome of the lesions. A therapeutic strategy based on the presence of 3p or 9p LOH would have led to overtreatment of six lesions but would have missed only 1 among the 18 progressing lesions. CONCLUSIONS: Baseline histology and 3p LOH analysis appear to be useful in predicting the outcome of high-grade precancerous lesions.

A simple method for the routine detection of somatic quantitative genetic alterations in colorectal cancer.

BACKGROUND & AIMS: Several quantitative genetic alterations have been suggested to have in colorectal cancer (CRC) either a prognostic or a therapeutic predictive value. Routine detection of these alterations is limited by the absence of simple methods. METHODS: The somatic quantitative multiplex polymerase chain reaction of short fluorescent fragments (QMPSF) is based on the simultaneous amplification under quantitative conditions of several dye-labeled targets both from tumor and nonmalignant tissues. For each patient, the resulting QMPSF fluorescent profiles are superimposed, and quantitative changes are simply detected by an increase or decrease of the corresponding fluorescent peaks. Two assays were developed and applied to 57 CRC: a "bar code" exploring several loci with known prognostic value and a "kinogram" studying the copy number change of kinase genes, against which inhibitors have been developed. RESULTS: The bar code revealed that the most frequent alterations were the gain of AURKA/20q13 (53%) and MYC/8q24 (39%) and heterozygous deletion of DCC/18q21.3 (39%) and TP53/17p13 (23%). The kinogram detected a gene copy number increase for AURKA, PTK2, MET, and EGFR in 53%, 37%, 33%, and 28% of the tumors, respectively. QMPSF results were validated by comparative genomic hybridization and multiplex real-time polymerase chain reaction on genomic DNA. CONCLUSIONS: The somatic QMPSF is a simple method able to detect simultaneously on a routine basis several quantitative changes in tumors. Its flexibility will allow the integration of clinically relevant genes. This high throughput method should be a valuable complementary tool of fluorescent in situ hybridization and comparative genomic hybridization.

Hyaluronectin modulation of lung metastasis in nude mice.

Hyaluronectin (HN) is a glycoprotein with a high affinity to hyaluronic acid (HA) and known to be a component of the extracellular matrix of tumours. Clinical studies have shown that a low level of HN correlates to tumours with poor prognosis, whereas a high level of HN correlates to tumours with good prognosis. We previously demonstrated in vitro that hyaluronidase activity, which promotes tumour progression and metastatic spread by degradation of HA into angiogenic oligosaccharides, was inhibited or promoted by HN, according to the level of HN-expression. This raises the question of the role played by HN in cancer, and particularly if high and low levels of HN-expression could trigger opposite effects on tumour growth and/or metastatic spread. To address this issue, we used a model of spontaneous lung fluorescent metastases that we characterised previously. We stably transfected the human HN cDNA into fluorescent H460MGFP cells and selected two clones characterised by different levels of HN-expression: HN110 and HN704, with a high and a low level of HN-expression, respectively. In vitro, we demonstrated that HN704 cell migration was significantly increased. Inoculation of clones to nude mice had no significant effect on tumour growth, but clearly revealed opposite effects on metastatic spread: HN110 significantly decreased the number of fluorescent metastases whereas HN704 significantly increased it. We also analysed HN, HA and hyaluronidase contents in sera and tumours. These results demonstrate that HN can play a role as either a suppressor or promoter of metastatic spread.

Contribution of the BOP1 gene, located on 8q24, to colorectal tumorigenesis.

The most common form of genomic instability observed in colorectal cancer is chromosomal instability (CIN), whose molecular bases remain to be determined. We have previously demonstrated that inactivation in human cells of several components of the Pes1-Bop1 complex (BOP1, GRWD1, PES1, ORC6L, and RPL3), involved in ribosome biogenesis, altered chromosome segregation. To determine the contribution to colorectal tumorigenesis of somatic alterations of genes involved in ribosome biogenesis, we screened 56 primary colorectal cancers, using quantitative multiplex PCR of short fluorescent fragments, a sensitive method for the detection of gene dosage alterations. We found that dosage increase of the BOP1 gene was a frequent event, being detected in 39% of the tumors, and we show that it is associated with an increase of BOP1 mRNA. Scanning of 8q24, on which BOP1 is located, revealed that in colorectal cancers, gene dosage increase of BOP1 can be independent from that of MYC and was more frequent than that affecting MYC. Finally, transient overexpression of BOP1 in human cells increased the percentage of multipolar spindles. Together with our previous results, the present study strongly suggests that deregulation of the BOP1 pathway contributes to colorectal tumorigenesis.