GJB5 association with BRAF mutation and survival in cutaneous malignant melanoma.

BACKGROUND: Gap-junctional intercellular communication is crucial for epidermal cellular homeostasis. Inability to establish melanocyte-keratinocyte contact and loss of the intercellular junction's integrity may contribute to melanoma development. Connexins, laminins and desmocollins have been implicated in the control of melanoma growth, where their reduced expression has been reported in metastatic lesions. OBJECTIVES: The aim of this study was to investigate connexin 31·1 (GJB5) expression and identify any association with BRAF mutational status, prognosis of patients with melanoma and mitogen-activated protein kinase (MAPK) inhibitor (MAPKi) treatment. METHODS: GJB5 expression was measured at RNA and protein level in melanoma clinical samples and established cell lines treated (or not) with BRAF and MEK inhibitors (MEKi), as well as in cell lines which developed MAPKi resistance. Findings were further validated and confirmed by analysis of independent datasets. RESULTS: Our analysis reveals significant downregulation of GJB5 expression in metastatic melanoma lesions compared with primary ones and in BRAF-mutated vs. BRAF-wildtype (BRAFWT ) melanomas. Likewise, GJB5 expression is significantly lower in BRAFV600E compared with BRAFWT cell lines and increases on MAPKi treatment. MAPKi-resistant melanoma cells display a similar expression pattern compared with BRAFWT cells, with increased GJB5 expression associated with morphological changes. Enhancement of BRAFV600E expression in BRAFWT melanoma cells significantly upregulates miR-335-5p expression with consequent downregulation of GJB5, one of its targets. Furthermore, overexpression of miR-335-5p in two BRAFWT cell lines confirms specific GJB5 protein downregulation. Reverse transcriptase quantitative polymerase chain reaction analysis also revealed upregulation of miR-335 in BRAFV600E melanoma cells, which is significantly downregulated in cells resistant to MEKi. Our data were further validated using the TCGA_SKCM dataset, where BRAF mutations associate with increased miR-335 expression and inversely correlate with GJB5 expression. In clinical samples, GJB5 underexpression is also associated with patient overall worse survival, especially at early stages. CONCLUSIONS: We identified a significant association between metastases/BRAF mutation and low GJB5 expression in melanoma. Our results identify a novel mechanism of gap-junctional protein regulation, suggesting a prognostic role for GJB5 in cutaneous melanoma.

High BRAF variant allele frequencies are associated with distinct pathological features and responsiveness to target therapy in melanoma patients.

BACKGROUND: BRAF mutant melanoma patients are commonly treated with anti-BRAF therapeutic strategies. However, many factors, including the percentage of BRAF-mutated cells, may contribute to the great variability in patient outcomes. PATIENTS AND METHODS: The BRAF variant allele frequency (VAF; defined as the percentage of mutated alleles) of primary and secondary melanoma lesions, obtained from 327 patients with different disease stages, was assessed by pyrosequencing. The BRAF mutation rate and VAF were then correlated with melanoma pathological features and patients' clinical characteristics. Kaplan-Meier curves were used to study the correlations between BRAF VAF, overall survival (OS), and progression-free survival (PFS) in a subset of 62 patients treated by anti-BRAF/anti-MEK therapy after metastatic progression. RESULTS: A highly heterogeneous BRAF VAF was identified (3%-90%). Besides being correlated with age, a higher BRAF VAF level was related to moderate lymphocytic infiltration (P = 0.017), to melanoma thickness according to Clark levels, (level V versus III, P = 0.004; level V versus IV, P = 0.04), to lymph node metastases rather than cutaneous (P = 0.04) or visceral (P = 0.03) secondary lesions. In particular, a BRAF VAF >25% was significantly associated with a favorable outcome in patients treated with the combination of anti-BRAF/anti-MEK drug (OS P = 0.04; PFS P = 0.019), retaining a significant value as an independent factor for the OS and the PFS in the multivariate analysis (P = 0.014 and P = 0.003, respectively). CONCLUSION: These results definitively support the role of the BRAF VAF as a potential prognostic and predictive biomarker in melanoma patients in the context of BRAF inhibition.

How liquid biopsies can change clinical practice in oncology.

Cell-free DNA fragments are shed into the bloodstream by tumor cells. The analysis of circulating tumor DNA (ctDNA), commonly known as liquid biopsy, can be exploited for a variety of clinical applications. ctDNA is being used to genotype solid cancers non-invasively, to track tumor dynamics and to detect the emergence of drug resistance. In a few settings, liquid biopsies have already entered clinical practice. For example, ctDNA is used to guide treatment in a subset of lung cancers. In this review, we discuss how recent improvements in the sensitivity and accuracy of ctDNA analyses have led to unprecedented advances in this research field. We further consider what is required for the routine deployment of liquid biopsies in the clinical diagnostic space. We pinpoint technical hurdles that liquid biopsies have yet to overcome, including preanalytical and analytical challenges. We foresee how liquid biopsies will transform clinical practice: by complementing (or replacing) imaging to monitor treatment response and by detecting minimal residual disease after surgery with curative intent.

Dissection of DLBCL microenvironment provides a gene expression-based predictor of survival applicable to formalin-fixed paraffin-embedded tissue.

Background: Gene expression profiling (GEP) studies recognized a prognostic role for tumor microenvironment (TME) in diffuse large B-cell lymphoma (DLBCL), but the routinely adoption of prognostic stromal signatures remains limited. Patients and methods: Here, we applied the computational method CIBERSORT to generate a 1028-gene matrix incorporating signatures of 17 immune and stromal cytotypes. Then, we carried out a deconvolution on publicly available GEP data of 482 untreated DLBCLs to reveal associations between clinical outcomes and proportions of putative tumor-infiltrating cell types. Forty-five genes related to peculiar prognostic cytotypes were selected and their expression digitally quantified by NanoString technology on a validation set of 175 formalin-fixed, paraffin-embedded DLBCLs from two randomized trials. Data from an unsupervised clustering analysis were used to build a model of clustering assignment, whose prognostic value was also assessed on an independent cohort of 40 cases. All tissue samples consisted of pretreatment biopsies of advanced-stage DLBCLs treated by comparable R-CHOP/R-CHOP-like regimens. Results: In silico analysis demonstrated that higher proportion of myofibroblasts (MFs), dendritic cells, and CD4+ T cells correlated with better outcomes and the expression of genes in our panel is associated with a risk of overall and progression-free survival. In a multivariate Cox model, the microenvironment genes retained high prognostic performance independently of the cell-of-origin (COO), and integration of the two prognosticators (COO + TME) improved survival prediction in both validation set and independent cohort. Moreover, the major contribution of MF-related genes to the panel and Gene Set Enrichment Analysis suggested a strong influence of extracellular matrix determinants in DLBCL biology. Conclusions: Our study identified new prognostic categories of DLBCL, providing an easy-to-apply gene panel that powerfully predicts patients' survival. Moreover, owing to its relationship with specific stromal and immune components, the panel may acquire a predictive relevance in clinical trials exploring new drugs with known impact on TME.

Digital PCR quantification of MGMT methylation refines prediction of clinical benefit from alkylating agents in glioblastoma and metastatic colorectal cancer.

BACKGROUND: O(6)-methyl-guanine-methyl-transferase (MGMT) silencing by promoter methylation may identify cancer patients responding to the alkylating agents dacarbazine or temozolomide. PATIENTS AND METHODS: We evaluated the prognostic and predictive value of MGMT methylation testing both in tumor and cell-free circulating DNA (cfDNA) from plasma samples using an ultra-sensitive two-step digital PCR technique (methyl-BEAMing). Results were compared with two established techniques, methylation-specific PCR (MSP) and Bs-pyrosequencing. RESULTS: Thresholds for MGMT methylated status for each technique were established in a training set of 98 glioblastoma (GBM) patients. The prognostic and the predictive value of MGMT methylated status was validated in a second cohort of 66 GBM patients treated with temozolomide in which methyl-BEAMing displayed a better specificity than the other techniques. Cutoff values of MGMT methylation specific for metastatic colorectal cancer (mCRC) tissue samples were established in a cohort of 60 patients treated with dacarbazine. In mCRC, both quantitative assays methyl-BEAMing and Bs-pyrosequencing outperformed MSP, providing better prediction of treatment response and improvement in progression-free survival (PFS) (P < 0.001). Ability of methyl-BEAMing to identify responding patients was validated in a cohort of 23 mCRC patients treated with temozolomide and preselected for MGMT methylated status according to MSP. In mCRC patients treated with dacarbazine, exploratory analysis of cfDNA by methyl-BEAMing showed that MGMT methylation was associated with better response and improved median PFS (P = 0.008). CONCLUSIONS: Methyl-BEAMing showed high reproducibility, specificity and sensitivity and was applicable to formalin-fixed paraffin-embedded tissues and cfDNA. This study supports the quantitative assessment of MGMT methylation for clinical purposes since it could refine prediction of response to alkylating agents.

Clinical and molecular features of attenuated adenomatous polyposis in northern Italy.

BACKGROUND: Attenuated familial adenomatous polyposis (AFAP) is characterized by the presence of 10-99 colorectal adenomas. The disease may be associated with mutations in either APC or MUTYH genes. We purposed to evaluate the contribution of adenomatous polyposis coli (APC) and MutY homologue (MUTYH) germline alterations to the AFAP phenotype and to identify genotype/phenotype correlations. METHODS: During counselling for familial adenomatous polyposis (FAP), 91 probands (and 107 affected individuals) who met the criteria of AFAP were identified. Eighty-two families were screened for constitutional mutations of the APC and MUTYH genes. RESULTS: MUTYH mutations were detected in 21 families (25.6 % of the 82 tested), and APC mutations in 7 (8.5 %). Overall, constitutional alterations were found in 34.1 % of the probands. Patients with APC mutations were younger at cancer onset and had a higher mean number of polyps (48.5 ± 33.0 in APC+ individuals vs. 35.7 ± 24.9 in MUTYH+ individuals, and 33.2 ± 18.4 in the "no mutation" group). Clinical features rendered the "no mutation" group closer to MUTYH+ than to the APC+ group. Colorectal cancer at diagnosis was detected in 40 % of AFAP individuals. CONCLUSIONS: AFAP is a new clinical entity with its frequency in the general population still undefined. The number of adenomas varies greatly, with an average of 30-40 lesions. The molecular basis of AFAP can be established in approximately 1/3 of the patients. Both MUTYH and APC genes are implicated in AFAP, though the role of MUTYH is of considerably greater relevance.

Malignant melanoma in patients with hereditary nonpolyposis colorectal cancer.

BACKGROUND: Malignant melanoma (MM) is the most aggressive skin cancer. Most MMs are sporadic, and in this setting an association with mismatch repair (MMR) gene mutations, typical of hereditary nonpolyposis colorectal cancer (HNPCC) tumours, has been proposed. OBJECTIVES: To characterize clinically and/or by molecular biology the patients with MM belonging to a cohort of 60 kindreds with HNPCC. Methods Patients with HNPCC with a diagnosis of MM were studied by immunohistochemistry (IHC) on tumour tissue using antibodies to MLH1, MSH2, p16, beta-catenin and E-cadherin, and by direct sequencing of MMR genes on germline DNA, and BRAF and NRAS on somatic DNA extracted from MM. RESULTS: Nine cutaneous MMs were detected in the tumour spectrum of eight families with HNPCC. The median age at diagnosis was 46 years. In one HNPCC family the diagnosis of MM was made in two first-degree relatives fitting the clinical definition of familial melanoma. IHC and sequencing analysis showed an MSH2 mutation in one patient with MM. CONCLUSIONS: Dermatological surveillance should be recommended to families in which MM is diagnosed in at least one member, especially at a young age. The combination of MMR gene mutations and abnormalities of p16 or other molecular pathways is needed to induce melanocytic carcinogenesis in a familial setting as well as in sporadic MM.

Impact of surgery on the development of duodenal cancer in patients with familial adenomatous polyposis.

PURPOSE: Precancerous duodenal lesions in patients with familial adenomatous polyposis can be detected with duodenoscopy and treatment may prevent the development of cancer. We proposed to determine the frequency, natural history, cumulative risk, and risk factors of the precancerous duodenal lesions in a series of patients diagnosed in northern Italy. METHODS: A prospective, endoscopic, follow-up protocol was performed in 50 patients examined by gastroduodenoscopy at two years of interval or less. The presence and severity of precancerous lesions of the duodenal mucosa were evaluated by Spigelman score. Twenty-five patients (50 percent) had proctocolectomy and ileoanal anastomosis, 15 (30 percent) had colectomy and ileorectal anastomosis, and 5 (10 percent) had proctocolectomy and definitive ileostomy from 0 to 3 years before the admission to the surveillance program. All patients showed more than a thousand adenomas in the colorectal mucosa. No patients with attenuated polyposis were found. RESULTS: At the first endoscopy, duodenal adenomas could be detected in 19 of 50 patients (38 percent), whereas at the end of the follow-up, 43 (86 percent) had duodenal lesions. The final mean Spigelman score increased during the follow-up period (P<0.001 respect to baseline values). No duodenal cancer could be detected. Eleven patients had or developed severe precancerous duodenal lesions (Stage IV) treated with endoscopic or surgical resection. The distribution of patients with Stage IV according to the surgery of the colon was: 2 of 25 treated with ileoanal anastomosis and 8 of 15 with ileorectal anastomosis (P=0.0024, Fisher's exact test). CONCLUSIONS: Patients with familial adenomatous polyposis are at risk of significant neoplasia. The natural history of precancerous lesions might be related to surgical treatment of colorectal neoplasms.

Prognostic significance of loss of heterozygosity at loci on chromosome 17p13.3-ter in sporadic breast cancer is evidence for a putative tumour suppressor gene.

Several studies indicate that the short arm of chromosome 17 is one of the most frequently altered regions in sporadic breast carcinomas (45-60%). In the present report the 17p13.3-ter locus in tumour DNA of breast cancer patients, along with their matching normal lymphocyte DNA, have been mapped with four markers (D17S5, D17S379, ABR and D17S34), spanning nearly 3 cM of the telomer. Sixty-five of 143 heterozygous tumours had lost at least one of the markers at the minimum region of loss (45%). High levels of loss of these distal markers on 17p13.3 are independent of TP53 mutations and are associated with tumour cell proliferation. A follow-up period of over 7 years demonstrates that loss of these markers correlates both with disease-free (P = 0.004) and overall survival (P = 0.007). In addition we show that for disease-free survival the prognostic power of this genetic alteration is second only to axillary lymph node involvement (3.1 vs 6.3 relative risk), and is a better predictor than the mutational status of TP53 (1.6 relative risk). Our results are further evidence of the presence, within the region, of at least a second tumour suppressor gene distal to TP53, that might be targeted by deletions.

The human ROX gene: genomic structure and mutation analysis in human breast tumors.

We have recently isolated a human gene, ROX, encoding a new member of the basic helix-loop-helix leucine zipper protein family. ROX is capable of heterodimerizing with Max and acts as a transcriptional repressor in an E-box-driven reporter gene system, while it was found to activate transcription in HeLa cells. ROX expression levels vary during the cell cycle, being down-regulated in proliferating cells. These biological properties of ROX suggest a possible involvement of this gene in cell proliferation and differentiation. The ROX gene maps to chromosome 17p13.3, a region frequently deleted in human malignancies. Here we report the genomic structure of the human ROX gene, which is composed of six exons and spans a genomic region of less than 40 kb. In an attempt to identify possible inactivating mutations in the ROX gene in human breast cancer, we performed a single-strand conformation polymorphism analysis of its coding region in 16 sporadic breast carcinomas showing loss of heterozygosity in the 17p13.3 region. No mutations were found in this analysis. Five nucleotide polymorphisms were identified in the ROX gene, three of which caused an amino acid substitution. These nucleotide changes were present in the peripheral blood DNAs of both the patients and the control individuals. In vitro translated assays did not show a significant decrease in the ability of the ROX mutant proteins to bind DNA or to repress transcription of a driven reporter gene in HEK293 cells. Despite experimental evidence that ROX might act as a tumor suppressor gene, our data suggest that mutations in the coding region of ROX are uncommon in human breast tumorigenesis.

High levels of loss at the 17p telomere suggest the close proximity of a tumour suppressor.

High levels of loss of distal markers on 17p13.3 in breast cancer suggested the presence within the region of at least one tumour-suppressor gene. Here we describe the derivation of two biallelic polymorphisms from the 17p telomeric yeast artificial chromosome (YAC) TYAC98. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and multiplex PCR analysis demonstrated that the high level of allelic imbalance observed in breast tumours represented loss of constitutional heterozygosity (LOH) and that this LOH extended to the telomere. Lung carcinoma (but not Wilms' tumour)-derived DNA again revealed a high level of loss of subtelomeric 17p sequences. Telomeric microsatellite polymorphisms from other chromosome arms did not show such elevated loss in either tumour type. This suggested that the 17p loss observed did not reflect a general telomeric instability and provided further evidence for the presence of a breast cancer tumour-suppressor gene in the distal region of 17p13.3.

Detailed mapping and loss of heterozygosity analysis suggests a suppressor locus involved in sporadic breast cancer within a distal region of chromosome band 17p13.3.

The chromosome region 17p13.3 is thought to encode a tumour suppressor gene involved in sporadic breast cancer and other malignancies. Physical ordering of markers has been carried out by a series of multicolour fluorescent in situ hybridisation (FISH) experiments, using isolated yeast artificial chromosomes (YACs) and cosmids. Eight polymorphic markers ordered within this new physical map and one external marker were used to investigate the pattern of loss of heterozygosity in a panel of 40 sporadic breast tumour patients. The data revealed a region of high loss (60%) within distal 17p13.3, defined by markers D17S926, D17S695 and D17S849 which mapped close together. A contig of YACs was constructed physically linking these three markers.

Evidence for a second tumor suppressor gene on 17p linked to high S-phase index in primary human breast carcinomas.

The short area of chromosome 17 is a frequent target for deletions in human tumors, including breast cancer. We have investigated by restriction fragment polymorphism analysis the pattern of loss of heterozygosity (LOH) at four loci on 17p13.1-17pter in a panel of 110 primary human breast carcinomas. A copy of the p53 gene was lost in 23% of the informative cases. Point mutations in the p53 gene were statistically associated with LOH at the same locus (p = 0.003) but not at other loci on 17p13.3-17pter. A second region bordered by the loci D17S5/D17S28 (17p13.3) and D17S34 (17pter) is also affected by LOH, independent of point mutations in the p53 gene. We propose the presence of a second tumor suppressor gene within this region. In support of this hypothesis is the significant association (p = 0.005) between LOH at the D17S5/D17S28, but not at the TP53 or D17S34 loci, and tumors having a high S-phase index.

Growth suppression of normal mammary epithelial cells by wild-type p53.

p53 mutations are frequent in human breast cancer. In order to understand the role of p53 in the context of the accumulation of mutations in breast cancer, a model of non transformed mammary cells was sought. The HC11 cells are immortalized, non transformed rodent mammary epithelial cells which synthesize milk proteins following stimulation with lactogenic hormones. p53 protein was readily detected in HC11 protein extracts with the PAb421 antibody. Two mutations were identified in the p53 cDNA from HC11 cells: a missense mutation at codon 138, substituting Trp for Cys, and a microdeletion, codon 123 to 130, of exon 5. The latter results from an intronic mutation of the splice acceptor site at the intron 4/exon 5 junction. The mutations affect separate p53 alleles, and no wt allele was found. Wt p53 was introduced into HC11 cells by means of a retroviral vector, under the control of a Cd(++)-inducible promoter. In the presence of CdSO4 a dramatic growth inhibition was observed. A temperature-sensitive mutant p53 gene was also transfected into HC11 cells. This resulted in a marked inhibition of cells growth at 32 degrees C, when the p53 is in the wt conformation, while no effect was observed at 37 degrees C, when the mutant conformation is predominant. wt p53-mediated inhibition of monolayer growth does not involve induction of programmed cell death and does not activate de novo synthesis of differentiation-specific milk proteins. We conclude that mutations in the p53 gene likely played a role in their immortalization. The HC11 cells provide a model for assessing the cooperative action of other mutations in mammary tumorigenesis.

The effect of fgf-3 int-2 on growth and transformation of mcf-10a normal human mammary epithelial-cells is distinct from fgf-1 and fgf-2.

Fibroblast growth factors (FGFs) are related polypeptides with mitogenic activity on cells of mesodermal and neuroectodermal origin. The Fgf-3 gene shares high homology with FGF-2 and its protein product can substitute FGF-2 as a growth factor. Other observations, however, indicate that Fgf-3 has specialized functions. We have investigated the effect of the expression and secretion of Fgf-3 on the growth and transformation of the human breast epithelial cell line MCF-10A. Overexpression of Fgf-3 stimulates proliferation of these cells in serum-free medium and induces anchorage-independent colony formation in soft agar. In contrast, these effects were not observed with purified FGF-1 and FGF-2 on either the parental or the Fgf-3-MCF-10A cells. Thus, Fgf-3 is distinct from FGF-1 and FGF-2 for its ability to induce cell proliferation and transformation of MCF-10A cells. This difference could be due, at least in part, to the expression of a specific set of FGF receptors with higher affinity for FGF-3 than FGF-1 or FGF-2.

Growth suppression of normal mammary epithelial cells by wild-type p53.

We examined the status of the p53 gene in the HC11 normal mammary epithelial cells. Two mutations were identified: a Cys to Trp change at codon 138 and a microdeletion of codon 123 to 130 resulting from mutation of the splice acceptor site. These two mutations were independent, and no wild-type p53 allele was found. Introduction of wt-p53 strongly inhibited growth in monolayer. Thus, the absence of wt-p53 can be sufficient for the immortalization of mammary cells.

Genetic and molecular heterogeneity of breast cancer cells.

We have undertaken a systematic study of primary human breast tumor DNAs to identify and characterize frequently occurring somatic mutations. Loss of heterozygosity (LOH) was found on chromosomes 1p (37%), 1q (20%), 3p (30%), 7 (41%), 13q (30%), 17p (49%), 17q (29%) and 18q (34%) in our tumor DNA panel. Specific subsets of tumors could be defined based on the particular collection of mutations they contained. One goal of these studies has been to determine whether there is a significant association between specific mutations and clinical parameters of the disease. We have found that LOH on chromosome 17p in tumor DNAs is associated with breast tumors having a high proliferative index and that LOH on chromosome 7 is associated with patients having a poor prognosis. Our analysis of chromosome 17 suggests that there may be as many as four tumor suppressor genes affected in primary human breast tumors.