RNA sequencing reveals PNN and KCNQ1OT1 as predictive biomarkers of clinical outcome in stage III colorectal cancer patients treated with adjuvant chemotherapy.

Five-year overall survival of stage III colorectal cancer (CRC) patients treated with standard adjuvant chemotherapy (ACHT) is highly variable. Genomic biomarkers and/or transcriptomic profiles identified lack of adequate validation. Aim of our study was to identify and validate molecular biomarkers predictive of ACHT response in stage III CRC patients by a transcriptomic approach. From a series of CRC patients who received ACHT, two stage III extreme cohorts (unfavorable vs. favorable prognosis) were selected. RNA-sequencing was performed from fresh frozen explants. Tumors were characterized for somatic mutations. Validation was performed in stage III CRC patients extracted from two GEO datasets. According to disease-free survival (DFS), 108 differentially expressed genes (104/4 up/downregulated in the unfavorable prognosis group) were identified. Among 104 upregulated genes, 42 belonged to olfactory signaling pathways, 62 were classified as pseudogenes (n = 17), uncharacterized noncoding RNA (n = 10), immune response genes (n = 4), microRNA (n = 1), cancer-related genes (n = 14) and cancer-unrelated genes (n = 16). Three out of four down-regulated genes were cancer-related. Mutational status (i.e., RAS, BRAF, PIK3CA) did not differ among the cohorts. In the validation cohort, multivariate analysis showed high PNN and KCNQ1OT1 expression predictive of shorter DFS in ACHT treated patients (p = 0.018 and p = 0.014, respectively); no difference was observed in untreated patients. This is the first study that identifies by a transcriptomic approach and validates PNN and KCNQ1OT1 as molecular biomarkers predictive of chemotherapy response in stage III CRC patients. After a further validation in an independent cohort, PNN and KCNQ1OT1 evaluation could be proposed to prospectively identify stage III CRC patients benefiting from ACHT.

Personalized laboratory medicine: a patient-centered future approach.

In contrast to population-based medical decision making, which emphasizes the use of evidence-based treatment strategies for groups of patients, personalized medicine is based on optimizing treatment at the level of the individual patient. The creation of molecular profiles of individual patients was made possible by the advent of "omics" technologies, based on high throughput instrumental techniques in combination with biostatistics tools and artificial intelligence. The goal of personalized laboratory medicine is to use advanced technologies in the process of preventive, curative or palliative patient management. Personalized medicine does not rely on changes in concentration of a single molecular marker to make a therapeutic decision, but rather on changes of a profile of markers characterizing an individual patient's status, taking into account not only the expected response to treatment of the disease but also the expected response of the patient. Such medical approach promises a more effective diagnostics with more effective and safer treatment, as well as faster recovery and restoration of health and improved cost effectiveness. The laboratory medicine profession is aware of its key role in personalized medicine, but to empower the laboratories, at least an enhancement in cooperation between disciplines within laboratory medicine will be necessary.

Phenotypic and molecular differences between giant-cell tumour of soft tissue and its bone counterpart.

AIMS: Giant-cell tumour (GCT) of soft tissue (GCT-ST) is a primary soft tissue neoplasm that is histologically similar to GCT of bone (GCT-B). Recently, it has been reported that >90% of GCT-Bs have a driver mutation in the H3F3A gene. As the relationship between GCT-ST and GCT-B is unclear, the aim of this study was to compare a series of GCT-STs and GCT-Bs with regard to the presence of H3F3A mutations and several immunophenotypic markers. METHODS AND RESULTS: Eight GCT-STs were retrieved from our institutional archives. Fifteen GCT-Bs served as controls. Direct sequencing for H3F3A mutations in coding regions between codons 1 and 42, including the hotspot codons (28, 35, and 37), was performed on DNA extracted from formalin-fixed paraffin-embedded tissue. Tumours were studied immunohistochemically for the expression of CD14, CD33, RANKL, RANK, p63, and the osteoblastic markers SATB2 and RUNX2. None of the seven GCT-STs that could be analysed showed H3F3A mutations, whereas 14 GCT-Bs (93.3%) were mutated. All eight GCT-STs were positive for RANK and RUNX2, whereas RANKL and SATB2 were detected in only two cases (25%). CD14 was detected only in mononuclear elements, whereas multinucleated giant cells and a proportion of the mononuclear population expressed CD33. Few mononuclear cells of GCT-STs expressed p63. In comparison, GCT-Bs showed higher expression of p63 (14 of 15 cases with >50% of positive mononuclear cells), RANKL, and SATB2, whereas CD14, CD33, RANK and RUNX2 were similarly expressed. CONCLUSIONS: Although GCT-ST and GCT-B are similar in histological appearance, our results indicate that they are immunophenotypically and genetically distinct.

Immunohistochemistry is highly sensitive and specific for the detection of NRASQ61R mutation in melanoma.

Testing for NRAS is now integral part in the assessment of metastatic melanoma patients because there is evidence that NRAS-mutated patients may be sensitive to MEK inhibitors, and RAS mutation is a common mechanism of acquired resistance during treatment with BRAF inhibitors. This study evaluated the sensitivity and specificity of immunohistochemical analysis using an N-Ras (Q61R) antibody to detect the presence of the NRASQ61R mutation in melanoma patients. A total of 98 primary cutaneous melanomas that have undergone examination of NRAS mutation were retrieved from a multicentric database. Formalin-fixed and paraffin-embedded melanoma tissues were analyzed for BRAF and NRAS mutations by independent, blinded observers using both conventional DNA molecular techniques and immunohistochemistry with the novel anti-human N-Ras (Q61R) monoclonal antibody (clone SP174). The antibody showed a sensitivity of 100% (14/14) and a specificity of 100% (83/83) for detecting the presence of an NRASQ61R mutation. Of the NRAS-mutated cases, none of the non-Q61R cases stained positive with the antibody (0/7). There were three cases with discordant NRAS mutational results. Additional molecular analysis confirmed the immunohistochemically obtained NRAS result in all cases, suggesting that a multiple analytical approach can be required to reach the correct sample classification. The reported immunohistochemical method is an accurate, rapid, and cost-effective method for detecting NRASQ61R mutation in melanoma patients, and represents a valuable supplement to traditional mutation testing. If validated in further studies, genetic testing would only be required for immunohistochemistry-negative patients to detect non-Q61R mutations.

Early changes in circulating tumor DNA (ctDNA) predict treatment response in metastatic KRAS-mutated colorectal cancer (mCRC) patients.

The detection of RAS mutations and co-mutations in liquid biopsy offers a novel paradigm for the dynamic management of metastatic colorectal cancer (mCRC) patients. Expanding the results of the prospective OMITERC (OMIcs application from solid to liquid biopsy for a personalized ThERapy of Cancer) project, we collected blood samples at specific time points from patients who received a first-line chemotherapy (CT) for KRAS-mutated mCRC. CTC quantification was performed by CellSearch® system. Libraries from cfDNA were prepared using the Oncomine™ Colon cfDNA Assay to detect tumour-derived DNA in cfDNA. The analysis involved >240 hotspots in 14 genes. Twenty patients with KRAS-mutated mCRC treated at the Medical Oncology Unit of Careggi University Hospital were prospectively enrolled. Nine patients had available data for longitudinal monitoring of cfDNA. After 6 weeks of first-line CT an increase of KRAS-mutated clone was reported in the only patient who did not obtain disease control, while all patients with decrease of KRAS clones obtained disease control. Overall, in patients with a short (<9 months) progression-free survival (PFS) we registered, at 6 weeks, an increase in cfDNA levels and in KRAS mutations or other co-mutations, i.e. PIK3CA, FBXW7, GNAS, and TP53. In selected cases, co-mutations were able to better anticipate radiological progressive disease (PD) than the increase of KRAS-mutated clones. In conclusion, our study confirms plasma ctDNA as a crucial tool for anticipating PD at an early time point and highlights the value of a comprehensive assessment of clonal dynamics to improve the management of patients with mCRC.

Genetic variants in miR-146a, miR-149, miR-196a2, miR-499 and their influence on relative expression in lung cancers.

BACKGROUND: The presence of sequence variants in miRNA genes may influence their processing, expression and binding to target mRNAs. Since single miRNA can have a large number of potential mRNA targets, even minor variations in its expression can have influences on hundreds of putative mRNAs. METHODS: Here, we evaluated 101 paired samples (cancer and normal tissues) from non-small cell lung carcinoma (NSCLC) patients to study the genotype distribution of single nucleotide polymorphisms (SNPs) in miR-146a (rs2910164 C-G), miR-149 (rs2292832 C-T), miR-196a2 (rs11614913 C-T) and miR-499 (rs3746444 G-A) and their influence on the expression of respective miRNAs. RESULTS: Relative expression of miR-146a, miR-149 and miR-499 were comparable in NSCLC and in paired control tissues. On the contrary, we clearly detected a significant increase (p<0.001) of miR-196a2 expression in NSCLC. In particular we found a significant association between miR-196a2 CC genotype and high expression, whereas TT geno-type showed a very low expression in comparison to both CT (p<0.005) and CC patients (p<0.01). We did not find any association between miR-149, miR-196a2 and miR-499 genotype and risk of NSCLC. Conversely, CG genotype of miR-146a appeared associated to an increased risk for NSCLC (p=0.042 and 1.77 OR). CONCLUSIONS: Our results seem to demonstrate that sequence variants of miR-196a2 can have an influence on its expression, while miR-146a can have a role in increasing the risk of NSCLC.

Atypical Spitzoid melanocytic tumors: a morphological, mutational, and FISH analysis.

BACKGROUND: Identification of the clinical behavior of atypical Spitzoid tumors with conflicting histopathologic features remains controversial. OBJECTIVE: We sought to assess whether molecular findings may be helpful in the diagnostic and prognostic assessment of atypical Spitzoid tumors. METHODS: A total of 38 controversial, atypical Spitzoid lesions (≥ 1 mm in thickness) were analyzed for clinicopathological features, chromosomal alterations by fluorescence in situ hybridization (FISH) analysis (RREB1/MYB/CCND1/CEP6), BRAF(V600E) mutation by allele-specific real-time polymerase chain reaction confirmed by sequencing, and H-RAS gene mutation by direct sequencing. RESULTS: Atypical Spitzoid lesions developed in 21 female and 17 male patients (mean age 22 years). Nine patients underwent sentinel lymph node biopsy and a sentinel lymph node micrometastasis was detected in 4 of these 9 cases. Four additional patients, who did not receive a sentinel lymph node biopsy, experienced bulky lymph node metastases and one experienced visceral metastases and death. Lesions from patients with lymph node involvement showed more deep mitoses (P < .01), less inflammation (P = .05), and more plasma cells (P = .04). FISH analysis demonstrated the presence of chromosomal alterations in 6 of 25 cases. Correlation with follow-up data showed that the only case with fatal outcome showed multiple chromosomal alterations by FISH analysis. BRAF(V600E) mutation was detected in 12 of 16 cases (75%) and H-RAS mutation on exon 3 was found in 3 of 11 cases (27%). LIMITATIONS: Our results require validation in a larger series with longer follow-up information. CONCLUSIONS: FISH assay may be of help in the prognostic evaluation of atypical Spitzoid tumors. Diagnostic significance of BRAF(V600E) and H-RAS mutations in this setting remains unclear.

From clinical trials to clinical practice: predictors of response to erlotinib in advanced non-small cell lung cancer patients pretreated with chemotherapy.

BACKGROUND: Inhibition of the epidermal growth factor receptor pathway with tyrosine kinase inhibitors can improve outcome of patients with advanced non-small cell lung cancer after first-line chemotherapy. The use of clinical characteristics and molecular markers may permit the identification of patients who are more likely to benefit from erlotinib. PATIENTS AND METHODS: Retrospective analysis of unselected patients with metastatic non-small cell lung cancer who had previously failed on at least one line of chemotherapy and treated at our institution with erlotinib (150 mg/day orally) until disease progression. Mutations of epidermal growth factor receptor (exon 19-21) and KRAS (codon 12-13) genes were screened with high-resolution melting analysis and identified with direct sequencing. RESULTS: Fifty-three patients were included in the study. The disease control rate was 38%. Median progression-free survival and median overall survival were 4 and 15 months, respectively. Skin rash, diarrhea and mucositis were the most common toxicities of erlotinib. In 19 patients, erlotinib dose was reduced for toxicity. The disease control rate and progression-free survival were significantly better in non-smokers, responders to chemotherapy and patients with epidermal growth factor receptor mutations. Overall survival was longer in patients with skin toxicity and epidermal growth factor receptor mutations. CONCLUSIONS: In our experience, epidermal growth factor receptor mutations, response to previous chemotherapy and non-smoking status were predictors of higher disease control rate and longer progression-free survival. Overall survival was significantly longer in patients with epidermal growth factor receptor mutations and skin toxicity.

Seladin-1 expression is regulated by promoter methylation in adrenal cancer.

BACKGROUND: Seladin-1 overexpression exerts a protective mechanism against apoptosis. Seladin-1 mRNA is variably expressed in normal human tissues. Adrenal glands show the highest levels of seladin-1 expression, which are significantly reduced in adrenal carcinomas (ACC). Since up to now seladin-1 mutations were not described, we investigated whether promoter methylation could account for the down-regulation of seladin-1 expression in ACC. METHODS: A methylation sensitive site was identified in the seladin-1 gene. We treated DNA extracted from two ACC cell lines (H295R and SW13) with the demethylating agent 5-Aza-2-deoxycytidine (5-Aza). Furthermore, to evaluate the presence of an epigenetic regulation also 'in vivo', seladin-1 methylation and its mRNA expression were measured in 9 ACC and in 5 normal adrenal glands. RESULTS: The treatment of cell lines with 5-Aza induced a significant increase of seladin-1 mRNA expression in H295R (fold increase, F.I. = 1.8; p = 0.02) and SW13 (F.I. = 2.9; p = 0.03). In ACC, methylation density of seladin-1 promoter was higher (2682 +/- 686) than in normal adrenal glands (362 +/- 97; p = 0.02). Seladin-1 mRNA expression in ACC (1452 +/- 196) was significantly lower than in normal adrenal glands (3614 +/- 949; p = 0.01). CONCLUSION: On this basis, methylation could be involved in the altered pattern of seladin-1 gene expression in ACC.

The pre-analytical phase of the liquid biopsy.

The term 'liquid biopsy', introduced in 2013 in reference to the analysis of circulating tumour cells (CTCs) in cancer patients, was extended to cell-free nucleic acids (cfNAs) circulating in blood and other body fluids. CTCs and cfNAs are now considered diagnostic and prognostic markers, used as surrogate materials for the molecular characterisation of solid tumours, in particular for research on tumour-specific or actionable somatic mutations. Molecular characterisation of cfNAs and CTCs (especially at the single cell level) is technically challenging, requiring highly sensitive and specific methods and/or multi-step processes. The analysis of the liquid biopsy relies on a plethora of methods whose standardisation cannot be accomplished without disclosing criticisms related to the pre-analytical phase. Thus, pre-analytical factors potentially influencing downstream cellular and molecular analyses must be considered in order to translate the liquid biopsy approach into clinical practice. The present review summarises the most recent reports in this field, discussing the main pre-analytical aspects related to CTCs, cfNAs and exosomes in blood samples for liquid biopsy analysis. A short discussion on non-blood liquid biopsy samples is also included.

Analytical Evaluation of an NGS Testing Method for Routine Molecular Diagnostics on Melanoma Formalin-Fixed, Paraffin-Embedded Tumor-Derived DNA.

Next Generation Sequencing (NGS) is a promising tool for the improvement of tumor molecular profiling in view of the identification of a personalized treatment in oncologic patients. To verify the potentiality of a targeted NGS (Ion AmpliSeq™ Cancer Hotspot Panel v2), selected melanoma samples (n = 21) were retrospectively analyzed on S5 platform in order to compare NGS performance with the conventional techniques adopted in our routine clinical setting (Sequenom MassARRAY system, Sanger sequencing, allele-specific real-time PCR). The capability in the identification of rare and low-frequency mutations in the main genes involved in melanoma (BRAF and NRAS genes) was verified and integrated with the results deriving from other oncogenes and tumor suppressor genes. The analytical evaluation was carried out by the analysis of DNA derived from control cell lines and FFPE (Formalin-Fixed, Paraffin-Embedded) samples to verify that the achieved resolution of uncommon mutations and low-frequency variants was suitable to meet the technical and clinical requests. Our results demonstrate that the amplicon-based NGS approach can reach the sensitivity proper of the allele-specific assays together with the high specificity of a sequencing method. An overall concordance among the tested methods was observed in the identification of classical and uncommon mutations. The assessment of the quality parameters and the comparison with the orthogonal methods suggest that the NGS method could be implemented in the clinical setting for melanoma molecular characterization.

Unbalanced GLA mRNAs ratio quantified by real-time PCR in Fabry patients' fibroblasts results in Fabry disease.

Total or partial deficiency of the human lysosomal hydrolase alpha-galactosidase A is responsible for Fabry disease, the X-linked inborn error of glycosphingolipid metabolism. Together with the predominant alpha-galactosidase A gene mRNA product encoding the lysosomal enzyme, a weakly regulated alternatively spliced alpha-galactosidase A transcript is expressed in normal tissues, but its overexpression, due to the intronic g.9331G>A mutation, leads to the cardiac variant. We report the molecular characterization of five Fabry patients including two siblings. Sequencing analysis of the alpha-galactosidase A gene coding region and intron/exon boundaries identified the new c.124A>G (p.M42V) genetic lesion as well as a known deletion in three patients, whereas in the two remaining patients, no mutations were identified. To evaluate possible alpha-galactosidase A gene transcription alterations, both predominant and alternatively spliced mRNAs were quantified by absolute real-time PCR on total RNA preparations from the patients' fibroblasts. An impressive reduction in the predominant alpha-galactosidase A transcript was detected in the last patients (Pt 4 and Pt 5). However, the alternatively spliced mRNA was dramatically overexpressed in one of them, carrying a new intronic lesion (g.9273C>T). These findings strongly suggest a correlation between this new intronic mutation and the unbalanced alpha-galactosidase A mRNAs ratio, which could therefore be responsible for the reduced enzyme activity causing Fabry disease. The real-time assay developed here to investigate the two alpha-galactosidase A mRNAs might play a crucial role in revealing possible genetic lesions and in confirming the pathogenetic mechanisms underlying Fabry disease.

Uncommon clinical presentations of pheochromocytoma and paraganglioma in two different patients affected by two distinct novel VHL germline mutations.

CONTEXT: The von Hippel-Lindau (VHL) syndrome is an inherited multitumour disorder characterized by clinical heterogeneity and high penetrance. Pheochromocytoma (Pheo) is present in 10%-15% of cases and can be isolated or associated with other lesions such as haemangioblastomas, kidney cysts or cancer and pancreatic lesions. In VHL patients, Pheos generally secrete norepinephrine and are located in the adrenals. Extra-adrenal Pheos (paragangliomas, PGLs) are rare. OBJECTIVE: While performing genetic testing in patients affected by apparently sporadic Pheos or PGLs, we found two novel different VHL germline mutations in two females who presented with two distinct very uncommon clinical pictures. One patient was studied for the presence of an adrenal incidentaloma and the other for the presence of a neck tumour. METHODS AND RESULTS: Patients coding regions and exon-intron boundaries of RET (exons 10, 11, 13-15), VHL, SDHD, SDHB and SDHC genes were amplified and sequenced. We identified two novel VHL point mutations: a L198V missense mutation in a 32-year-old female affected by a right adrenal compound and mixed tumour constituted by an epinephrine secreting Pheo, a ganglioneuroma and an adrenocortical adenoma, and a T152I missense mutation in a 24-year-old female affected by a left carotid body tumour. No other lesions were found in the patients or in the VHL mutation positive relatives. CONCLUSIONS: These cases enlarge the list of VHL mutations and add new insights in the clinical variability of VHL disease, thus confirming the importance of genetic testing in patients affected by apparently sporadic Pheos or PGLs.

High-resolution melting analysis for rapid detection of KRAS, BRAF, and PIK3CA gene mutations in colorectal cancer.

High-resolution melting analysis (HRMA) provides a valid approach to efficiently detect DNA genetic and somatic mutations. In this study, HRMA was used for the screening of 116 colorectal cancers (CRCs) to detect hot-spot mutations in the KRAS and BRAF oncogenes. Mutational hot spots on the PIK3CA gene, exons 9 and 20, were also screened. Direct sequencing was used to confirm and characterize HRMA results. HRMA revealed abnormal melting profiles in 65 CRCs (56.0%), 16 of them harboring mutations in 2 different genes simultaneously. The frequency of mutations was 17.2% for PIK3CA (11.2% in exon 9 and 6.0% in exon 20), 43.1% for KRAS exon 2, and 9.5% in exon 15 of the BRAF gene. We found a significant association between PIK3CA and KRAS mutations (P = .008), whereas KRAS and BRAF mutations were mutually exclusive (P = .001). This report describes a novel approach for the detection of PIK3CA somatic mutations by HRMA.

GM1 gangliosidosis: molecular analysis of nine patients and development of an RT-PCR assay for GLB1 gene expression profiling.

The human GLB1 gene produces two alternatively spliced transcripts that encode the lysosomal enzyme beta-galactosidase (GLB1) and the elastin binding protein (EBP). Mutations at the GLB1 locus, which are responsible for the storage disorder GM1 gangliosidosis, may affect either both proteins or GLB1 only. The EBP, when affected, contributes to specific features of GM1 gangliosidosis patients, such as cardiomyopathy and connective-tissue abnormalities. Here we report the development of reliable and quantitative assays based on real-time PCR for assessing the levels of GLB1 and EBP transcripts in patients' samples. We also report the characterisation of GLB1 gene mutations in nine GM1 gangliosidosis patients in order to correlate the genetic lesions with mRNA levels and phenotypes. Mutation analysis identified four new (c.1835_1836delCC; p.Arg148Cys; c.1068+1G>T; and p.Pro549Leu), five known (p.Arg59His; p.Arg201His; p.Gly123Arg; c.245+1G>A; and c.75+2insT) mutations and one new polymorphism (c.1233+8T>C). Comparative analysis of the patients' phenotypes enabled a more thorough correlation between GLB1 mutations and specific clinical manifestations. GLB1 and EBP mRNA levels were both reduced in three patients carrying the splicing defects. The accurate and fast method for the detection of alternatively spliced transcripts of the GLB1 gene could be applied to other disease-causing lysosomal genes that encode multiple mRNAs.

Influence of 17q gain and promoter polymorphisms on mRNA expression of somatostatin receptor type 2 in neuroblastoma.

BACKGROUND: Neuroblastoma, the most frequent solid extracranial tumor in children, is characterized by a wide spectrum of clinical behaviours. We previously reported that high expression of somatostatin receptor type-2 (sst2) mRNA is associated to increased overall and event free survival. Several genetic abnormalities are detected in neuroblastomas, frequently involving balanced and/or unbalanced gain on the long arm on chromosome 17, the same region containing sst2 gene. METHODS: In this study we detected balanced and/or unbalanced 17q gain in 50 neuroblastomas. Since two polymorphisms in sst2 promoter (-57 C>G and -83 A>G) were previously described as responsible for an in vitro reduction of sst2 mRNA expression, promoter sequencing was also performed in the same samples. The results were compared to sst2 mRNA expression, measured by real-time RT-PCR. RESULTS: The frequency of 17q gain (14/50 neuroblastomas) was significantly associated to sst2 mRNA over-expression (Fischer's exact test: p=0.0012). The sst2 expression was significant higher both in balance and unbalance 17q amplifications (ANOVA: p=0.04). Conversely, we found a reduction of sst2 mRNA in neuroblastomas with -57 C>G promoter polymorphism (ANOVA: p=0.03). CONCLUSION: We highlighted that 17q gain and promoter polymorphisms can play a role into the regulation of sst2 expression in neuroblastomas.

Clodronate acts on human osteoclastic cell proliferation, differentiation and function in a bioreversible manner.

Background. Clodronate is used in high bone resorption diseases. Its action was defined as "cytotoxic" based on the induced cellular ATP loss, without any experimental verification of reversibility. In the present report the reversibility of clodronate action was tested on cultured human osteoclastic cell cultures. As "in vitro" bioeffects of clodronate are reversible, this compound should not be defined as "cytotoxic".Introduction. Bisphosphonates are pyrophosphate analogs able to inhibit osteoclast-mediated bone resorption widely used in the treatment of diseases with high bone turnover. Several evidences have shown that bisphosphonates can be divided into two groups with distinct molecular mechanisms of action depending on the nature of the R(2)side chain. The nitrogen-containing bisphosphonates act on osteoclasts by preventing protein prenylation, while non-nitrogen-containing bisphosphonates, like clodronate, are metabolized intracellularly to a ß-γ-methylene analog of ATP that induces inhibition of the ADP/ATP translocase.Materials and Methods. In order to evaluate clodronate effects on osteoclastic cells and the bioreversibility of its action, we have used a human preosteoclastic (FLG 29.1) cell line and primary cultures of human osteoclast-like (HOC) cells. Functional and differentiative modifications were evaluated with immunocytochemical tartrate-resistant acid phosphatase activity (TRAcP) assay and with rapid quantitative detection of the complex "matrix metalloproteinase 9/tissue inhibitor of metalloproteinase" (MMP9/TIMP1) by RT-PCR analysis based on "TaqMan" technology. The apoptosis phenomenon were detected by DNA ladder analysis and quantified by counting apoptotic cells with Transmission Electron Microscopy (TEM) analysis. Adenosine-5'-[ ß - γ -dichloromethylene] triphosphate (AppCCl(2)p) was detected and identified in cell extract by HPLC-ESI-MS-MS Mass Spectrometry. Intracellular ATP modulation in the presence of clodronate was evaluated by luciferin-luciferase assay. The Mann-Whitney "U" test was conducted for statistical analysis.Results. We found that clodronate inhibited both proliferation and differentiative features of cells of the osteoclastic lineage. Furthermore, treatment of both cell types with clodronate caused apoptosis, generation of measurable levels of AppCCl(2)p, and reduction of intracellular ATP levels. Addition of ATP to the culture medium caused an inhibition of the biological actions of clodronate on the human osteoclastic cell lineage.Conclusions. These data indicate that intracellular accumulation of the metabolite AppCCl(2)p is the likely route by which clodronate inhibits osteoclastic function and this effect is reversed by ATP.

Histone 3.3 mutations in giant cell tumor and giant cell-rich sarcomas of bone.

Mutually exclusive histone 3.3 gene mutations have been recognized in chondroblastoma and giant cell tumor of bone (GCTB), which may be useful for differential diagnostic purposes in morphologically ambiguous cases. Although more than 90% of GCTBs present histone 3.3 variants exclusively in the H3F3A gene, chondroblastoma is mutated mainly in H3F3B. In this study, we examined a series of giant cell-rich primary bone tumors, aiming to evaluate the possible diagnostic role of histone 3.3 mutations in the differential diagnosis between GCTB and giant cell-rich sarcomas. Sixteen cases of nonmetastatic GCTB, 9 GCTBs with lung metastases, and 35 giant cell-rich sarcomas were selected from our institutional archives. Eight chondroblastomas were used as controls. Direct sequencing for the presence of H3F3A and H3F3B variants in coding region between codons 1 and 42, including the hotspot codons (28, 35, and 37), was performed on DNA extracted from formalin-fixed, paraffin-embedded tissue using conventional polymerase chain reaction and fast coamplification at lower denaturation temperature-polymerase chain reaction. Overall, 24 GCTBs (96%) presented a mutation in the H3F3A gene (15 of 16 nonmetastatic and 9 of 9 metastatic). Five sarcomas harbored an H3F3A mutation (3 p.G35W, 1 p.G35L, and 1 p.G35E), and these were all secondary malignant GCTBs. In conclusion, we confirm that H3F3A mutational testing may be a useful adjunct to differentiate GCTB from giant cell-rich sarcomas. Although the presence of H3F3A mutations does not exclude with certainty a diagnosis of sarcoma, the possibility of a malignant evolution of GCTB should also be considered.

Isolation by size of epithelial tumor cells in peripheral blood of patients with breast cancer: correlation with real-time reverse transcriptase-polymerase chain reaction results and feasibility of molecular analysis by laser microdissection.

The aim of this study is the counting and the immunomorphological and molecular characterization of circulating tumor cells (CTCs) by the isolation by size of epithelial tumor cells (ISET) method in the peripheral blood of patients with breast cancer. An evaluation of the method's ability to reveal the presence of occult carcinoma cells in blood of a patient with breast cancer was performed and the results compared with those obtained by quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay for the evaluation of cytokeratin-19 (CK-19) mRNA expression. The feasibility of molecular analysis of CTCs after laser microdissection of filters used in ISET was illustrated, referring to HER-2 amplification. Blood samples drawn from 44 patients with breast cancer were preoperatively analyzed by ISET. From the same samples, total RNA was extracted and submitted to quantitative real-time RT-PCR for the detection of CK-19 mRNA-positive cells using TaqMan technology. HER-2 amplification was measured by real-time RT-PCR on DNA extracted from cells recovered by laser microdissection from 7 selected ISET-positive filters. Of 44 samples, 12 (27%) showed the presence of epithelial cells on the filter (mean +/- SE: 8.5 +/- 2.4 cells per milliliter of blood). A statistically significant agreement (P = .001) was observed between real-time RT-PCR results and those obtained by ISET. With regard to HER-2 amplification, a good correspondence was found between the results obtained from microdissected CTCs and those obtained using DNA extracted from the primary tumor (R = 0.918; P < .01), as well as the immunohistochemistry results. The ISET method allows for the collection of breast carcinoma cells by filtration despite their smaller dimension relative to other carcinoma cell types. The sensitivity and specificity of the method is comparable with those obtained using the quantitative real-time RT-PCR assay for the evaluation of CK-19 mRNA expression. Moreover, the laser microdissection technique allows for the recovery of nucleic acids for further molecular analysis and CTC characterization.

Quantitative analysis of carbonic anhydrase IX mRNA in human non-small cell lung cancer.

Hypoxia is associated with malignant progression and poor outcome in human cancers. The effects of hypoxia are mediated by a series of genomic changes that enable tumor cells to survive or escape their oxygen deficient environment. Recent studies indicated that carbonic anhydrase IX (CA IX) is an intrinsic marker of hypoxia. In the present study we investigated with quantitative RT-PCR the expression of CA IX mRNA in 93 non-small cell lung carcinomas (NSCLC) and in their paired not affected tissues. CA IX mRNA was expressed in 100% NSCLC and in 76% of paired not affected tissues, even if tumoral CA IX expression was found constantly higher (p < 0.02) than that found in normal tissues. The increase of CA IX mRNA expression in cancer tissues was significantly correlated to the increase of corresponding protein, as determined with conventional immunoblotting (p = 0.027). In addition the expression of CA IX mRNA in NSCLC samples was significantly correlated to VEGF (p = 0.002) and MMP-9 (p = 0.002) mRNAs. Whereas CA IX mRNA expression was not associated to any clinical-pathological parameters in our patients, global survival analysis of cancer-related death revealed that high expression of CA IX mRNA predicted unfavorable outcome (p = 0.001) and shorter disease free survival interval (p = 0.004). A multivariate analysis showed that CA IX expression was the strongest prognostic parameter (p = 0.000) in comparison to other conventional predictive markers. In addition, differences emerged on the basis of clinical-pathological parameters: in fact separate Kaplan-Meyer analyses of patients indicated that whereas high levels of CA IX mRNA expression were not predictive of worse prognosis in early NSCLC (G1, T1, Stage 1 and pN- patients), this parameter appeared highly significant in advanced NSCLC (G2-G3, T2-T3, Stage 2-3 and pN+ patients). Finally we demonstrated that CA IX expression was not able to discriminate different survival probability in adenocarcinomas, whereas the same parameter was highly predictive in squamous (p = 0.03) and adenosquamous cell carcinomas (p = 0.001).