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.

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.

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.

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.

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.

Mutational analysis of single circulating tumor cells by next generation sequencing in metastatic breast cancer.

Circulating Tumor Cells (CTCs) represent a "liquid biopsy" of the tumor potentially allowing real-time monitoring of cancer biology and therapies in individual patients.The purpose of the study was to explore the applicability of a protocol for the molecular characterization of single CTCs by Next Generation Sequencing (NGS) in order to investigate cell heterogeneity and provide a tool for a personalized medicine approach.CTCs were enriched and enumerated by CellSearch in blood from four metastatic breast cancer patients and singularly isolated by DEPArray. Upon whole genome amplification 3-5 single CTCs per patient were analyzed by NGS for 50 cancer-related genes.We found 51 sequence variants in 25 genes. We observed inter- and intra-patient heterogeneity in the mutational status of CTCs.The highest number of somatic deleterious mutations was found in the gene TP53, whose mutation is associated with adverse prognosis in breast cancer.The discordance between the mutational status of the primary tumor and CTCs observed in 3 patients suggests that, in advanced stages of cancer, CTC characteristics are more closely linked to the dynamic modifications of the disease status.In one patient the mutational profiles of CTCs before and during treatment shared only few sequence variants.This study supports the applicability of a non-invasive approach based on the liquid biopsy in metastatic breast cancer patients which, in perspective, should allow investigating the clonal evolution of the tumor for the development of new therapeutic strategies in precision medicine.

Denosumab treated giant cell tumour of bone: a morphological, immunohistochemical and molecular analysis of a series.

AIMS: Denosumab, a fully human monoclonal antibody directed against RANKL, has recently been introduced in the treatment strategy of giant cell tumour of bone (GCTB). Aim of this study was to investigate the phenotypical modifications induced by denosumab treatment in a series of 15 GCTB. METHODS: The tumours were characterised for histone 3.3 mutations, and studied immunohistochemically for the modifications of RANKL, RANK, SATB2 and RUNX2 expression, as well as of tumour proliferative activity and angiogenesis. RESULTS: Nine of 11 tumours investigated presented a histone 3.3 mutation in H3F3A, and 2 of these for which the analysis was carried out in pretreatment and post-treatment specimens showed the same mutation in both. Denosumab induced the disappearance of osteoclast-like giant cells, leaving residual spindle neoplastic cells arranged in a storiform pattern, with deposition of trabecular collagen matrix and osteoid, which tended to maturation in the peripheral portions of the lesion. RANK and RANKL expression was variable, with no significant variation after treatment. Moreover, we did not observe any significant modification of the expression of the osteoblastic markers SATB2 and RUNX2. Denosumab treatment determined a significant reduction of the proliferative index and of tumour angiogenesis (p=0.001, Wilcoxon rank-sum test). CONCLUSIONS: These results indicate that denosumab induces a partial maturation towards the osteoblastic phenotype of the neoplastic cells of GCTB, with production of fibrous and osteoid matrix, but with minor immunophenotypical changes. Finally, we first report an antiangiogenic activity of denosumab in GCTB, possibly mediated by a RANKL-dependent pathway.

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.

KIT genetic alterations in anorectal melanomas.

BACKGROUND: Mucosal melanomas (MM) represent a heterogeneous tumour population that exhibits site-specific molecular profiles. AIMS: In a multicentre retrospective study, we investigated KIT aberrations in primary anorectal (AR) melanomas compared with melanoma metastatic to the gastrointestinal (GI) tract. METHODS: Primary AR MM (n=31) and GI metastatic melanoma (n=27) were studied for KIT mutations on exons 11, 13, 17 and 18 by high-resolution melting analysis, direct sequencing and c-KIT expression by immunohistochemistry. Selected cases were also investigated for increased KIT gene copy number by fluorescent in situ hybridisation. RESULTS: Functional KIT mutations were demonstrated in 11/31 (35.5%) of AR melanomas and in 1/26 (3.8%) of GI melanoma metastases (p=0.004). A significant difference emerged between primary and metastatic MM with regards to KIT-positive immunostaining (p=0.002). Immunohistochemical c-KIT protein overexpression did not correlate with KIT mutational status. Increased KIT copy number was demonstrated in 5/20 AR primary cases. CONCLUSIONS: The rate of functional mutations in KIT is significantly higher in AR MM than in GI metastatic melanoma. KIT protein overexpression does not correlate with KIT mutations and cannot be used for screening purposes. Recognising the molecular heterogeneity of MM helps to identify patients who require a different therapeutic approach.

Implementation of a companion diagnostic in the clinical laboratory: the BRAF example in melanoma.

A companion diagnostic test provides information that is essential for the safe and effective use of a corresponding therapeutic product as indicated in the drug instructions. The implementation of a companion diagnostic follows the rules of a molecular test for somatic mutations in a routine clinical laboratory environment and needs guidance on practical aspects, including the choice of the proper analytical method and the procedures for internal and external quality controls. Selection of the appropriate assay for detection of genetic alterations depends on several factors: the type of mutation under study, the sample to be assayed and its preparation procedure. In addition, the results of a molecular assay require a complex interpretation process of the analytical data as the patient's genotype, the translation of the identified variant into a predicted phenotype and knowledge on restrictions of the method used. In relation to these aspects herein we report an opinion paper of the Working Group Personalized Laboratory Medicine jointly constituted by the European Federation of Laboratory Medicine (EFLM) and by the European Society of Pharmacogenomics and Theranostics (ESPT) using, as an example, the BRAF genotype analysis in tumor tissue samples for identification of melanoma patients that can benefit treatment with BRAF inhibitors. The manuscript is focused on the following aspects: i) medical rationale, ii) methodologies of analysis, iii) laboratory performance evaluation and iv) the laboratory specific report for the clinicians. The critical evaluation of these aspects would be useful for the implementation of a companion diagnostic in the clinical laboratory.

SPIDIA-RNA: second external quality assessment for the pre-analytical phase of blood samples used for RNA based analyses.

One purpose of the EC funded project, SPIDIA, is to develop evidence-based quality guidelines for the pre-analytical handling of blood samples for RNA molecular testing. To this end, two pan-European External Quality Assessments (EQAs) were implemented. Here we report the results of the second SPIDIA-RNA EQA. This second study included modifications in the protocol related to the blood collection process, the shipping conditions and pre-analytical specimen handling for participants. Participating laboratories received two identical proficiency blood specimens collected in tubes with or without an RNA stabilizer. For pre-defined specimen storage times and temperatures, laboratories were asked to perform RNA extraction from whole blood according to their usual procedure and to return extracted RNA to the SPIDIA facility for further analysis. These RNA samples were evaluated for purity, yield, integrity, stability, presence of interfering substances, and gene expression levels for the validated markers of RNA stability: FOS, IL1B, IL8, GAPDH, FOSB and TNFRSF10c. Analysis of the gene expression results of FOS, IL8, FOSB, and TNFRSF10c, however, indicated that the levels of these transcripts were significantly affected by blood collection tube type and storage temperature. These results demonstrated that only blood collection tubes containing a cellular RNA stabilizer allowed reliable gene expression analysis within 48 h from blood collection for all the genes investigated. The results of these two EQAs have been proposed for use in the development of a Technical Specification by the European Committee for Standardization.

BRAF and KIT somatic mutations are present in amelanotic melanoma.

The genotypic profile of rare amelanotic melanomas (AMs) has been poorly investigated, thus preventing either an accurate identification as a distinctive melanoma subtype or therapy stratification. Here, we investigated the presence of the BRAF(V600E) mutation by real-time quantitative PCR and KIT mutations (exons 11 and 17) by sequencing analysis in 33 AMs. AMs included 'truly' amelanotic lesions (n = 19), with no melanin pigmentation upon dermoscopic inspection and hypomelanotic lesions (n = 14), by definition partially pigmented lesions showing a melanin pigmentation area of less than 25% of the total surface area. The frequency of the BRAF(V600E) mutation was 70.3% in the 33 cases, a percentage that increased to 89% when only the subgroup of thin melanomas (≤ 1 mm in thickness, n = 9) was considered. KIT mutations were found in 12.1% of AMs, all of which developed in nonacral sites. The identification of a relatively high frequency of BRAF(V600E) and KIT mutations in AMs may have important consequences for implementation of the novel targeted therapies now available to treat this life-threatening disease.

The somatic affairs of BRAF: tailored therapies for advanced malignant melanoma and orphan non-V600E (V600R-M) mutations.

BRAF V600R-M-D are uncommon mutations, not included in the experimental protocols of BRAF selective inhibitors. We report the evaluation of correlations among different types of BRAF somatic mutations in melanoma and their management with BRAF inhibitors. 21 patients with BRAF mutated metastatic melanoma were enrolled in the protocol with BRAF inhibitors for compassionate use at the University of Modena. Hot spot V600E mutations were found in 19 patients. V600R mutation and double (V600E -V600M) mutation were identified in two melanomas. In one case, V600K mutation was found. Two screening failures were noted. Mean progression free survival at follow-up of to 8 weeks, was 7.6 months. Five patients had a very short follow-up and the experimental protocol is still ongoing, so we cannot provide complete follow-up data. However, all of them are still under treatment and disease progression free. An objective response with few side effects was observed in all patients. in vitro studies with the aim of testing drug sensitivity.

Monitoring the susceptibility to oseltamivir of Influenza A(H1N1) 2009 virus by nested-PCR and pyrosequencing during the pandemic and in the season 2010-2011.

For the early detection of the H275Y mutation as a marker of oseltamivir resistance in A(H1N1) pandemic strains, a sensitive and specific pyrosequencing assay was developed. This assay analyses a region 99nts long, encompassing the H275Y site, amplified by a nested PCR. Seventy-five respiratory specimens, obtained from 62 patients during the pandemic and in the 2010-2011 influenza season, in Tuscany, were tested. Resistant strains were demonstrated in 10 patients. In three other patients, resistant and sensitive variants were found. This pyrosequencing assay may be a useful method for monitoring the spread of resistant influenza H1N1 2009 strains.

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.

High-resolution melting analysis: a new molecular approach for the early detection of Diplodia pinea in Austrian pine.

The differentiation of Diplodia pinea from closely related species, such as Diplodia scrobiculata and Diplodia seriata, and its detection in plant tissue, represented a critical issue for a long time. Molecular screening tools have recently been developed to address this topic. In this study we applied one of the most sensitive and rapid diagnostic screening method so far developed, called High-Resolution Melting Analysis (HRMA), to detect D. pinea in Austrian pine (Pinus nigra). HRMA exploits differences in the melting behaviour of PCR products to rapidly identify DNA sequence variants without the need for cumbersome post-PCR methods. We developed a HRMA method to detect specific fungal sequences in the mitochondrial small subunit ribosome gene (mt SSU rDNA). The reliability of this technique was firstly assessed on DNA extracted from pure cultures of D. pinea and closely related species. Amplicon differences were screened by HRMA and the results confirmed by direct DNA sequencing. Subsequently, HRMA was tested on DNA from symptomatic and symptomless pine shoots, and the presence of the fungus was also confirmed by both conventional and molecular quantitative approaches. The HRMA allowed the distinction of D. pinea from closely related species, showing specific melting profiles for the each pathogen. This new molecular technique, here tested in a plant-fungus pathosystem for the first time, was very reliable in both symptomatic and symptomless shoots. HRMA is therefore a highly effective and accurate technique that permits the rapid screening of pathogens in the host.

Association between single nucleotide polymorphisms in the XRCC1 and RAD51 genes and clinical radiosensitivity in head and neck cancer.

PURPOSE: Individual variability in radiosensitivity is large in cancer patients. Single nucleotide polymorphisms (SNPs) in genes involved in DNA repair and in protection against reactive oxygen species (ROS) could be responsible for such cases of radiosensitivity. We investigated the association between the occurrence of acute reactions in 101 patients with squamous cell carcinoma of the head and neck (SCCHN) after radiotherapy (RT) and five genetic polymorphisms: XRCC1 c.1196A>G, XRCC3 c.722C>T, RAD51 (c.-3429G>C, c.-3392G>T), and GSTP1 c.313A>G. MATERIALS AND METHODS: Genetic polymorphisms were detected by high resolution melting analysis (HRMA). The development of acute reactions (oral mucositis, skin erythema and dysphagia) associated with genetic polymorphisms was modeled using Cox proportional hazards, accounting for biologically effective dose (BED). RESULTS: Development of grade ≥2 mucositis was increased in all patients (chemo-radiotherapy and radiotherapy alone) with XRCC1-399Gln allele (HR=1.72). The likelihood of developing grade ≥2 dysphagia was higher in carriers of RAD51 c.-3429 CC/GC genotypes (HR=4.00). The presence of at least one SNP or the co-presence of both SNPs in XRCC1 p.Gln399Arg /RAD51 c.-3429 G>C status were associated to higher likelihood of occurrence of acute toxicities (HR=2.03). CONCLUSIONS: Our findings showed an association between genetic polymorphisms, XRCC1 c.1196A>G and RAD51 c.-3429 G>C, and the development of radiation-induced toxicities in SCCHN patients.

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.