Isabella Bernardis

Impact of mutational status on outcomes in myelofibrosis patients treated with ruxolitinib in the COMFORT-II study.

The JAK1/JAK2 inhibitor ruxolitinib produced significant reductions in splenomegaly and symptomatic burden and improved survival in patients with myelofibrosis (MF), irrespective of their JAK2 mutation status, in 2 phase III studies against placebo (COMFORT-I) and best available therapy (COMFORT-II). We performed a comprehensive mutation analysis to evaluate the impact of 14 MF-associated mutations on clinical outcomes in 166 patients included in COMFORT-II. We found that responses in splenomegaly and symptoms, as well as the risk of developing ruxolitinib-associated anemia and thrombocytopenia, occurred at similar frequencies across different mutation profiles. Ruxolitinib improved survival independent of mutation profile and reduced the risk of death in patients harboring a set of prognostically detrimental mutations (ASXL1, EZH2, SRSF2, IDH1/2) with an hazard ratio of 0.57 (95% confidence interval: 0.30-1.08) vs best available therapy. These data indicate that clinical efficacy and survival improvement may occur across different molecular subsets of patients with MF treated with ruxolitinib.

Targeted cancer exome sequencing reveals recurrent mutations in myeloproliferative neoplasms

With the intent of dissecting the molecular complexity of Philadelphia-negative myeloproliferative neoplasms (MPN), we designed a target enrichment panel to explore, using next-generation sequencing (NGS), the mutational status of an extensive list of 2000 cancer-associated genes and microRNAs. The genomic DNA of granulocytes and in vitro-expanded CD3+T-lymphocytes, as a germline control, was target-enriched and sequenced in a learning cohort of 20 MPN patients using Roche 454 technology. We identified 141 genuine somatic mutations, most of which were not previously described. To test the frequency of the identified variants, a larger validation cohort of 189 MPN patients was additionally screened for these mutations using Ion Torrent AmpliSeq NGS. Excluding the genes already described in MPN, for 8 genes (SCRIB, MIR662, BARD1, TCF12, FAT4, DAP3, POLG and NRAS), we demonstrated a mutation frequency between 3 and 8%. We also found that mutations at codon 12 of NRAS (NRASG12V and NRASG12D) were significantly associated, for primary myelofibrosis (PMF), with highest dynamic international prognostic scoring system (DIPSS)-plus score categories. This association was then confirmed in 66 additional PMF patients composing a final dataset of 168 PMF showing a NRAS mutation frequency of 4.7%, which was associated with a worse outcome, as defined by the DIPSS plus score.

Epidemiology and clinical relevance of mutations in postpolycythemia vera and postessential thrombocythemia myelofibrosis: A study on 359 patients of the AGIMM group.

Transformation to secondary myelofibrosis (MF) occurs as part of the natural history of polycythemia vera (PPV‐MF) and essential thrombocythemia (PET‐MF). Although primary (PMF) and secondary MF are considered similar diseases and managed similarly, there are few studies specifically focused on the latter. The aim of this study was to characterize the mutation landscape, and describe the main clinical correlates and prognostic implications of mutations, in a series of 359 patients with PPV‐MF and PET‐MF. Compared with PV and ET, the JAK2V617F and CALR mutated allele burden was significantly higher in PPV‐MF and/or PET‐MF, indicating a role for accumulation of mutated alleles in the process of transformation to MF. However, neither the allele burden nor the type of driver mutation influenced overall survival (OS), while absence of any driver mutation (triple negativity) was associated with significant reduction of OS in PET‐MF, similar to PMF. Of the five interrogated subclonal mutations (ASXL1, EZH2, SRSF2, IDH1, and IDH2), that comprise a prognostically detrimental high molecular risk (HMR) category in PMF, only SRSF2 mutations were associated with reduced survival in PET‐MF, and no additional mutation profile with prognostic relevance was highlighted. Overall, these data indicate that the molecular landscape of secondary forms of MF is different from PMF, suggesting that unknown mutational events might contribute to the progression from chronic phase disease to myelofibrosis. These findings also support more extended genotyping approaches aimed at identifying novel molecular abnormalities with prognostic relevance for patients with PPV‐MF and PET‐MF. Am. J. Hematol. 91:681–686, 2016.

Amplicon-based next-generation sequencing: an effective approach for the molecular diagnosis of epidermolysis bullosa.

Epidermolysis bullosa (EB) is caused by mutations in genes that encode proteins belonging to the epidermal–dermal junction assembly. Due to the extreme clinical/genetic heterogeneity of the disease, the current methods available for diagnosing EB involve immunohistochemistry of biopsy samples and transmission electron microscopy followed by single‐candidate gene Sanger sequencing (SS), which are labour‐intensive and expensive clinical pathways.

Unravelling the Complexity of Inherited Retinal Dystrophies Molecular Testing: Added Value of Targeted Next-Generation Sequencing

To assess the clinical utility of targeted Next-Generation Sequencing (NGS) for the diagnosis of Inherited Retinal Dystrophies (IRDs), a total of 109 subjects were enrolled in the study, including 88 IRD affected probands and 21 healthy relatives. Clinical diagnoses included Retinitis Pigmentosa (RP), Leber Congenital Amaurosis (LCA), Stargardt Disease (STGD), Best Macular Dystrophy (BMD), Usher Syndrome (USH), and other IRDs with undefined clinical diagnosis. Participants underwent a complete ophthalmologic examination followed by genetic counseling. A custom AmpliSeq™ panel of 72 IRD-related genes was designed for the analysis and tested using Ion semiconductor Next-Generation Sequencing (NGS). Potential disease-causing mutations were identified in 59.1% of probands, comprising mutations in 16 genes. The highest diagnostic yields were achieved for BMD, LCA, USH, and STGD patients, whereas RP confirmed its high genetic heterogeneity. Causative mutations were identified in 17.6% of probands with undefined diagnosis. Revision of the initial diagnosis was performed for 9.6% of genetically diagnosed patients. This study demonstrates that NGS represents a comprehensive cost-effective approach for IRDs molecular diagnosis. The identification of the genetic alterations underlying the phenotype enabled the clinicians to achieve a more accurate diagnosis. The results emphasize the importance of molecular diagnosis coupled with clinic information to unravel the extensive phenotypic heterogeneity of these diseases.

Homozygous familial hypobetalipoproteinemia: A Turkish case carrying a missense mutation in apolipoprotein B.

The autosomal co-dominant disorder familial hypobetalipoproteinemia (FHBL) may be due to mutations in the APOB gene encoding apolipoprotein B (apoB), the main constituent peptide of chylomicrons, very low and low density lipoproteins. We describe an 11month-old child with failure to thrive, intestinal lipid malabsorption, hepatic steatosis and severe hypobetalipoproteinemia, suggesting the diagnosis of homozygous FHBL, abetalipoproteinemia (ABL) or chylomicron retention disease (CMRD). The analysis of candidate genes showed that patient was homozygous for a variant (c.1594 C>T) in the APOB gene causing arginine to tryptophan conversion at position 505 of mature apoB (Arg505Trp). No mutations were found in a panel of other potential candidate genes for hypobetalipoproteinemia. In vitro studies showed a reduced secretion of mutant apoB-48 with respect to the wild-type apoB-48 in transfected McA-RH7777 cells. The Arg505Trp substitution is located in the βα1 domain of apoB involved in the lipidation of apoB mediated by microsomal triglyceride transfer protein (MTP), the first step in VLDL and chylomicron formation. The patients condition improved in response to a low fat diet supplemented with fat-soluble vitamins. Homozygosity for a rare missense mutation in the βα1 domain of apoB may be the cause of both severe hypobetalipoproteinemia and intestinal lipid malabsorption.

Genomic alterations at the basis of treatment resistance in metastatic breast cancer: Clinical applications

The standard of care for breast cancer has gradually evolved from empirical treatments based on clinical-pathological characteristics to the use of targeted approaches based on the molecular profile of the tumor. Consequently, an increasing number of molecularly targeted drugs have been developed. These drugs target specific alterations, called driver mutations, which confer a survival advantage to cancer cells. To date, the main challenge remains the identification of predictive biomarkers for the selection of the optimal treatment. On this basis, we evaluated a panel of 25 genes involved in the mechanisms of targeted treatment resistance, in 16 primary breast cancers and their matched recurrences, developed during treatment. Overall, we found a detection rate of mutations higher than that described in the literature. In particular, the most frequently mutated genes were ERBB2 and those involved in the PI3K/AKT/mTOR and the MAPK signaling pathways. The study revealed substantial discordances between primary tumors and metastases, stressing the need for analysis of metastatic tissues at recurrence. We observed that 85.7% of patients with an early-stage or locally advanced primary tumor showed at least one mutation in the primary tumor. This finding could explain the subsequent relapse and might therefore justify more targeted adjuvant treatments. Finally, the mutations detected in 50% of relapsed tissues could have guided subsequent treatment choices in a different way. This study demonstrates that mutation events may be present at diagnosis or arise during cancer treatment. As a result, profiling primary and metastatic tumor tissues may be a major step in defining optimal treatments.