Marina Vignoli

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.

Human IL2RA null mutation mediates immunodeficiency with lymphoproliferation and autoimmunity.

Cell-surface CD25 expression is critical for maintaining immune function and homeostasis. As in few reported cases, CD25 deficiency manifests with severe autoimmune enteritis and viral infections. To dissect the underlying immunological mechanisms driving these symptoms, we analyzed the regulatory and effector T cell functions in a CD25 deficient patient harboring a novel IL2RA mutation. Pronounced lymphoproliferation, mainly of the CD8+ T cells, was detected together with an increase in T cell activation markers and elevated serum cytokines. However, Ag-specific responses were impaired in vivo and in vitro. Activated CD8+STAT5+ T cells with lytic potential infiltrated the skin, even though FOXP3+ Tregs were present and maintained a higher capacity to respond to IL-2 compared to other T-cell subsets. Thus, the complex pathogenesis of CD25 deficiency provides invaluable insight into the role of IL2/IL-2RA-dependent regulation in autoimmunity and inflammatory diseases.

The p.G23S CDKN2A founder mutation in high-risk melanoma families from Central Italy.

We have investigated the frequency and spectrum of CDKN2A/CDK4 mutations in 23 cutaneous melanoma families from Central Italy (Tuscany). Three distinct mutations were identified in five families. One mutation, p.G23S, was present in three families. Several lines of evidence indicate that p.G23S is a pathogenic mutation: it is located in the functionally important first ankyrinic domain of p16, it was not detected in a sample of 100 control individuals, and it was present in all tested affected individuals from the three families. Haplotype analysis showed a common ancestral origin of the p.G23S mutation. Our data show that the p.G23S mutation is an important cause of hereditary melanoma in Tuscany.

CACP syndrome: identification of five novel mutations and of the first case of UPD in the largest European cohort

Camptodactyly-Arthropathy-Coxa vara-Pericarditis (CACP) syndrome is a rare autosomal recessive disorder caused by mutations in PRG4 gene that encodes for proteoglycan 4, a mucin-like glycoprotein that is the major lubricant for joints and tendon surfaces. The molecular studies reported so far have described the identification of 15 mutations associated with this syndrome and the majority of them were found in families of Arabian origin. Here we report the molecular investigation of the largest European cohort that comprises 13 patients, and allowed the identification of 5 novel mutations and of the first case of CACP syndrome resulting from uniparental disomy of chromosome 1.

Genomic rearrangements of the CDKN2A locus are infrequent in Italian malignant melanoma families without evidence of CDKN2A/CDK4 point mutations.

Predisposition to familial cutaneous malignant melanoma has been associated with mutations in the CDKN2A and CDK4 genes. However, only a small subgroup of melanoma pedigrees harbour CDKN2A or CDK4 germline mutations. It is possible that other types of CDKN2A rearrangements, not detectable by routine PCR-based approaches, are involved in a fraction of melanoma cases negative for point sequence changes. In order to gain insights on the possible role of CDKN2A large deletions or duplications in melanoma susceptibility in the Italian population, we screened a series of 124 cutaneous malignant melanoma families referred to five national medical/cancer genetics centres. All probands were negative for point mutations in CDKN2A and CDK4. All samples were tested by MLPA (multiplex ligation-dependent probe amplification), and the results were confirmed by real-time quantitative PCR in a subset of 53 cases. No genomic rearrangements were detected in this series, one of the largest so far investigated. These data suggest that large deletions/duplications in the CDKN2A locus are infrequently involved in the development of familial melanoma in the Italian population. Based on these results, routine search for these rearrangements in CDKN2A- and CDK4-mutation negative melanoma families is not warranted, although it would be reasonable to pursue it in selected cases with very strong family history and/or showing linkage to 9p21.

Langerhans cell histiocytosis in IPEX syndrome: Possible role for natural regulatory T cells?

pletely inhibited after immunoabsorption of the antibodies on purified human epidermal filaggrin but not on bovine serum albumin used as a control [(6) and Fig. 1a]. In addition, it is now clear that at least part of the profilaggrin present in the human buccal mucosa epithelium is citrullinated. In fact, it is extracted in the absence of a denaturing agent, and it is recognized by the human autoantibody called ‘antiperinuclear factor’. The latter is specifically found in the serum of patients with rheumatoid arthritis and belongs to the family of autoantibodies that exclusively recognize epitopes generated by citrullination of arginine residues on the targeted antigenic proteins (8, 9). In conclusion, these data clearly demonstrate that profilaggrin is expressed by the most differentiated keratinocytes of the oral cavity mucosae, the filaggrin monomers being generated only in the orthokeratinized epithelium of the hard palate.

Inherited Multiple Autoimmunity: Molecular and Clinical Characterization of IPEX and IPEX-Like Syndromes

describes the study design to facilitate the enrollment of patients treated in clinical settings. Study: All females who become pregnant during IgHy treatment (treated while pregnant or in the 30 days prior to conception) will be encouraged to participate. As soon as the patient becomes aware of the pregnancy, treatment with IgHy will be stopped. Subjects will receive an alternative gammaglobulin treatment and all other medical care will be performed as is standard. The overall duration of the study is approximately 6 years and there is no minimum sample size pre-specified. Data from medical records on the routine assessments during the pregnancy will be collected. In those subjects consenting to blood drawing, samples will be taken to assess antibodies against rHuPH20 every 3 months. Data will be assessed on the development of the fetus in utero, and on the growth and development of the infant for 2 years after delivery. Descriptive methods will be used to analyze and report data from this IgHy pregnancy registry. ESID-0098 Non-Interventional Post-Authorization Safety Study (PASS) on the Long-Term Safety of HYQVIA® (IGHY) K. Fielhauer, N. Nikolov, C. Hermann, A. Nagy, R.I. Schiff Bioscience, Baxter Healthcare, Vienna, Austria Bioscience, Baxter Healthcare SA, Zurich, Switzerland Bioscience, Baxter Healthcare, Westlake Village, USA Introduction: IgHy (recombinant human hyaluronidase [rHuPH20]-facilitated subcutaneous [SC] infusion of immunoglobulin [IG]) is a novel treatment that has been approved as a replacement therapy in adults (≥18 years) with primary immunodeficiency (PID) syndromes, myeloma, and chronic lymphocytic leukemia. This is a non-interventional, prospective, uncontrolled, multi-center, open label, post-authorization safety study to be conducted in the EEA to acquire additional data (including the assessment of anti-rHuPH20 antibodies) on the long-term safety of IgHy. This abstract describes the study design to facilitate the evaluation of patients treated in a

GUT IMMUNE RECONSTITUTION IN IPEX SYNDROME AFTER HSCT

Adoptive cell transfer (ACT) of transgenic T lymphocytes expressing tumor specific T cell receptor (TCR) is a promising therapy. However, little is known about the fate of engineered T cells and the dynamics of host immunity. We comprehensively monitored the transgenic and endogenous immune response in 8 melanoma patients over 8–10 time points in an ACT therapy of transgenic F5 MART-1 TCR T cells, using highly multiplexed clinical immune diagnostics methods (Ma, C. et al. Nature Medicine 17, 738–743 (2011)). We found that the TCR engineered T cells sustained a 2–3 week duration anti-tumor response. After day 30 a second wave endogenous multi-clonal melanoma specific T cell response emerged. The melanoma-specific T cells within the ACTwent through a process of in vivo functional changes that correlated with clinical antitumor activity. The appearance and the persistence of the second wave immune response provided indications of long term therapeutic efficacy.