Multiple primary sporadic gastrointestinal stromal tumors in the adult: an underestimated entity.

PURPOSE: Gastrointestinal stromal tumors (GIST) are commonly regarded as solitary tumors. The occurrence of multiple lesions is considered an extraordinary event restricted to pediatric GISTs and rare hereditary conditions. Beyond these well-defined situations, the presentation of multiple synchronous lesions is commonly viewed as the result of the metastatic spreading of a single primary GIST. Based on this axiom, patients with multifocal disease are classified as advanced stage and treated as such. Whether, indeed, the detection of several lesions in sporadic adult GIST patients may be suggestive of phenomena of tumor multiplicity still needs to be clarified. EXPERIMENTAL DESIGN: From a multicentric series of 442 consecutive cases, 26 of which with advanced disease, we selected 5 patients who presented up to three distinct GIST nodules. Five additional cases with similar characteristics were also contributed by two other institutions. The clonal relationship between the synchronous lesions was assessed by comparing KIT/PDGFRA mutation and microsatellite pattern. RESULTS: An independent origin of the synchronous lesions was established in 6 of 10 cases. Notably, in one patient, one lesion arose in the peritoneum, which is ordinarily regarded as a site of metastasis. CONCLUSIONS: Our data indicate that a significant fraction of GIST patients with multifocal presentation are actually affected by multiple primary tumors, suggesting that mesenchymal GIST precursor cells of these individuals are somehow primed to transformation. Thus, in the presence of multifocal GIST manifestations, an accurate characterization of the different tumor sites should be undertaken for a proper patient staging and therapy planning.

Identification of novel chimpanzee MHC class I and II alleles using an improved sequence-based typing strategy.

The chimpanzee (Pan troglodytes) is a valuable model for the study of infection with hepatitis C virus and hepatitis B virus, to which only humans and chimpanzees are susceptible. Because the cellular immune response plays a crucial role in host defense against these viruses, the analysis of major histocompatibility complex (MHC) (Patr) class I and II allele diversity in chimpanzees is essential for immune response analysis and vaccine development. In the present study, we report a novel, rapid, and sensitive sequence-based typing strategy to identify polymorphisms of the principal Patr class I (Patr-A and Patr-B) and Patr class II (Patr-DQB1 and Patr-DRB1) alleles. Using this method we identified seven novel Patr alleles in 17 chimpanzees, one of them present in 3 chimpanzees. Furthermore, we detected heterozygosity more rapidly and with higher sensitivity than was done with previous techniques that were based on reverse transcription and amplification of messenger RNA followed by molecular cloning and sequencing.

Atypical lymphoproliferation progressing into B-cell lymphoma in rheumatoid arthritis treated with different biological agents: clinical course and molecular characterization.

A patient with rheumatoid arthritis (RA) developed an atypical lymphoproliferative disorder (LPD) after methotrexate and cyclosporine A, which regressed after suspension of both drugs. After subsequent treatment with rituximab, the LPD was still undetectable. Anti-tumor necrosis factor a therapy was used when the arthritis relapsed, but an aggressive B-cell non Hodgkin's lymphoma developed. Molecular analyses showed an oligoclonal B-cell expansion at the LPD step. A minor clone with significant sequence homology to B-cell lymphomas arising in Sjogren's syndrome and mixed cryoglobulinemia syndrome, given rise to the non-Hodgkin's lymphoma. Treatment of rheumatoid arthritis associated with lymphoproliferation represents a clinical challenge, and common pathogenetic pathways to lymphoma may occur in different autoimmune diseases.

KIT, PDGFRA, and BRAF mutational spectrum impacts on the natural history of imatinib-naive localized GIST: a population-based study.

The mutation status of KIT or PDGFRA notoriously affects the response of advanced gastrointestinal stromal tumors (GISTs) to tyrosine kinase inhibitors. Conversely, it is currently still unclear whether mutation status impinges on the prognosis of localized, untreated GISTs. Hence, at present, this variable is not included in decision making for adjuvant therapy. A series of 451 primary localized GISTs were analyzed for KIT, PDGFRA, and BRAF mutations. Univariable and multivariable analyses and a backward selection procedure were used to assess the impact of mutation status on overall survival and to identify prognostically homogenous groups. Mutation was a significant prognostic indicator of overall survival in naive, localized GISTs (P<0.001): KIT-mutated patients had a worse outcome than PDGFRA-mutated or triple-negative (KIT, PDGFRA, BRAF wild-type) cases. Multivariable Cox regression models allowed us to identify 3 molecular risk groups: group I exhibited the best outcome and included PDGFRA exon 12, BRAF, and KIT exon 13-mutated cases; group II, of intermediate clinical phenotype (HR=3.06), included triple-negative, KIT exon 17, PDGFRA exon 18 D842V, and PDGFRA exon 14-mutated cases; group III displayed the worst outcome (hazard ratio=4.52), and comprised KIT exon 9 and exon 11 and PDGFRA exon 18 mutations apart from D842V. This study highlights the prognostic impact of mutation status on the natural course of GIST and suggests that the molecular prognostic grouping may complement the conventional clinicopathologic risk stratification criteria in decision making for adjuvant therapy.

Overexpression of TWIST2 correlates with poor prognosis in head and neck squamous cell carcinomas.

Head and neck squamous cell carcinomas (HNSCC) are a heterogeneous group of tumors with variable presentation and clinical behavior. Despite improvements in surgical and radiation therapy techniques, the 5-year survival rate has not improved significantly over the past decades. Thus, there is an urgent need to identify novel markers that may allow for the development of personalized therapeutic approaches. In the present study we evaluated the prognostic role of the expression of genes related to the induction of epithelial mesenchymal transition (EMT). To this aim, a consecutive series of 69 HNSCC were analyzed for the expression of TWIST1, TWIST2, SNAI1, SNAI2, E-Cadherin, N-Cadherin and Vimentin.TWIST1, TWIST2, SNAI1 and SNAI2 were significantly overexpressed in HNSCC, with TWIST2, SNAI1 and SNAI2 being more markedly increased in tumors compared to normal mucosae. The expression of TWIST1 and SNAI2 was associated with upregulation of mesenchymal markers, but failed to correlate with pathological parameters or clinical behaviour. In contrast, we found that upregulation of TWIST2, which was independent of the activation of a mesenchymal differentiation program, correlated with poor differentiation grade (p=0.016) and shorter survival (p=0.025), and identifies a subset of node-positive oral cavity/pharynx cancer patients with very poor prognosis (p less than 0.001). Overall our study suggests that the assessment of TWIST2 expression might help to stratify HNSCC patients for risk of disease progression, pointing to TWIST2 as a potential prognostic marker.

Molecular and clinicopathologic characterization of gastrointestinal stromal tumors (GISTs) of small size.

Although Gastrointestinal stromal tumors (GISTs) affect about 0.0014% of the population, GISTs smaller than 1 cm (microGISTs) are detectable in about 20% to 30% of elderly individuals. This suggests that microGISTs likely represent premalignant precursors that evolve only in a minute fraction of cases toward overt GISTs. We sought histopathologic and molecular explanations for the infrequent clinical progression in small GISTs. To investigate the mechanisms of GIST progression and identify subsets with differential malignant potential, we carried out a thorough characterization of 170 GISTs <2 cm and compared their KIT/PDGFRA status with overt GISTs. The proliferation was lower in microGISTs compared with GISTs from 1 to 2 cm (milliGISTs). In addition, microGISTs were more frequently incidental, gastric, spindle, showed an infiltrative growth pattern, a lower degree of cellularity, and abundant sclerosis. The progression was limited to 1 ileal and 1 rectal milliGISTs. KIT/PDGFRA mutations were detected in 74% of the cases. The overall frequency of KIT/PDGFRA mutation and, particularly, the frequency of KIT exon 11 mutations was significantly lower in small GISTs compared with overt GISTs. Five novel mutations, 3 in KIT (p.Phe506Leu, p.Ser692Leu, p.Glu695Lys) 2 in PDGFRA (p.Ser847X, p.Ser667Pro), plus 4 double mutations were identified. Small GISTs share with overt GIST KIT/PDGFRA mutation. Nevertheless, microGISTs display an overall lower frequency of mutations, particularly canonical KIT mutations, and also carry rare and novel mutations. These molecular features, together with the peculiar pathologic characteristics, suggest that the proliferation of these lesions is likely sustained by weakly pathogenic molecular events, supporting the epidemiologic evidence that microGISTs are self-limiting lesions.

Salivary gland B cell lymphoproliferative disorders in Sjögren's syndrome present a restricted use of antigen receptor gene segments similar to those used by hepatitis C virus-associated non-Hodgkins's lymphomas.

Sjögren's syndrome (SS) represents a pathological model of the evolution from polyclonal B lymphocyte activation to oligoclonal/monoclonal B cell expansion, which may culminate in the development of a malignant lymphoproliferative disease. The different phases of this process are usually marked by the appearance of antigen-driven activated B cell clones, which are commonly IgM-positive and with rheumatoid factor (RF) activity. However, the agent(s) able to trigger B cell proliferation is still unknown. A similar pathogenetic mechanism exist in mixed cryoglobulinemia, another autoimmune disease that often evolves to non-Hodgkin's lymphoma (NHL) and in which hepatitis C virus (HCV) infection has been demonstrated to play an etiopathogenetic role. In the present study, we cloned and sequenced the antigen receptor (IgR) variable region genes of SS-associated monoclonal non-neoplastic lymphoproliferations and compared them with those of our previous reported HCV-associated NHL, to derive clues on the antigen(s) that sustains SS. The results obtained showed remarkable homologies between the antigen combinatory regions of the IgR expressed by both diseases. These homologies concern: a) the specific combinations of heavy and light variable region genes; b) the limited length of complementarity-determining regions (CDR3); c) the homology with antibodies with RF activity; d)the amino acid sequences of CDR3 in which common somatic mutations are present that possibly determine the antigen-binding specificity. In conclusion, although there are significant differences between SS and HCV-associated lymphoproliferative diseases, they share many molecular characteristics, which suggest an immunological cross-reactivity or molecular mimicry among the agents that underlie these disorders.

Differentiation between non-Hodgkin's lymphoma recurrence and second primary lymphoma by VDJ rearrangement analysis.

Relapses in non-Hodgkin's lymphomas (NHL) could be due to the reappearance of the initial neoplasm or new primary tumours. Discrimination between the two events would allow a more targeted therapeutic approach. VDJ rearrangement was used as marker of clonality in metachronous biopsy specimens from 10 patients with relapsed B-NHL. Complimentary determining region 3 was amplified and sequenced. D-JH was identical in eight matched primary/secondary tumours, confirming the diagnosis of recurrence. In contrast, primary and secondary tumours in two patients were of different clonal origin. Our data indicate that VDJ analysis is a fundamental tool for identification of relapses in NHL.

Extrasalivary lymphoma development in Sjögren's syndrome: clonal evolution from parotid gland lymphoproliferation and role of local triggering.

OBJECTIVE: To characterize Sjögren's syndrome (SS)-related B cell lymphoproliferation at the premalignant stage and during the evolution to B cell lymphoma, and to better understand the pathobiologic mechanisms associated with clonal expansion and the possible influence of different microenvironments on neoplastic transformation. METHODS: We analyzed sequential parotid and lung biopsy specimens that were obtained from a single patient with SS at multiple time points over a 7-year period. Polymerase chain reaction DNA amplification, cloning, and sequencing of the immunoglobulin heavy chain variable region showed clonality, somatic mutations, intraclonal heterogeneity, and genealogic relationships of the B cell clones in the different biopsy specimens. RESULTS: The evolution of a nonmalignant B cell clone that was present in the parotid gland and evolved into a B cell lymphoma was documented. During such a process, one subclone was selected that accumulated somatic mutations in a pattern consistent with the preservation of antigen receptor functionality, possibly attributable to continued hypermutation and selection. Intraclonal diversity indicated the presence of local triggers in both the parotid and lung microenvironments. CONCLUSION: Molecular followup of B cell lymphoproliferation in SS, from nonmalignant stage to overt B cell lymphoma, indicated a role for B cell receptor engagement in clonal survival. The outgrowth of one subclone, with malignant transformation in the lung, a target organ different from the initial site of the lymphoproliferative process (the parotid gland), indicates that resident stimuli in different microenvironments may locally sustain ongoing lymphoproliferation and B cell transformation.