Alpha Exon Research Articles

Multifocal gastrointestinal stromal tumor with osseous metaplasia: a case report

BackgroundGastrointestinal stromal tumor is considered the most common mesenchymal neoplasm of the gastrointestinal tract. The majority of gastrointestinal stromal tumor cases are located in the stomach and usually affects older adults. Most of gastrointestinal stromal tumor cases are sporadic; however, few have a syndromic association, including Carney triad, Carney–Stratakis syndrome, familial gastrointestinal stromal tumor syndrome, and neurofibromatosis type 1.Case presentationHerein, we report a rare case of a 54-year-old Middle-Eastern female with multifocal gastrointestinal stromal tumor mixed type (epithelioid and spindle cell type) with osseous metaplasia. Fluoresce in situ hybridization analysis of platelet-derived growth factor receptor alpha revealed deletion in 42% of the tumor cells studied. Interestingly, next generation sequencing revealed platelet-derived growth factor receptor alpha exon 12 mutation (p.Y555C) and exon 14 mutation (p.N659Y).ConclusionsIn conclusion, osseous metaplasia in GIST is a very rare event and only few cases are reported in the literature. The number of reported cases is inadequate to confirm the pathogenesis and the prognosis.

A tamoxifen-inducible Cre knock-in mouse for lens-specific gene manipulation

Mouse models are valuable tools in studying lens biology and biochemistry, and the Cre-loxP system is the most used technology for gene targeting in the lens. However, numerous genes are indispensable in lens development. The conventional knockout method either prevents lens formation or causes simultaneous cataract formation, hindering the studies of their roles in lens structure, growth, metabolism, and cataractogenesis during lens aging. An inducible Cre-loxP mouse line is an excellent way to achieve such a purpose. We established a lens-specific Cre ERT2 knock-in mouse (LCEK), an inducible mouse model for lens-specific gene targeting in a spatiotemporal manner. LCEK mice were created by in-frame infusion of a P2A-CreERT2 at the C-terminus of the last coding exon of the gene alpha A crystallin (Cryaa). LCEK mice express tamoxifen-inducible Cre recombinase uniquely in the lens. Through ROSAmT/mG and two endogenous genes (Gclc and Rbpj) targeting, we found no Cre recombinase leakage in the lens epithelium, but 50–80% leakage was observed in the lens cortex and nucleus. Administration of tamoxifen almost completely abolished target gene expression in both lens epithelium and cortex but only mildly enhanced gene deletion in the lens nucleus. Notably, no overt leakage of Cre activity was detected in developing LCEK lens when bred with mice carrying loxP floxed genes that are essential for lens development. This newly generated LCEK line will be a powerful tool to target genes in the lens for gene functions study in lens aging, posterior capsule opacification (PCO), and other areas requiring precision gene targeting.

First Report of Cobweb Disease in Oudemansiella raphanipes Caused by Cladobotryum varium in Beijing, China.

Oudemansiella raphanipes is an edible mushroom with medicinal properties,which has been recently cultivated throughout China (Hao et al. 2016). In October 2019, a disease with symptoms similar to that of cobweb disease (Carrasco et al. 2017) was observed in O. raphanipes in the Tongzhou District, Beijing, China, infecting 25% of the fruiting bodies (Fig. 1A, B). White cotton-like net of hyphae were present typically on the casing soil or on the stipe of the fruiting bodies; they gradually spread to the pileus, covering the fruiting body, which eventually wilted and died (Fig. 1C, D), resulting in yield reduction and economic loss. Cultures were obtained by aseptically transferring the diseased fruiting bodies onto potato dextrose agar (PDA) at 25 °C; they were deposited in the culture collection (ID: JZBQA1) of the Beijing Academy of Agricultural and Forestry Sciences, China. The colonies were pale white/white, with an occasional formation of yellow diffusing pigments on the reverse side (Fig. 1E-G). Conidiophores were Cladobotryum-like, phialides were solitary or commonly divergent in whorls of 2-3 (-4), lageniform to subulate, 20-63.5 (-66) × (3.8-) 4-5.3 (-9) μm (n = 40) (Fig. 1H, I); conidia were hyaline, oval to ellipsoidal, with one or two septa, (10.4-) 11.4-20 (-22) × 6.6-9.5 (-10) μm (n = 40) (Fig. 1J); chlamydospores were globose or ellipsoidal (Fig. 1K). The morphological characteristics were consistent with that of Cladobotryum varium (Back et al. 2012a, b; Sun et al. 2019). For species-level fungal identification, genomic DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, USA). The internal transcribed spacer (ITS) regions, translation elongation factor 1 alpha exon (TEF1-α), RNA polymerase II subunit b (RPB2), and RNA polymerase I largest subunit (RPB1) genes were amplified using the primer pairs ITS1/ITS4 (White et al. 1990), EF1-983F/2218R (Rehner and Buckley 2005), RPB2-5F/7cR (Liu et al. 1999), and RPB1F1 (5'-GCCGATGAAGTTGGTCTA-3')/RPB1R1 (5'-TATGTTGCGGTGAGCCTT-3'), respectively. A BLAST nucleotide search showed 99.34% (449/452 bp), 99.24% (914/921 bp), 98.08% (1,022/1,042 bp), and 99.66% (588/590 bp) homology, respectively, with those of the ex-type culture of Hypomyces aurantius TFC 95-171 (FN859425.1, FN868743.1, FN868679.1, and FN868805.1). The four sequences were deposited in GenBank (accession numbers: MW534093, MW560066, MW560064, and MW560065). Phylogenetic trees based on the assessed gene loci revealed that the JZBQA1 strain was closely related to C. varium (Fig. 2). A in vivo pathogenicity test was performed using the fruiting bodies (Fig. 1L, O). Spore suspension (108 spores/mL) of the JZBQA1 strain or sterile distilled water was sprayed on six healthy fruiting bodies, maintained in an artificial climate chamber at 24-26°C. Cobweb-like features were observed on the fruiting bodies treated with the spore suspension 2-3 days post-inoculation; while those treated with water did not exhibit such features (Fig. 1L, O). The same pathogen was re-isolated and confirmed from the infected fruiting bodies by integrated analysis of morphological characteristics and gene sequencing data. Cladobotryum spp. infects different varieties of cultivated edible mushrooms, resulting in the development of cobweb diseases (Cao et al. 2020; Carrasco et al. 2017). Cladobotryum varium is the causal agent of cobweb disease in Flammulina velutipes and Hypsizygus marmoreus (Back et al. 2012a, b). To our knowledge, this is the first report of cobweb disease caused by C. varium in O. raphanipes. This finding is a valuable contribution to the knowledge of cobweb disease development in edible fungi.

Association of PML-RARα Fusion mRNA Type With Pretreatment Hematologic Characteristics But Not Treatment Outcome in Acute Promyelocytic Leukemia: An Intergroup Molecular Study

In each case of acute promyelocytic leukemia (APL) one of three PML-RAR alpha mRNA types is produced, depending on the break/fusion site in the PML gene that is linked to a common RAR alpha gene segment: a short (S)-form type, PML exon 3 RAR alpha exon 3; a long (L)-form type, PML exon 6 RAR alpha exon 3; or a variable (V)-form type, variably deleted PML exon 6 RAR alpha exon 3. We evaluated whether PML-RAR alpha mRNA type is associated with distinct pretreatment clinical characteristics and therapeutic outcome in previously untreated adult APL patients registered to protocol INT 0129 by the Eastern Cooperative Oncology Group, the Southwest Oncology Group, and the Cancer and Leukemia Group B. Of 279 clinically eligible cases, 230 were molecularly evaluable, and of these, 111 were randomized to receive remission induction therapy with all-trans retinoic acid (ATRA) and 119 with conventional chemotherapy. Nine cases not excluded by central pathology review were PML-RAR alpha negative, and notably, none of five of these cases treated with ATRA achieved complete remission (CR). Among 221 PML-RAR alpha-positive cases, there were 82 S-form cases (37%), 121 L-form cases (55%), and 18 V-form cases (8%). Before any antileukemic therapy, the S-form type, compared with the L-form type, was associated with higher values for the white blood cell (WBC) count (median 2,500/microL v 1,600/microL; P = .009), the percentage of blood blasts plus promyelocytes (median 29% v 8.5%; P = .03), and the absolute blood blasts plus promyelocytes (884/microL v 126/microL; P = .019). Also, an increased percentage of S-form versus L-form cases had the M3 variant phenotype, 24% v 12% (P = .036). There were no differences between S-form and L-form cases in either CR rate (79% v 69%; P = .14) or disease free survival distribution (multivariate analysis adjusting for the association of S-form type and higher WBC count; P = .40). We conclude that the S-form type is associated with previously-identified adverse risk WBC parameters but that the identification of the S-form or L-form type of PML-RAR alpha mRNA, per se, does not predict clinical outcome or add to the value of an increased WBC count as a negative prognostic indicator in APL patients.

A severe case of acidosis with concomitant use of avapritinib and metformin requiring emergent hemodialysis

Introduction: Avapritinib (Ayvakit), a tyrosine kinase inhibitor (TKI), has been recently approved for the treatment of adult patients with unresectable or metastatic gastrointestinal stromal tumor (GIST) who have a platelet-derived growth factor receptor alpha (PDGFRA) exon 18 mutation. New therapeutic agents for malignancy, specifically TKIs, have allowed for major advances in treatment options. Case Report: A 56-year-old male with past medical history significant for GIST of the stomach, who was recently started on a new trial medication ayvakit, presented with nausea, vomiting, and feeling ill. His initial laboratory work was remarkable for blood glucose level of 19 mg/dL, creatinine of 10.41 mg/dL, potassium of 4.3 mmol/L, anion gap of 41 mmol/L, and arterial blood gas with pH of 6.707, pO2 163 mmHg, pCO2 14 mmHg, and HCO3 of 2 mmol/L on 2 L of nasal cannula oxygen. The patient was in hypovolemic shock secondary to dehydration, hence requiring vasopressors for a short duration. Among the patient’s home medications, it is of significance to mention metformin as a daily medication. He emergently underwent hemodialysis for severe acidosis, with significant improvement in his clinical status. Conclusion: Acidosis can occur as a side effect of therapy or synergistic effect of multiple medications. Avapritinib usage along with medications that are known to have potential to cause acidosis, such as metformin, needs to be cautiously administered. Understanding the potential side effects of new therapy in combination with the patient’s established medications can facilitate the recognition of potential causal relationships.

Avapritinib for metastatic or unresectable gastrointestinal stromal tumors.

Avapritinib is a tyrosine kinase inhibitor (TKI) that has recently received Food and Drug Administration (FDA) approval for the treatment of metastatic or unresectable gastrointestinal stromal tumors harboring a platelet-derived growth factor receptor alpha (PDGFRA) exon 18 mutation. Mutations in the activation loop of PDGFRA or KIT confer resistance to conventional TKIs due to structural changes in the receptor. Avapritinib was developed to selectively target these mutations, thereby offering a new treatment option for patients in whom imatinib, sunitinib, and regorafenib have failed. This review covers the basic science and preclinical studies that guided avapritinib's development, in addition to the data currently available from early clinical studies as well as those later-stage trials that led to its approval.

Association of hypoxia inducible factor-1 alpha exon 12 mutation in diabetic patients with and without diabetic foot ulcer

Hypoxia inducible factor 1 alpha (HIF-1α) is a key regulator of the genes involved in the cellular response to hypoxia. This study aims to determine the HIF-1α gene polymorphism and its association with protein expression in diabetic subjects with and without diabetic foot ulcers (DFU). A total of 529 patients with T2DM (N = 185), DFU (N = 199) and Control (N = 145) were accounted for the study. PCR–RFLP experiment was carried out in order to find the allelic and genotypic comparison of HIF-1α gene in various groups of patients. There was a highly increased frequency of GA, RR value of 3.533(2.099–5.950) with p-value of 0.0001 on DFU patients when compared to that of control subjects with risk allele of GA, RR value of 1.756 (1.294–2.384) with p-value of 0.00001. Thus, we found that there was a significant association of HIF-1α polymorphism in exon 12 among DFU patients when compared to control groups. The circulatory HIF-1α protein expression study indicated a decreased expression in DFU levels when compared to T2DM and control. Overall, the study showed that there is an association of HIF-1α polymorphism (G1970A) in diabetes and DFU patients when compared to the healthy group.

"Squirrel" Primer-Based PCR Assay for Direct and Targeted Sanger Sequencing of Short Genomic Segments.

Currently, short DNA segments of sub-100 bp can be sequenced either directly by next-generation sequencing and pyrosequencing, which are expensive, or indirectly, via Sanger sequencing combined with the cumbersome and failure-prone plasmid cloning. To circumvent these issues, we have generated a novel sequencing-purposed PCR assay using long-tailed primers (squirrel primers) to Sanger sequence directly sub-100 bp genomic amplicons. Squirrel primers, 40-65 nt in length, were used to amplify 51-93 bp long genomic sequences of KRAS exons 2 and 3, BRAF exon 15, PI3K catalytic subunit alpha exon 20, and phosphatase and tensin homolog exon 3 from colorectal cancer (CRC) cell lines and preamplified clinical CRC samples with known mutation status by PCR. Following this, a short second pair of primers that bind at the 5' region of the long tails was used for sequencing on the 3130 × l ABI Prism Genetic Analyzer. The sequencing data were analyzed via FinchTV software. High-quality sequencing data were obtained from 51 to 93 bp long genomic sequences with our novel PCR assay, with capture of all of the target sequences in all of the samples in both the forward and reverse directions and confirmation of the mutation status of the CRC samples. Whereas the sequencing quality was independent of the template type, it showed a squirrel primer tail length-dependent pattern. Our novel PCR assay for direct and targeted Sanger sequencing of short genomic segments has potential applications in focused molecular/genetic profiling of cancer in research and diagnostics fields in which fragmented DNA, such as circulating tumor DNA and archival tissue DNA, are used as starting templates.

Seven wood-inhabiting new species of the genus Trichoderma (Fungi, Ascomycota) in Viride clade.

More than 200 recent collections of Trichoderma from China were examined and 16 species belonging to the Viride clade were identified based on integrated studies of phenotypic and molecular data. Among them, seven wood-inhabiting new species, T. albofulvopsis, T. densum, T. laevisporum, T. sinokoningii, T. sparsum, T. sphaerosporum and T. subviride, are found. They form trichoderma- to verticillium-like conidiophores, lageniform to subulate phialides and globose to ellipsoidal conidia, but vary greatly in colony features, growth rates, and sizes of phialides and conidia. To explore their taxonomic positions, the phylogenetic tree including all the known species of the Viride clade is constructed based on sequence analyses of the combined RNA polymerase II subunit b and translation elongation factor 1 alpha exon genes. Our results indicated that the seven new species were well-located in the Koningii, Rogersonii and Neorufum subclades as well as a few independent terminal branches. They are clearly distinguishable from any existing species. Morphological distinctions and sequence divergences between the new species and their close relatives were discussed.

Succinate dehydrogenase deficient gastrointestinal stromal tumor: a clinicopathologic analysis of eight cases

To study the clinicopathologic features, diagnosis and differential diagnosis of succinate dehydrogenase (SDH) deficient gastrointestinal stromal tumors (GISTs) as a unique tumor subtype. SDHB and SDHA immunohistochemistry was performed in 120 gastric GISTs, in addition to CD117, DOG-1, CD34, smooth muscle actin (SMA), desmin, S-100 protein, cytokeratin (CK) and Ki-67. Subset of the cases was further evaluated for the presence of mutations in CKIT exons 9, 11, 13 and 17 mutations and platelet derived growth factor receptor alpha(PDGFRA) exons 12 and 18. Eight of 120 (6.6%) GIST cases were found SDH-deficient including 3 male and 5 female patients (median age of 36.2 years; ranging 16 to 65 years of age). The tumors involved antrum (6 cases), lesser curvature (1 case) and fundus (1 case). Macroscopically, the dominant tumor masses varied from 3 to 10 cm in diameter with a multinodular or plexiform pattern involving the gastric wall. Microscopically,tumor cells had predominantly epithelioid morphology, with occasional mixed spindle cell nodules. Lymphovascular invasion was identified in 5 cases. Immunohistochemistry for SDHB was negative in all 8 cases, and SDHA was negative in 5 cases. All 8 SDHB negative cases also expressed CD117, DOG-1 and CD34, but were negative for SMA, desmin, S-100 and CK. All 8 cases were found to have wild-type CKIT and PDGFRA genes. Available clinical follow-up were obtained in 7 cases, ranging from 2 to 60 months (median follow-up 23.3 months), and all patient were alive. Three cases were found to have liver metastases at their first diagnosis, and one developed omental and mesenteric metastases in 17 months. SDH-deficient GIST is a distinct subtype of GIST, with a predilection to occur in young and female patients. Characteristic pathological findings include multinodular gastric wall involvement, epithelioid cell morphology, frequently lymphovascular invasion with occasional lymph node and liver metastases, but an overall indolent clinical behavior. Immunohistochemistry for SDHB is required for the diagnosis.

Clinical utility of the new American Joint Committee on Cancer staging system for gastrointestinal stromal tumors

The objectives of the current study were to assess the reliability of the new revision of the American Joint Committee on Cancer (AJCC) staging system for gastrointestinal stromal tumors (GISTs) based on the National Comprehensive Cancer Network-Armed Forces Institute of Pathology risk classification and to analyze the factors that influence after resection for primary GISTs in 2 AJCC groups: patients with GISTs originating from the stomach and omentum (G-GISTs) and patients with other primary GISTs located mainly in the small bowel (nongastric GISTs [NG-GISTs]). The authors prospectively analyzed a group of 640 patients with primary, CD117-positive GISTs who underwent surgery with curative intention (R0/R1 resection), including 340 G-GISTs (55.5%) and 300 NG-GISTs (44.5%). Factors were explored that had an effect on disease-free survival time (DFS), which was calculated from the date of radical operation to the date of recurrence or last follow-up. The median follow-up was 39 months. Compared with NG-GISTs, G-GISTs were characterized by a significantly lower median size (5.3 cm and 8.5 cm, respectively; P < .0001) and lower mitotic activity (median, 3 in 50 high-power fields [HPF] vs 5 in 50 HPF; P < .0001), and they were diagnosed in older patients (median age, 62 years vs 57 years; P = .002). The most commonly detected mutations in G-GIST were those located in KIT exon 11 (60.5%) and platelet-derived growth factor receptor alpha (PDGFRA) exon 18 (19%) versus KIT exons 11 and 9 in NG-GISTs (72% and 17.4%, respectively). The prognosis of patients who had G-GISTs was significantly better compared that of patients who had NG-GISTs, with 5-year DFS rates of 69% (median, 83 months) versus 43% (median, 33 months), respectively (P < .00001). The most significant prognostic factors that correlated with shorter DFS in both G-GISTs and NG-GISTs were primary tumor size >5 cm and >10 cm (P < .0001) and mitotic index >5 in 50 HPF and >10 in 50 HPF (P < .0001). The 5-year DFS rates in G-GISTs according to AJCC stage categories were as follows: 96% for stage IA tumors, 92% for stage IB tumors, 51% for II tumors, 22% for stage IIIA tumors, and 22% for stage IIIB tumors (P < .0001). The 5-year DFS rates in NG-GISTs according to AJCC categories were as follows: 92% for stage I tumors, 66% for stage II tumors, 28% for IIIA tumors, and 16% for IIIB tumors (P < .0001). The high prognostic significance of the AJCC classification also was confirmed for overall survival data, including the impact of therapy with tyrosine kinase inhibitors. The reliability of AJCC risk classification after resection of primary GIST was confirmed for DFS and overall survival. Patients with primary G-GISTs had a better prognosis than patients with NG-GISTs. In both groups, primary tumor size and mitotic activity were the most important prognostic factors in terms of DFS.

European species of Hypocrea part II: species with hyaline ascospores

To date 75 species of Hypocrea/Trichoderma forming teleomorphs are recognised in Europe. The 56 hyaline-spored species are here described in detail and illustrated in colour plates, including cultures and anamorphs. This number includes 16 new holomorphs, two new teleomorphs and nine anamorphs of species previously described as teleomorphs. Phylogenetic placement and relationships of the species are shown on the strict consensus tree, based on sequences of RNA polymerase II subunit b (rpb2) and translation elongation factor 1 alpha (tef1) exon, comprising 135 species of the genus Hypocrea/Trichoderma. All available holotypes of species described from Europe including some from North America have been examined. A dichotomous key to the species is provided primarily utilising ecological and morphological traits of the teleomorphs and, where necessary, morphology of the anamorphs and cultures, and growth rates. Species descriptions are subdivided among five chapters, arranged primarily according to the larger phylogenetic clades, viz. section Trichoderma with 13 species, the pachybasium core group with 13 species including four species with stipitate stromata (‘Podostroma’), species forming large effused stromata with 10 species including the section Hypocreanum, 9 species of the Brevicompactum, Lutea and Psychrophila clades, and 11 residual species of various smaller clades or of unknown phylogenetic placement. Finally, a list comprising dubious names and species excluded from Hypocrea that are relevant for Europe, or species claimed to occur in Europe by other authors is provided. Hypocrea minutispora is by far the most common species in Europe. For H. moravica, H. subalpina and H. tremelloides the anamorphs are newly described. The anamorphs of the latter two species and H. sambuci produce hyaline conidia on unusual structures new to Trichoderma. These three species form a new subclade of the morphologically strikingly different section Longibrachiatum, which is currently only represented by H. schweinitzii in Europe as a holomorph. The subclade is not named yet formally due to low statistical support. H. fungicola f. raduli is described as the new species H. austriaca, while H. hypomycella was found not to belong to Hypocrea. The typification of H. pilulifera, H. tremelloides and H. lutea has been clarified. Gliocladium deliquescens, the anamorph of H. lutea, is combined in Trichoderma. Species are epitypified where appropriate. Anamorph names are established prospectively to avoid numerous new combinations in future when they may be possibly used as holomorphic names if the ICBN is altered accordingly.

Sex steroids in uterine endometrial cancers.

Some uterine endometrial cancers conserve estrogen dependency in advancement. However, the concept of advancement in tumor is complicated, because it involves simple growth in primary tumor and secondary spreading. The expression manner of estrogen receptor alpha exon 5 splicing variant, ER beta, progesterone receptor-A (N-terminus deletion mutant) is associated with metastatic potential in uterine endometrial cancers. Increased estrogen-related receptor alpha expression is related to tumor advancement with the loss of estrogen dependency. Steroid receptor coactivator-3 contributes to tumor progression and can be used as a treatment target for advanced uterine endometrial cancers. Estrogen responsive oncogenes, c-jun and c-Ha-ras, are not modi-fied by progestin in uterine endometrial cancer cells and are considered to be an instinct phenotype as such cancers. By contrast, metastatic potential of estrogen-dependent uterine endometrial cancers can be partially controlled by progestin via metastasis-related genes, E-cadherin/catenins, plasminogen activator inhibitor-1, vascular endothelial growth factor. Thus, sex steroids related phenomena are impress-ive in the advancement of uterine endometrial cancers.

A quality control program for mutation detection in KIT and PDGFRA in gastrointestinal stromal tumours

Although most gastrointestinal stromal tumours (GIST) carry oncogenic mutations in KIT exons 9, 11, 13 and 17, or in platelet-derived growth factor receptor alpha (PDGFRA) exons 12, 14 and 18, around 10% of GIST are free of these mutations. Genotyping and accurate detection of KIT/PDGFRA mutations in GIST are becoming increasingly useful for clinicians in the management of the disease. To evaluate and improve laboratory practice in GIST mutation detection, we developed a mutational screening quality control program. Eleven laboratories were enrolled in this program and 50 DNA samples were analysed, each of them by four different laboratories, giving 200 mutational reports. In total, eight mutations were not detected by at least one laboratory. One false positive result was reported in one sample. Thus, the mean global rate of error with clinical implication based on 200 reports was 4.5%. Concerning specific polymorphisms detection, the rate varied from 0 to 100%, depending on the laboratory. The way mutations were reported was very heterogeneous, and some errors were detected. This study demonstrated that such a program was necessary for laboratories to improve the quality of the analysis, because an error rate of 4.5% may have clinical consequences for the patient.

Novel and recurrent p14ARF mutations in Italian familial melanoma

CDKN2A and CDK4 are the only known high-penetrant genes conferring proneness to cutaneous melanoma. The CDKN2A locus consists of four exons and encodes several alternate transcripts, two of which are p16(INK4a) and p14(ARF), and originate from different open reading frames. Exon 1alpha is specific for p16(INK4a), while exon 1beta characterizes p14(ARF). Most CDKN2A mutations are located in exons 1alpha and 2, while exon 1beta variations have been identified in rare melanoma-prone pedigrees. In a previous study, we investigated 155 Italian melanoma cases, including 94 familial melanomas (FAMs) and 61 sporadic multiple primary melanomas (MPMs), for p16(INK4a)/CDK4 germline alterations and identified 15 p16(INK4a) and 1 CDK4 point mutations. In the present work, we extended our search to p14(ARF) mutations and CDKN2A deletions in the remaining samples. We identified the recurrent g.193+1G> A mutation in two FAM cases, while an additional pedigree displayed the previously undescribed variant g.161G> A. Multiplex ligation-dependent probe amplification (MLPA) screening for copy variations resulted negative in all cases. In Italy, the overall frequency of p14(ARF) mutations is 3.2% in FAM and 0% in sporadic MPM. Re-evaluation of our patients' cohort emphasizes that the chance of identifying CDKN2A/CDK4 mutations in FAM is mainly influenced by the number of affected family members and the presence of one or more MPM cases. Accordingly, mutation rate rises to 61% in selected cases. Further studies are expected in order to investigate CDKN2A rarer mutations, including atypical deletions and inherited epimutations.

Multiple gastrointestinal stromal tumors in neurofibromatosis type 1: Report of a case

This report presents a case of multiple gastrointestinal stromal tumors (GIST) with neurofibromatosis type 1 (NF1). A 68-year-old woman was admitted to the hospital because of a tumor close to the head of the pancreas. Imaging studies revealed submucosal tumors of the duodenum. The retroperitoneal tumor was diagnosed before surgery. Besides the main tumor in the duodenum, multiple small submucosal tumors were found in the duodenum and upper part of the jejunum during the operation. All of these tumors were resected. The histological diagnosis of all these tumors was GISTs. These tumors were immunohistochemically positive for KIT, but they demonstrated no mutation in c-kit exons 9, 11, 13, and 17, and platelet-derived growth factor receptor alpha exons 12 and 18. No recurrence occurred for a year after surgery.

Low-grade Epithelial-Myoepithelial Carcinoma of Bartholin Gland: Report of 2 Cases of a Distinctive Neoplasm Arising in the Vulvovaginal Region

We report 2 cases of a distinctive neoplasm arising from Bartholin gland and presenting as a vulval or vaginal mass. The tumors occurred in patients aged 44 and 51 years and were 2 and 3 cm in maximum dimension. In both cases, normal Bartholin gland tissue was identified adjacent to the lesion. The neoplasms were unencapsulated and largely well circumscribed but with a focally infiltrative edge. They were composed of tubular, trabecular, or insular arrangements with a double layer of inner cuboidal cells with round nuclei and outer cells with ovoid nuclei and clear cytoplasm, corresponding to epithelial and myoepithelial cells, respectively. Luminal eosinophilic colloid-like material was present. In both cases, a minor proportion of the neoplasm consisted of cribriform arrangements, creating an appearance reminiscent of adenoid cystic carcinoma, although the overall morphology was not typical of that lesion. Mitotic figures were identified in both cases, the mitotic count being 1 and 5/10 high-power fields. Immunohistochemically, the inner cells were positive with epithelial markers, including broad-spectrum cytokeratins and epithelial membrane antigen, and the outer cell layer was positive with myoepithelial markers p63, calponin, and alpha-smooth muscle actin. Both neoplasms exhibited diffuse strong immunoreactivity of the epithelial cells with c-kit. Activating mutations in KIT exons 9, 11, 13, and 17 and in platelet-derived growth factor receptor alpha exons 12, 14, and 18 were searched for by polymerase chain reaction and direct sequencing but were not identified. We believe this represents a low-grade carcinoma arising from Bartholin gland composed of a dual population of epithelial and myoepithelial cells and closely resembling the salivary gland neoplasm termed epithelial-myoepithelial carcinoma. We propose the term low-grade epithelial-myoepithelial carcinoma of Bartholin gland.

A gastrointestinal stromal tumor of the stomach morphologically resembling a neurofibroma: demonstration of a novel platelet-derived growth factor receptor α exon 18 mutation

Gastrointestinal stromal tumors are increasingly being recognized because of their characteristic expression of KIT (CD 117). Most KIT-positive gastrointestinal stromal tumors have activating mutations in the c-kit gene. A subgroup of gastrointestinal stromal tumors are negative for KIT expression, and in these tumors, activating mutations in platelet-derived growth factor receptor alpha are common. Most platelet-derived growth factor receptor alpha mutation-positive gastrointestinal stromal tumors show an epithelioid histology and are located in the stomach. Herein, we describe an unusual gastric stromal tumor. The tumor was negative for KIT expression and the morphology did not show an epithelioid pattern but rather was composed of bland spindle cells reminiscent of a neurofibroma. Molecular analysis revealed a somatic mutation in platelet-derived growth factor receptor alpha exon 18 (D842F). Aside from demonstrating a new platelet-derived growth factor receptor alpha mutation, this case illustrates the usefulness of molecular testing as a diagnostic tool and clearly indicates the wide range of morphology that can be observed in gastrointestinal stromal tumors.

Revised spectrum of mutations in sarcoglycanopathies

To define the spectrum of mutations in alpha-, beta-, gamma-, and delta-sarcoglycan (SG) genes, we analyzed these genes in 69 probands with clinical and biological criteria compatible with the diagnosis of autosomal recessive limb-girdle muscular dystrophy. For 48 patients, muscle biopsies were available and multiplex western blot analysis of muscle proteins showed significant abnormalities of alpha- and gamma-SG. Our diagnostic strategy includes multiplex western blot, sequencing of SG genes, multiplex quantitative-fluorescent PCR and RT-PCR analyses. Mutations were detected in 57 patients and homozygous or compound heterozygous mutations were identified in 75% (36/48) of the patients with abnormal western blot, and in 52% (11/21) of the patients without muscle biopsy. Involvement of alpha-SG was demonstrated in 55.3% of cases (26/47), whereas gamma- and beta-SG were implicated in 25.5% (12/47) and in 17% (8/47) of cases, respectively. Interestingly, we identified 25 novel mutations, and a significant proportion of these mutations correspond to deletions (identified in 14 patients) of complete exon(s) of alpha- or gamma-SG genes, and partial duplications (identified in 5 patients) of exon 1 of beta-SG gene. This study highlights the high frequency of exonic deletions of alpha- and gamma-SG genes, as well as the presence of a hotspot of duplications affecting exon 1 of the beta-SG gene. In addition, protein analysis by multiplex western blot in combination with mutation screening and genotyping results allowed to propose a comprehensive and efficient diagnostic strategy and strongly suggested the implication of additional genes, yet to be identified, in sarcoglycanopathy-like disorders.

Severe Obesity and Insulin Resistance due to Deletion of the Maternal Gsα Allele Is Reversed by Paternal Deletion of the Gsα Imprint Control Region

The G protein alpha-subunit G(s)alpha mediates receptor-stimulated cAMP production and is imprinted with reduced expression from the paternal allele in specific tissues. Disruption of the G(s)alpha maternal (but not paternal) allele leads to severe obesity, hypertriglyceridemia, and insulin resistance in mice and obesity in patients with Albright hereditary osteodystrophy. Paternal deletion of a G(s)alpha imprint control region (1A) leads to loss of tissue-specific G(s)alpha imprinting. To determine whether the metabolic abnormalities resulting from disruption of the G(s)alpha maternal allele could be reversed by loss of paternal G(s)alpha imprinting, females with a heterozygous G(s)alpha exon 1 deletion were mated to males with heterozygous deletion of the imprint control region (1A) to generate mice with maternal G(s)alpha deletion (E1(m-)), paternal 1A deletion (1A(p-)), double mutants (E1(m-):1A(p-)), and wild type. E1(m-) mice developed obesity, glucose intolerance, insulin resistance, and hypertriglyceridemia, which were all normalized by the paternal 1A deletion in E1(m-):1A(p-) mice. Obesity in E1(m-) was associated with reduced energy expenditure and sympathetic nerve activity, and these were also normalized in E1(m-):1A(p-) mice. 1A(p-) mice had reduced body weight associated with proportional decreases in fat and lean mass as well as increased activity levels. The metabolic phenotype resulting from maternal G(s)alpha deletion is rescued by a genetic lesion that leads to loss of tissue-specific G(s)alpha imprinting, consistent with this phenotype being a direct consequence of G(s)alpha imprinting in one or more specific tissues.