Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2.

BACKGROUND: Somatic mutations in the Janus kinase 2 gene (JAK2) occur in many myeloproliferative neoplasms, but the molecular pathogenesis of myeloproliferative neoplasms with nonmutated JAK2 is obscure, and the diagnosis of these neoplasms remains a challenge. METHODS: We performed exome sequencing of samples obtained from 151 patients with myeloproliferative neoplasms. The mutation status of the gene encoding calreticulin (CALR) was assessed in an additional 1345 hematologic cancers, 1517 other cancers, and 550 controls. We established phylogenetic trees using hematopoietic colonies. We assessed calreticulin subcellular localization using immunofluorescence and flow cytometry. RESULTS: Exome sequencing identified 1498 mutations in 151 patients, with medians of 6.5, 6.5, and 13.0 mutations per patient in samples of polycythemia vera, essential thrombocythemia, and myelofibrosis, respectively. Somatic CALR mutations were found in 70 to 84% of samples of myeloproliferative neoplasms with nonmutated JAK2, in 8% of myelodysplasia samples, in occasional samples of other myeloid cancers, and in none of the other cancers. A total of 148 CALR mutations were identified with 19 distinct variants. Mutations were located in exon 9 and generated a +1 base-pair frameshift, which would result in a mutant protein with a novel C-terminal. Mutant calreticulin was observed in the endoplasmic reticulum without increased cell-surface or Golgi accumulation. Patients with myeloproliferative neoplasms carrying CALR mutations presented with higher platelet counts and lower hemoglobin levels than patients with mutated JAK2. Mutation of CALR was detected in hematopoietic stem and progenitor cells. Clonal analyses showed CALR mutations in the earliest phylogenetic node, a finding consistent with its role as an initiating mutation in some patients. CONCLUSIONS: Somatic mutations in the endoplasmic reticulum chaperone CALR were found in a majority of patients with myeloproliferative neoplasms with nonmutated JAK2. (Funded by the Kay Kendall Leukaemia Fund and others.).

Development of tissue-engineered models of oral dysplasia and early invasive oral squamous cell carcinoma.

BACKGROUND: Current organotypic models of dysplasia and oral squamous cell carcinoma (OSCC) lack the complexity that mimics in vivo tissue. Here we describe a three-dimensional in vitro model of the oral epithelium that replicates tumour progression from dysplasia to an invasive phenotype. METHODS: The OSCC cell lines were seeded as a cell suspension (D20, Cal27) or as multicellular tumour spheroids (FaDu) with oral fibroblasts on to a de-epidermised acellular dermis to generate tissue-engineered models and compared with patient biopsies. RESULTS: The D20 and Cal27 cells generated a model of epithelial dysplasia. Overtime Cal27 cells traversed the basement membrane and invaded the connective tissue to reproduce features of early invasive OSCC. When seeded onto a model of the normal oral mucosa, FaDu spheroids produced a histological picture mimicking carcinoma in situ with severe cellular atypia juxtaposed to normal epithelium. CONCLUSION: It is possible to culture in vitro models with the morphological appearance and histological characteristics of dysplasia and tumour cell invasion seen in vivo using native dermis. Such models could facilitate study of the molecular processes involved in malignant transformation, invasion and tumour growth as well as in vitro testing of new treatments, diagnostic tests and drug delivery systems for OSCC.

The range and demographics of salivary gland tumours diagnosed in a UK population.

Salivary gland tumours are relatively rare and comprise a diverse range of neoplasms. The aim of this study was to determine the range and demographics of all histologically diagnosed salivary tumours in a European population. All entries for salivary gland tumours from 1974 to 2005 inclusive were retrieved and analysed for each diagnosis including number of specimens, male:female ratio and age range. These data were then analysed for the distribution of benign and malignant salivary tumours in major and minor salivary glands. 58,880 specimens were received; of these, 741 cases (1.3% of all specimens) were diagnosed as salivary gland tumours with a male to female ratio of 0.7:1. There were 481 (64.9%) benign and 260 (35.1%) malignant neoplasms, with the most common tumours being pleomorphic adenoma and mucoepidermoid carcinoma, respectively. Our study provides demographic data on a large series of salivary gland tumours in a European population. Accurate diagnosis is essential as salivary lesions have diverse clinical and prognostic outcomes. This study has confirmed that some tumours have a predilection for certain sites and that the risk of malignant disease is also greater at specific sites within the oral cavity.

FIP1L1-PDGFRA in chronic eosinophilic leukemia and BCR-ABL1 in chronic myeloid leukemia affect different leukemic cells.

We investigated genetically affected leukemic cells in FIP1L1-PDGFRA+ chronic eosinophilic leukemia (CEL) and in BCR-ABL1+ chronic myeloid leukemia (CML), two myeloproliferative disorders responsive to imatinib. Fluorescence in situ hybridization specific for BCR-ABL1 and for FIP1L1-PDGFRA was combined with cytomorphology or with lineage-restricted monoclonal antibodies and applied in CML and CEL, respectively. In CEL the amount of FIP1L1-PDGFRA+ cells among CD34+ and CD133+ cells, B and T lymphocytes, and megakaryocytes were within normal ranges. Positivity was found in eosinophils, granulo-monocytes and varying percentages of erythrocytes. In vitro assays with imatinib showed reduced survival of peripheral blood mononuclear cells but no reduction in colony-forming unit growth medium (CFU-GM) growth. In CML the BCR-ABL1 fusion gene was detected in CD34+/CD133+ cells, granulo-monocytes, eosinophils, erythrocytes, megakaryocytes and B-lymphocytes. Growth of both peripheral blood mononuclear cells and CFU-GM was inhibited by imatinib. This study provided evidence for marked differences in the leukemic masses which are targeted by imatinib in CEL or CML, as harboring FIP1L1-PDGFRA or BCR-ABL1.

A survey of oral and maxillofacial pathology specimens submitted by general dental practitioners over a 30-year period.

OBJECTIVES: To determine the range and frequency of diagnoses in specimens submitted for histopathological examination by general dental practitioners (GDPs). METHODS: A retrospective analysis was carried out of all cases submitted by GDPs for the period 1974-2003, using a Foxpro Windows database. The data were collated into 10 diagnostic categories each comprising number of diagnoses, percentage of each diagnosis within a diagnostic category and each diagnosis as a percentage of total cases. RESULTS: GDPs submitted 6,666 cases out of a total of 53,474 for this period. While the total number of specimens increased four-fold over the 30-year period, specimens from GDPs increased from 7% to 17%. The range of diagnoses increased from 18 to 45. Of the 617 GDPs who submitted material, 279 (45%) submitted less than two specimens each in 30 years. Nine malignant neoplasms were diagnosed. Other significant pathology included 320 benign neoplasms as well as diagnoses ranging from mucosal lesions such as lichen planus to odontogenic cysts. CONCLUSIONS: It is clear that GDPs have provided an increased number of biopsy specimens over the last three decades. This reflects an increasing demand by GDPs for a diagnostic oral histopathology service and their use of this service should be encouraged.

Profound parental bias associated with chromosome 14 acquired uniparental disomy indicates targeting of an imprinted locus.

Acquired uniparental disomy (aUPD) is a common finding in myeloid malignancies and typically acts to convert a somatically acquired heterozygous mutation to homozygosity. We sought to identify the target of chromosome 14 aUPD (aUPD14), a recurrent abnormality in myeloid neoplasms and population cohorts of elderly individuals. We identified 29 cases with aUPD14q that defined a minimal affected region (MAR) of 11.2 Mb running from 14q32.12 to the telomere. Exome sequencing (n=7) did not identify recurrently mutated genes, but methylation-specific PCR at the imprinted MEG3-DLK1 locus located within the MAR demonstrated loss of maternal chromosome 14 and gain of paternal chromosome 14 (P<0.0001), with the degree of methylation imbalance correlating with the level of aUPD (r=0.76; P=0.0001). The absence of driver gene mutations in the exomes of three individuals with aUPD14q but no known haematological disorder suggests that aUPD14q may be sufficient to drive clonal haemopoiesis. Analysis of cases with both aUPD14q and JAK2 V617F (n=11) indicated that aUPD14q may be an early event in some cases but a late event in others. We conclude that aUPD14q is a recurrent abnormality that targets an imprinted locus and may promote clonal haemopoiesis either as an initiating event or as a secondary change.

Benzodiazepine withdrawal syndromes.

We report eight cases of benzodiazepine withdrawal syndromes seen in a general psychiatric hospital. These consisted of acute organic brain syndrome, grand mal convulsions and abstinence syndromes. All of the cases were using benzodiazepines in prescribed therapeutic doses. These problems appear to be more common than are generally acknowledged.

Solitary fibrous tumour of the cheek: An unusual presentation of a rare soft tissue tumour.

This case report discusses the unusual presentation and ultrasound features of a solitary fibrous tumour of the face. Solitary fibrous tumour is an uncommon form of soft tissue tumour which, although seen predominantly within the lung pleura, can occur throughout the body in sites such as the peritoneum, mediastinum and head and neck. Ultrasound is an excellent imaging modality in the assessment of soft tissue masses in the head and neck. The ultrasound features demonstrated by this example of solitary fibrous tumour are reviewed. This report also highlights that ultrasound alone is ultimately limited in reaching a definitive diagnosis. The roles of other investigations such as ultrasound-guided biopsy and cross-sectional imaging are discussed.

Detection and molecular monitoring of FIP1L1-PDGFRA-positive disease by analysis of patient-specific genomic DNA fusion junctions.

To evaluate current detection methods for FIP1L1-PDGFRA in hypereosinophilic syndrome (HES), we developed a means to rapidly amplify genomic break points. We screened 202 cases and detected genomic junctions in all samples previously identified as RT-PCR positive (n=43). Genomic fusions were amplified by single step PCR in all cases whereas only 22 (51%) were single step RT-PCR positive. Importantly, FIP1L1-PDGFRA was detected in two cases that initially tested negative by RT-PCR or fluorescence in situ hybridization. Absolute quantitation of the fusion by real-time PCR from genomic DNA (gDNA) using patient-specific primer/probe combinations at presentation (n=13) revealed a 40-fold variation between patients (range, 0.027-1.1 FIP1L1-PDGFRA copies/haploid genome). In follow up samples, quantitative analysis of gDNA gave 1-2 log greater sensitivity than RQ-PCR of cDNA. Minimal residual disease assessment using gDNA showed that 11 of 13 patients achieved complete molecular response to imatinib within a median of 9 months (range, 3-17) of starting treatment, with a sensitivity of detection of up to 1 in 10(5). One case relapsed with an acquired D842V mutation. We conclude that detection of FIP1L1-PDGFRA from gDNA is a useful adjunct to standard diagnostic procedures and enables more sensitive follow up of positive cases after treatment.

Fine needle aspiration cytology as an additional tool in the diagnosis of odontogenic keratocyst.

OBJECTIVE: The aim of this study was to assess the use of fine needle aspiration cytology (FNAC) in diagnosis of odontogenic keratocyst (OKC), as well as to describe the cytological and immunohistochemical features. METHODS: Eight consecutive patients submitted to FNAC and diagnosed with OKC were included in this study. FNAC was performed using 24-gauge needles attached to a 10-ml syringe, supported by a mechanical-syringe holder to facilitate aspiration. All cases provided a liquid or viscous content for smears that were either air-dried for Diff-Quick staining or immediately fixed in 95% alcohol and stained by the Papanicolaou technique. Incisional biopsies were carried out to confirm the diagnosis. Immunohistochemical reactions against anti-pan-cytokeratin (CK), CK14 and CK19 were performed in 3 microm sections obtained from cell blocks and biopsy specimens. RESULTS: Cytologically many isolated or groups of keratinocytes with normal or ill defined nuclei were seen, besides numerous anucleated squamous cells and keratinous debris. Immunohistochemically, the keratin lamellae were positive for pan-cytokeratin and CK19, but negative for CK14. In biopsy specimens, CK14 expression was restricted to basal cells, while only the superficial cells were positive for CK19. CONCLUSIONS: In summary, FNAC is useful, reliable and safe tool for the preoperative diagnosis of OKC.

An analysis of oral and maxillofacial pathology found in adults over a 30-year period.

BACKGROUND: The aim of this study was to determine the range of histologically diagnosed lesions in 44,000 oral and maxillofacial pathology specimens, from adults 17 years and older, submitted for diagnosis to our laboratory over a 30-year period (1973-2002). MATERIALS: All entries for specimens from the patients were retrieved and compiled into 12 diagnostic categories. RESULTS: During the period, 44,007 specimens comprised a male-to-female ratio of 0.9:1. The diagnostic category with the largest number of specimens was mucosal pathology (36.0%) followed by odontogenic cysts (13.8%). Malignant tumours accounted for 5.4% of all specimens and benign tumours 4.6%. CONCLUSION: This survey showed that while the majority of diagnoses are benign, approximately one in 19 cases required major head and neck surgery for malignant disease.

An analysis of oral and maxillofacial pathology found in children over a 30-year period.

AIM: The vast majority of oral diseases are confined to oral tissues, but numerous underlying systemic conditions may present with signs and symptoms within the oral cavity. Since the epidemiology of diseases is variable between regions, the authors carried out Europe's first paediatric-based survey of oral and maxillofacial pathology specimens submitted for diagnosis. DESIGN: All entries for specimens from children between the ages of 0 and 16 years during the 30-year period from 1973 to 2002 were retrieved and compiled into 12 diagnostic categories. RESULTS: During the study period, 4406 (8.2%) specimens came from children between the ages of 0 and 16 years, with a male to female ratio of 1.01. The diagnostic category with the largest number of specimens was tooth pathology (22.1%), followed by salivary gland disease (19.1%) and mucosal pathology (12.1%). In all, there were 114 benign tumours of nonodontogenic origin, 43 odontogenic tumours and 31 malignant tumours. The most frequently diagnosed lesions were mucous extravasation cysts, which accounted for over 16% of cases. Periapical pathology in the form of a radicular cyst, residual cyst or chronic periapical granuloma formed almost 13% of all cases. CONCLUSIONS: This survey shows that, while nearly 10% of specimens submitted to the authors' laboratory are from children under 16 years of age, the majority of lesions are of a benign nature, requiring minimal intervention; less than 1% of cases comprise malignant lesions. Odontogenic tumours are relatively rare in this age group; however, certain lesions such as adenomatoid odontogenic tumour and ameloblastic fibroma occur predominantly in children and, therefore, remain an important diagnostic consideration.

Range and demographics of odontogenic cysts diagnosed in a UK population over a 30-year period.

BACKGROUND: The aim of this study was to determine the range of all histologically diagnosed odontogenic cysts along with age range, sex distribution and site of presentation over a 30-year period. METHODS: All entries for odontogenic cysts occurring during 1975-2004 inclusive were retrieved and analysed for demographic data. RESULTS: A total of 55,446 specimens were received, of these 7121 (12.8%) specimens were diagnosed as odontogenic cysts. Radicular cyst was the most common diagnosis (52.3%), followed by dentigerous cyst (18.1) and odontogenic keratocysts (11.6%). CONCLUSIONS: Our study provides demographic data on a large series of odontogenic cysts in a European population. This is one of the largest series reported to date. Cysts such as the paradental cyst have a predilection for certain ages, sexes and sites. Odontogenic keratocysts and glandular odontogenic cysts have a marked propensity to recur as well as behave aggressively. It is essential that such lesions are detected as early as possible to minimize any necessary surgery.

Cough and hypereosinophilia due to FIP1L1-PDGFRA fusion gene with tyrosine kinase activity.

Eosinophil-associated conditions, such as asthma and eosinophilic bronchitis, have been associated with chronic persistent cough, usually responding to corticosteroid therapy. This case study reports a case of persistent cough associated with gastro-oesophageal reflux (GOR) and hypereosinophilia. Treatment of GOR with proton pump inhibitors and fundoplication did not control the cough. However, high dose prednisolone, but not inhaled corticosteroids, did. The presence of the FIP1L1-PDGFRA fusion gene in myeloid cells was confirmed by fluorescence in situ hybridisation analysis using CHIC2 deletion as a surrogate marker. The cough and other disease features were subsequently suppressed by the tyrosine kinase inhibitor, imatinib. This is the first case of persistent cough caused by hypereosinophilic syndrome characterised by FIP1L1-PDGFRA fusion gene and aberrant tyrosine kinase activity.

An integrated approach to scanning microscope data acquisition.

This paper describes an integrated data acquisition system built for a cryogenic STEM which is currently under construction in the authors' laboratory. Advantage was taken of modern digital and microprocessor techniques to construct a versatile and expandable system in the minimum time. The system is able to operate in conjunction with an external image processing computer or as an independent system with built-in image processing facilities.

Desaturation of linoleic acid from exogenous lipids by isolated chloroplasts.

When [14C]diacylgalactosylglycerol was added to isolated pea or lettuce chloroplasts linolenate synthesis was seen. The desaturation of [14C]linoleate in diacylgalactosylglycerol to [14C]linolenate was stimulated by the addition of a soluble protein fraction containing lipid-exchange activity. Other [14C]acyl lipids were ineffective, except that [14C]phosphatidylcholine in the presence of UDP-galactose and sn-glycerol 3-phosphate could also supply [14C]linoleate for desaturation. These results are consistent with a role of diacylgalactosylglycerol in linolenate synthesis, as indirectly suggested by labelling experiments.

Oncogenic derivatives of platelet-derived growth factor receptors.

Platelet-derived growth factor receptors (PDGFRs) and their ligands, platelet-derived growth factors (PDGFs) play critical roles in mesenchymal cell migration and proliferation. In embryogenesis the PDGFR/PDGF system is essential for the correct development of the kidney, cardiovascular system, brain, lung and connective tissue. In adults, PDGFR/PDGF is important in wound healing, inflammation and angiogenesis. Abnormalities of PDGFR/PDGF are thought to contribute to a number of human diseases, and especially malignancy. Constitutive activation of the PDGFRalpha or PDGFRbeta receptor tyrosine kinases is seen in myeloid malignancies as a consequence of fusion to diverse partner genes, and activating mutations of PDGFRalpha are seen in gastrointestinal tumours (GISTs). Autocrine signalling as a consequence of PDGF-B overexpression is clearly implicated in the pathogenesis of dermatofibrosarcoma protruberans (DFSP) and overexpression of PDGFRs and/or their ligands has been described in many solid tumours. PDGFR signalling is inhibited by imatinib mesylate, and this compound has clear clinical activity in patients with myeloid malignancies, GIST and DFSP.