Fermiology and Superconductivity of Topological Surface States in PdTe_{2}.

We study the low-energy surface electronic structure of the transition-metal dichalcogenide superconductor PdTe_{2} by spin- and angle-resolved photoemission, scanning tunneling microscopy, and density-functional theory-based supercell calculations. Comparing PdTe_{2} with its sister compound PtSe_{2}, we demonstrate how enhanced interlayer hopping in the Te-based material drives a band inversion within the antibonding p-orbital manifold well above the Fermi level. We show how this mediates spin-polarized topological surface states which form rich multivalley Fermi surfaces with complex spin textures. Scanning tunneling spectroscopy reveals type-II superconductivity at the surface, and moreover shows no evidence for an unconventional component of its superconducting order parameter, despite the presence of topological surface states.

Application of The Paris System to atypical urine cytology samples: correlation with histology and UroVysion® FISH.

OBJECTIVES: To evaluate whether atypical urine cytology cases may be stratified more objectively using The Paris System (TPS) and whether reclassified cases correlate with histology and UroVysion® results. METHODS: Atypical urine cytology cases subjected to UroVysion® testing over a period of 6 years were reviewed. Each case was reclassified according to TPS and correlated with histology and UroVysion® results. RESULTS: A total of 91 cases were identified; 70.3% were reclassified as 'negative for high-grade urothelial carcinoma (HGUC)' and 14.3% as 'atypical urothelial cells (AUC)'. The histological correlation was available in 45 cases. In the 'negative for HGUC' category, 67.9% had no histological evidence of malignancy, but 17.9% were diagnosed as HGUC. In the 'AUC' category, histology revealed urothelial carcinoma in 70% of the cases (of these, 71.4% were high grade). There was no histological evidence of malignancy in 30% of cases; notably, all of which were from patients under surveillance. The sensitivity and specificity of UroVysion® were 85.7% and 33.3% in the 'AUC' group and 62.5% and 100% in the 'negative for HGUC' group. CONCLUSIONS: The Paris System is an objective template for reporting urine cytology specimens, and is particularly useful in identifying HGUC cases and refining the category of 'AUC'.

ALK rearrangements in EBUS-derived transbronchial needle aspiration cytology in lung cancer.

OBJECTIVES: Patients with non-small cell lung cancer (NSCLC) positive for anaplastic lymphoma kinase (ALK) gene rearrangements may be treated successfully with the ALK inhibitor crizotinib. ALK copy-number abnormalities have also been described. In this study, we evaluated the suitability of fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) to determine ALK status in endobronchial ultrasound (EBUS)-derived cytology samples. METHODS: Samples were obtained from 55 consecutive patients with NSCLC who had undergone EBUS-transbronchial needle aspiration (TBNA) according to our standard clinical protocols. All tumours had been screened previously for epithelial growth factor receptor (EGFR) and v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations. FISH, using commercially available ALK rearrangement-specific probes, was employed to assess ALK status. IHC using the ALK-1 monoclonal antibody (DAKO) was also performed. RESULTS: FISH analysis was successful in 52 of 55 samples (94.5%); ALK rearrangement was demonstrated in 3 of 52 samples from patients with NSCLC (5.7%). ALK amplification was observed in 3 of 52 patient samples (5.7%) and an increase in ALK copy number was found in 28 of 52 patient samples (53.8%). IHC on cell blocks demonstrated ALK expression in one of three samples with ALK rearrangement. One patient sample had concomitant ALK rearrangement and KRAS mutation. CONCLUSIONS: We found FISH to be superior to IHC using the ALK-1 monoclonal antibody for the detection of ALK rearrangement in EBUS-TBNA cytology specimens in NSCLC, and also that ALK rearrangement can co-exist with KRAS mutation in the same tumour.

Autoimmune cytopenias in the 22q11.2 deletion syndrome.

We describe two cases of recurrent autoimmune cytopenias, which were subsequently diagnosed with a 22q11.2 deletion/DiGeorge syndrome. The cases are of particular interest as both possessed limited clinical features of this syndrome, and the investigation of haematological abnormalities led to the establishment of a definitive genetic diagnosis.

Genetic susceptibility to Hodgkin's disease and secondary neoplasias: FISH analysis reveals patients at high risk of developing secondary neoplasia.

BACKGROUND: Cytotoxic drugs administered before high-dose therapy (HDT) represent a significant factor in the development of leukemic complications in patients with lymphoid malignancies. This retrospective study was used to detect evidence of abnormal therapy-related myelodysplasia/secondary acute myeloid leukaemia (tMDS/sAML) clones before HDT in a subset of patients who subsequently developed secondary neoplasia. PATIENTS AND METHODS: 230 patients with non-Hodgkin's lymphoma (NHL) underwent HDT comprising cyclophosphamide and total body irradiation (TBI) with autologous hematopoietic progenitor-cell support. Thirty-three patients have developed tMDS/sAML and 20 of these were screened for the presence of emerging therapy-related abnormalities before HDT. A further 24 patients without evidence of secondary neoplasia were screened using fluorescence in situ hybridisation (FISH). RESULTS: Significant levels of abnormal cells were identified in 20/20 patients screened who have developed secondary neoplasia compared with only three of 24 patients in the HDT control group who have not. The latter three patients have since died. CONCLUSIONS: The triple FISH assay was developed to detect loss of chromosomal material from 5q31, 7q22 and 13q14. It can potentially identify those patients at risk of alkylating agent-induced leukaemia before they proceed to HDT. Used in a prospective manner, the triple FISH assay could permit more informed clinical management.

The characterisation of the lymphoma cell line U937, using comparative genomic hybridisation and multi-plex FISH.

The cell line U937, which has been used extensively for studies of myeloid differentiation, bears the t(10;11)(p13;q14) translocation which results in a fusion between the MLLT10 (myeloid/lymphoid or mixed-lineage leukemia [trithorax, Drosophila, homolog]; translocated to 10; alias AF10) gene and the Ap-3-like clathrin assembly protein, PICALM (Clathrin assembly lymphoid myeloid leukaemia). Apart from this translocation, very little is known about the other genetic alterations in this cell line that may represent significant events in disease progression. In this study, conventional G-banding, CGH and M-FISH have been used to characterise fully all of the cytogenetic alterations present in the U937 cell line. M-FISH analysis confirmed the presence of the t(10;11) and an apparently normal copy of both chromosomes 10 and 11. A t(1;5) translocation was observed as well as several unbalanced rearrangements. CGH detected amplifications resulting from duplications of 2q, 6p and 13q. These changes could result in fusion gene products involved in carcinogenesis or the positions of putative oncogenes and tumour suppressor genes. A good correlation between conventional G-banding, CGH and M-FISH was observed.

Localisation of a novel region of recurrent amplification in follicular lymphoma to an approximately 6.8 Mb region of 13q32-33.

Follicular lymphoma (FL) is characterised by the presence of the t(14;18)(q32;q21) and represents approximately 25% of new cases of non-Hodgkin's lymphoma. While the t(14;18) is a well-documented rearrangement, the role of secondary cytogenetic abnormalities in the development and progression of these tumours remains unclear. Comparative genomic hybridisation was used to characterise changes in DNA copy number in tumour DNA from patients with this malignancy. The mean numbers of deletion and amplification events found in each of the 45 samples studied were 1.8 and 2.3, respectively. Regions of recurrent (>10% tumour samples) gain involved chromosomes 2p13-16 (16%), 7 (20%), 12 (16%), 13q21-33 (18%), 18 (27%), and X (36%) and frequent losses localised to 6q (29%) and 17p (20%). Amplification of chromosome 13 represents a novel finding in FL. The minimal amplified region was refined to a 6.8-Mb interval of 13q32-33 between the BAC clones 88K16 and 44H20 by fluorescence in situ hybridisation studies using metaphase chromosomes derived from tumour material. There are a number of reports in the literature suggesting that amplification of chromosome 13 also occurs in other human cancers. The location of the putative oncogene on 13q described here in follicular and transformed lymphoma may also be important in the evolution of many other malignancies.

Identification and molecular characterisation of a CALM-AF10 fusion in acute megakaryoblastic leukaemia.

The t(10;11)(p13;q14-21) is a non-random translocation described in acute lymphoblastic and myeloid leukaemias. It results in the fusion of the gene CALM, which encodes a clathrin assembly protein, on 11q14 to the gene AF10, a putative transcription factor on 10p13. Here we describe for the first time, the occurrence of a CALM-AF10 fusion in a case of acute megakaryoblastic leukaemia. Fluorescence in situ hybridisation and reverse transcriptase polymerase chain reaction were used to confirm the presence of a CALM-AF10 fusion. A novel splice variant of CALM missing nt 1927-2091 was also detected. Though CALM is a cytoplasmic protein, the chimaeric fusion product is able to localise to both the nucleus and cytoplasm. Analysis of the fusion variants suggests, however, that the critical fusion product is likely to be cytoplasmic and contain the interactive leucine zipper of AF10.

Detection of chromosome abnormalities pre-high-dose treatment in patients developing therapy-related myelodysplasia and secondary acute myelogenous leukemia after treatment for non-Hodgkin's lymphoma.

PURPOSE: To assess whether pre-high-dose therapy (HDT)-related factors play a critical role in the development of therapy-related myelodysplasia (tMDS) or secondary acute myelogenous leukemia (sAML). PATIENTS AND METHODS: Twenty-nine of 230 patients with a primary diagnosis of non-Hodgkin's lymphoma (NHL) developed tMDS/sAML after HDT comprising cyclophosphamide and total-body irradiation (TBI) supported by autologous hematopoietic progenitor cells. G-banding and fluorescence in-situ hybridization (FISH) were used to detect clonal cytogenetic abnormalities. RESULTS: The majority of patients showed complex karyotypes at diagnosis of tMDS/sAML containing, in particular, complete or partial loss of chromosomes 5 and/or 7. Using single locus-specific FISH probes, significant levels of clonally abnormal cells were found before HDT in 20 of 20 tMDS/sAML patients screened, compared with three of 24 patients screened who currently have not developed tMDS/sAML, at a median follow-up of 5.9 years after HDT. CONCLUSION: Prior cytotoxic therapy may play an important etiologic role and may predispose to the development of tMDS/sAML. Using a triple FISH assay designed to detect loss of chromosomal material from 5q31, 7q22, or 13q14, significant levels of abnormal cells can be detected before HDT and may predict which patients are at increased risk of developing secondary disease. Further prospective evaluation of this FISH assay is warranted to determine its predictive power in this setting.

The use of real-time quantitative polymerase chain reaction and comparative genomic hybridization to identify amplification of the REL gene in follicular lymphoma.

Using comparative genomic hybridization (CGH), aberrations in DNA copy number were studied before and after transformation of follicular lymphoma to diffuse large B-cell lymphoma in six patients (15 lymph node biopsies in total). The most common and also the most discrete and intense amplification occurring in four out of 15 biopsies from three different patients was of 2p13-16. Using real-time quantitative polymerase chain reaction (RQ-PCR), REL amplification was found to be implicated at this locus. This technique also identified amplified REL in a further two biopsies, presumably below the detection level of CGH. REL amplification was quantified by comparing it, in most cases, with three endogenous reference genes, albumin, beta2-microglobulin and CD8alpha, that lie close to REL on 2p. There was no correlation apparent between 2p13-16 amplification or REL amplification and transformation. This study shows the usefulness of coupling CGH, for detecting recurring abnormalities, with the real-time PCR technique for rapid gene dosage quantification and confirms that the REL gene is a potential candidate in the pathogenesis of a particular subset of follicular lymphomas.

Cytogenetic and molecular evidence of marrow involvement in extramedullary acute myeloid leukaemia.

A diagnosis of granulocytic sarcoma was made in a 2-year-old child based on the detection of myelomonocytic blasts in tissue obtained from a subcutaneous nodule with no evidence of concomitant disease in the bone marrow. The child responded to systemic chemotherapy and is in remission 3 years later. An identical clone with an in frame fusion of the MLL and AF10 genes was identified from both tissue and bone marrow samples. The generation of an in frame MLL-AF10 fusion requires complex intra- and interchromosomal exchanges between chromosomes 10 and 11. In this case, an intrachromosomal rearrangement of chromosome 5 was also observed. This case illustrates the presence of systemic disease in extramedullary leukaemia, its response to systemic rather than topical therapy and suggests that the events leading to chromosomal translocations in leukaemia may be part of a generalized intracellular event.

Characterization of acute promyelocytic leukemia cases lacking the classic t(15;17): results of the European Working Party. Groupe Français de Cytogénétique Hématologique, Groupe de Français d'Hematologie Cellulaire, UK Cancer Cytogenetics Group and BIOMED 1 European Community-Concerted Action "Molecular Cytogenetic Diagnosis in Haematological Malignancies".

Acute promyelocytic leukemia (APL) is typified by the t(15;17), generating the PML-RAR alpha fusion and predicting a beneficial response to retinoids. However, a sizeable minority of APL cases lack the classic t(15;17), prompting the establishment of the European Working Party to further characterize this group. Such cases were referred to a workshop held in Monza, Italy and subjected to morphologic, cytogenetic, and molecular review, yielding 60 evaluable patients. In the majority (42 of 60), molecular analyses revealed underlying PML/RAR alpha rearrangements due to insertions (28 of 42) or more complex mechanisms, including 3-way and simple variant translocations (14 of 42). Metaphase fluorescence in situ hybridization (FISH) demonstrated that insertions most commonly led to formation of the PML-RAR alpha fusion gene on 15q. In 11 of 60 workshop patients, PLZF/RAR alpha rearrangements were identified, including 2 patients lacking the t(11;17)(q23;q21). In one case with a normal karyotype, FISH analysis revealed insertion of RAR alpha into 11q23, and PLZF-RAR alpha was the sole fusion gene formed. Two patients were found to have t(5;17), one with a diffuse nuclear NPM staining pattern and with NPM-RAR alpha and RAR alpha-NPM transcripts detected. In the other with an unbalanced der(5)t(5;17)(q13;q21) and a nucleolar NPM localization pattern, an NPM/RAR alpha rearrangement was excluded, and FISH revealed deletion of one RAR alpha allele. In the remaining 5 workshop patients, no evidence was found for a rearrangement of RAR alpha, indicating that in rare instances, alternative mechanisms could mediate the differentiation block that typifies this disease. This study highlights the importance of combining morphologic, cytogenetic, and molecular analyses for optimal management of APL patients and better understanding of the pathogenesis of the disease. (Blood. 2000;96:1297-1308)

Therapy-related myelodysplasia and secondary acute myelogenous leukemia after high-dose therapy with autologous hematopoietic progenitor-cell support for lymphoid malignancies.

PURPOSE: To evaluate the incidence of and risk factors for therapy-related myelodysplasia (tMDS) and secondary acute myelogenous leukemia (sAML), after high-dose therapy (HDT) with autologous bone marrow or peripheral-blood progenitor-cell support, in patients with non-Hodgkin's lymphoma (NHL). PATIENTS AND METHODS: Between January 1985 and November 1996, 230 patients underwent HDT comprising cyclophosphamide therapy and total-body irradiation, with autologous hematopoietic progenitor-cell support, as consolidation of remission. With a median follow-up of 6 years, 27 (12%) developed tMDS or sAML. RESULTS: Median time to development of tMDS or sAML was 4.4 years (range, 11 months to 8.8 years) after HDT. Karyotyping (performed in 24 cases) at diagnosis of tMDS or sAML revealed complex karyotypes in 18 patients. Seventeen patients had monosomy 5/5q-, 15 had -7/7q-, seven had -18/18q-, seven had -13/13q-, and four had -20/20q-. Twenty-one patients died from complications of tMDS or sAML or treatment for tMDS or sAML, at a median of 10 months (range, 0 to 26 months). Sixteen died without evidence of recurrent lymphoma. Six patients were alive at a median follow-up of 6 months (range, 2 to 22 months) after diagnosis of tMDS or sAML. On multivariate analysis, prior fludarabine therapy (P =.009) and older age (P =.02) were associated with the development of tMDS or sAML. Increased interval from diagnosis to HDT and bone marrow involvement at diagnosis were of borderline significance (P =.05 and.07, respectively). CONCLUSION: tMDS and sAML are serious complications of HDT for NHL and are associated with very poor prognosis. Alternative strategies for reducing their incidence and for treatment are needed.

A new recurrent translocation, t(5;11)(q35;p15.5), associated with del(5q) in childhood acute myeloid leukemia. The UK Cancer Cytogenetics Group (UKCCG)

Partial deletion of the long arm of chromosome 5, del(5q), is the cytogenetic hallmark of the 5q-syndrome, a distinct subtype of myelodysplastic syndrome-refractory anemia (MDS-RA). Deletions of 5q also occur in the full spectrum of other de novo and therapy-related MDS and acute myeloid leukemia (AML) types, most often in association with other chromosome abnormalities. However, the loss of genetic material from 5q is believed to be of primary importance in the pathogenesis of all del(5q) disorders. In the present study, we performed fluorescence in situ hybridization (FISH) studies using a chromosome 5-specific whole chromosome painting probe and a 5q subtelomeric probe to determine the incidence of cryptic translocations. We studied archival fixed chromosome suspensions from 36 patients with myeloid disorders (predominantly MDS and AML) and del(5q) as the sole abnormality. In 3 AML patients studied, this identified a translocation of 5q subtelomeric sequences from the del(5q) to the short arm of an apparently normal chromosome 11. FISH with chromosome 11-specific subtelomeric probes confirmed the presence of 11p on the shortened 5q. Further FISH mapping confirmed that the 5q and 11p translocation breakpoints were the same in all 3 cases, between the nucleophosmin (NPM1) and fms-related tyrosine kinase 4 (FLT4) genes on 5q35 and the Harvey ras-1-related gene complex (HRC) and the radixin pseudogene (RDPX1) on 11p15.5. Importantly, all 3 patients with the cryptic t(5;11) were children: a total of 3 of 4 AML children studied. Two were classified as AML-M2 and the third was classified as M4. All 3 responded poorly to treatment and had short survival times, ranging from 10 to 18 months. Although del(5q) is rare in childhood AML, this study indicates that, within this subgroup, the incidence of cryptic t(5;11) may be high. It is significant that none of the 24 MDS patients studied, including 11 confirmed as having 5q-syndrome, had the translocation. Therefore, this appears to be a new nonrandom chromosomal translocation, specifically associated with childhood AML with a differentiated blast cell phenotype and the presence of a del(5q).