Clinical validation of a type-specific real-time quantitative human papillomavirus PCR against the performance of hybrid capture 2 for the purpose of cervical cancer screening.

To be acceptable for use in cervical cancer screening, a new assay that detects DNA of high-risk human papillomavirus (hrHPV) types must demonstrate high reproducibility and performance not inferior to that of a clinically validated HPV test. In the present study, a real-time quantitative PCR (qPCR) assay targeting the E6 and E7 genes of hrHPV was compared with Hybrid Capture 2 (hc2) in a Belgian cervical cancer screening setting. In women >30 years old, the sensitivity and specificity for intraepithelial neoplasias of grade 2 or worse (93 cases of cervical intraepithelial neoplasias of grade 2 or worse (CIN2+) and 1,207 cases of no CIN or CIN1) were 93.6% and 95.6%, respectively, and those of hc2 were 83.9% and 94.5%, respectively {relative sensitivity of qPCR/hc2 = 1.12 [95% confidence interval (CI), 1.01 to 1.23]; relative specificity = 1.01 [95% CI, 0.99 to 1.03]}. A score test showed that the sensitivity (P < 0.0001) and specificity (P < 0.0001) of the qPCR assay were not inferior to those of hc2 at the required thresholds of 90% and 98%, respectively. The overall agreement of hrHPV positivity between the two runs of the qPCR tests was 98.7% (95% CI, 97.5 to 99.4%), with a kappa value of 0.96 (95% CI, 0.83 to 1.00). The qPCR assay used in this study can be considered a reliable HPV assay that fulfills the clinical validation criteria defined for use in cervical cancer screening.

Translocation t(1;6)(p35.3;p25.2): a new recurrent aberration in "unmutated" B-CLL.

Although reciprocal chromosomal translocations are not typical for B-cell chronic lymphocytic leukemia (B-CLL), we identified the novel t(1;6)(p35.3;p25.2) in eight patients with this disorder. Interestingly, all cases showed lack of somatically mutated IgV(H). Clinical, morphological, immunologic, and genetic features of these patients are described. Briefly, the age ranged from 33 to 81 years (median: 62.5 years) and the sex ratio was 6M:2F. Most of the patients (6/8) presented with advanced clinical stage. Therapy was required in seven cases. After a median follow-up of 28 months, five patients are alive and three died from disease evolution. Three cases developed transformation into diffuse large B-cell lymphoma. Translocation t(1;6) was found as the primary karyotypic abnormality in three patients. Additional chromosomal aberrations included changes frequently found in unmutated B-CLL, that is, del(11)(q), trisomy 12 and 17p aberrations. Fluorescence in situ hybridization analysis performed in seven cases allowed us to map the t(1;6) breakpoints to the 1p35.3 and 6p25.2 chromosomal bands, respectively. The latter breakpoint was located in the genomic region coding for MUM1/IRF4, one of the key regulators of lymphocyte development and proliferation, suggesting involvement of this gene in the t(1;6). Molecular characterization of the t(1;6)(p35.3;p25.2), exclusively found in unmutated subtype of B-CLL, is in progress.

Congenital dyserythropoietic anaemia type II: a case study.

A 13-year-old girl with chronic anaemia showed features of congenital dyserythropoietic anaemia (CDA) type II. The main clinical and haematological findings were splenomegaly, a mild microcytic anaemia, and numerous bizarre and binucleate normoblasts in the bone marrow. The acidified serum lysis test (Ham's test) performed with 5 normal sera was positive. The patient's red blood cells showed a markedly increased expression of the i red blood cell antigen.

Simultaneous occurrence of myelodysplastic syndrome and monoclonal B lymphocytes with a different clonal origin.

Bone marrow and peripheral blood from a myelodysplastic syndrome patient with trisomy 13 and monoclonal B lymphocytes (without evidence of systemic lymphoma) were investigated for clonal lymphoid lineage involvement using interphase fluorescence in situ hybridization (FISH) and X-chromosome inactivation assay (HUMARA) on CD19+ and CD34+ sorted cells. Trisomy 13 was detected in 55% of CD34+ cells and in 5.5% of CD19+ cells, the latter being comparable to the negative control specimen. X-chromosome inactivation showed both CD34+ and CD19+ cells to be monoclonal, though their inactivated X-chromosome was different. The results strongly suggested that both populations of CD34+ and CD19+ cells have originated from a different progenitor stem cell.

Standardisation of multiplex fluorescent short tandem repeat analysis for chimerism testing.

To evaluate the origin of cells after allogeneic haematopoietic stem cell transplantation we optimised and evaluated two commercially available systems (AmpFlSTR Profiler Plus and GenePrint Powerplex-16) which are based on multiplex fluorescent short tandem repeat (STR) analysis. A standard procedure for interpretation of electropherographs was found essential to obtain reproducible results. On the basis of the relative length of donor and recipient alleles, TYPE-I (no shared alleles are used to calculate chimerism), TYPE-II (one shared and one unshared allele is used to calculate chimerism) or TYPE-III (not informative) allelic distribution types were distinguished. Also, stutter peaks were recognised as an important criterion to exclude a marker for analysis. Intralaboratory and multicentre evaluation of the AmpFlSTR Profiler Plus system showed that mixed blood samples could be determined with an absolute deviation of less than 2%. A sensitivity threshold was set at 5% for TYPE-I and 10% for TYPE-II markers since relative imprecision increases at low chimerism values. No significant difference of calculated chimerism values was observed between STR markers shared between both systems. By monitoring 26 allogeneic peripheral blood stem cell transplants, the applicability of the proposed method was demonstrated.

The concept of typical and atypical chronic lymphocytic leukaemia.

Subdivision of CLL into typical and atypical subtypes, as proposed by the FAB group in 1989, is not yet widely accepted and its clinical significance is still debated. In recent years, however, a strong correlation was found between atypical morphology trisomy 12 and an aberrant immunophenotype. In the first part of this review we discuss current concepts and generally accepted data on morphology, immunophenotype, genetic abnormalities, clinical features and prognostic factors in CLL. Subsequently, based on our own series and other recently published data, we analyse the validity and clinical impact of classifying CLL into typical and atypical entities and demonstrate that they may represent two closely related but different entities.

Trisomy 6 is the hallmark of a dysplastic clone in bone marrow aplasia.

Clonal hematopoiesis with trisomy 6 as the sole karyotypic change was revealed by cytogenetics in two cases of aplastic anemia. In both patients, dyserythropoiesis was characterized by asynchrony of maturation between nucleus and cytoplasm, binucleated elements, and intercytoplasmic connections. In addition to conventional cytogenetics, the size of the trisomic clone was evaluated by fluorescence in situ hybridization on fixed cells at diagnosis and in the course of the disease by using an alpha-satellite centromeric probe for chromosome 6. Moreover, in situ hybridization on bone marrow smears showed that dysplastic erythrocytes as well as myeloid cells belonged to the trisomic clone. Trisomy 6 identifies a subgroup of hematologic disorders with bone marrow hypo-aplasia and dyserythropoiesis.

Trisomy 3q11-q29 is recurrently observed in B-cell non-Hodgkin's lymphomas associated with cold agglutinin syndrome.

Three cases of low-grade B-cell non-Hodgkin's lymphoma associated with cold agglutinin syndrome, cytogenetically characterized by partial trisomy 3, are presented in this report. Our data suggest that the long arm of chromosome 3 might be of particular importance in the pathogenesis of this subgroup of lymphomas.

Lymph node histology in typical and atypical chronic lymphocytic leukemia.

According to the French-American-British (FAB) proposal on the classification of chronic lymphoid leukemia (CLL), the disorder can be subdivided into typical and atypical CLL. We recently demonstrated the prognostic significance of this subgrouping and based on these results we suggested that typical and atypical CLL represent two closely related, but different entities. These results prompted us to investigate 42 patients diagnosed with CLL based on the results of lymph node biopsy in order to identify the histologic counterpart of the CLL variants. A first group of 14 cases showed a monomorphic proliferation of small round lymphocytes associated with the occurrence of small pseudofollicles. All these cases were diagnosed as typical CLL on peripheral blood (13 cases) or bone marrow smear (1 case). The remaining 28 cases showed aberrant histologic features characterized by the presence of large numbers of paraimmunoblasts and prolymphocytes, forming very large pseudofollicles, and/or by nuclear irregularities of the neoplastic cells. Based on peripheral blood smears (22 cases) or bone marrow smears (six cases), two cases showed no peripheral blood involvement, 21 cases were diagnosed as atypical CLL, and five as typical CLL. From these data we can conclude that a histologic counterpart of the CLL variants recognized in the FAB proposal does exist; moreover, our data may explain reports on lymph node involvement by CLL composed of small cleaved cells and clarify the occurrence of pseudofollicles in cases described as mantle cell lymphomas.

FISH identifies different types of duplications with 12q13-15 as the commonly involved segment in B-cell lymphoproliferative malignancies characterized by partial trisomy 12.

Clinical, cytogenetic, fluorescence in situ hybridization (FISH), and Southern blot data of 18 patients with different subtypes of B-cell non-Hodgkin's lymphoma, cytogenetically characterized by partial trisomy 12, are presented. These chromosomal changes occurred predominantly in clinically progressive chronic lymphocytic leukemia, mixed cell type, and advanced-stage follicle center cell lymphoma at the time of relapse or transformation into diffuse large cell lymphoma. Partial trisomy 12 consistently included the long arm of chromosome 12, either completely or partially, and resulted from dup(12q) or other rearrangements involving chromosome 12. The duplications were cytogenetically identified as dup(12)(q13q23), dup(12)(q13q22), or dup(12)(q13q15) in follicle center cell lymphoma or t(14;18)-positive diffuse large cell lymphoma; dup(12)(q13q22) or dup(12)(q13q24) in chronic lymphocytic leukemia; and dup(12)(q13q21) in a case of t(14;18)-negative diffuse large cell lymphoma. FISH, using library probes and a panel of YAC probes, mapped along the long arm of chromosome 12, confirmed the cytogenetic results in all cases analyzed except for three cases of t(14;18)-positive follicle center lymphoma or diffuse large cell lymphoma with dup(12q). In these cases, FISH showed similar, possibly identical, duplications, which involved a region more centromeric (12q11-21) than assumed by karyotypic analysis (12q13-22 or 12q13-23) and included alphoid DNA sequences, a combination hitherto unknown. In addition, commonly duplicated regions of chromosome 12 could be defined: 12q11-21, including alphoid DNA sequences for follicle center cell lymphoma or t(14;18)-positive diffuse large cell lymphoma, 12q13-22 for chronic lymphocytic leukemia, and 12p13-q15 for marginal zone cell lymphoma, all of which overlapped in 12q13-15. Whether these regions, especially 12q13-15, may contain genes which are important in malignant transformation or disease progression of B-cell lymphoproliferative malignancies characterized by complete or partial trisomy 12 remains to be determined.

Further characterization of morphologically defined typical and atypical CLL: a clinical, immunophenotypic, cytogenetic and prognostic study on 390 cases.

We analysed a group of 390 patients, diagnosed with chronic lymphocytic leukaemia (CLL). Cases were subclassified as morphologically typical and atypical CLL according to the criteria of the FAB proposal. Typical CLL cases were mostly diagnosed at a low-risk stage (Binet A/Rai 0), required no immediate treatment and expected a long survival; atypical CLL cases mostly presented at a more advanced risk stage (Binet B/Rai I-II), usually required immediate treatment and their survival was shorter. Moreover, clinical staging was of prognostic significance in typical but not in atypical cases. In typical CLL, del(11q) was the most common chromosomal abnormality (21%) whereas in atypical CLL trisomy 12 was found in about 65% of the cases documented with an abnormal karyotype. Although chromosomal abnormalities were associated with a poor survival in typical CLL, they are of no prognostic significance in atypical CLL. Based on these data, we conclude that subtyping CLL by morphology enables the identification of two groups of cases, each characterized by a specific clinical presentation, different cytogenetic abnormalities and prognostic parameters. We speculate that these two groups may represent two related, but different, diseases with different prognostic parameters and a different survival.

Genetic abnormalities in chronic lymphocytic leukemia and their clinical and prognostic implications.

Clonal chromosome abnormalities can be detected in approximately 50% of patients with chronic lymphocytic leukemia (CLL). The most common changes are trisomy 12, followed by structural abnormalities of 13q, 11q, 6q, and 14q. By fluorescence in situ hybridization (FISH), these aberrations can be demonstrated even in cases with insufficient mitotic yield or a normal karyotype. The biologic consequences of trisomy 12 are unknown, but a gene dosage effect is suspected and studies on partial trisomy 12 indicate that the region 12q13 to 12q22 might be of particular pathogenetic importance. Trisomy 12 is strongly associated with atypical lymphocyte morphology and seems to be a secondary event in leukemogenesis, as shown by combined immunophenotyping and interphase FISH. Structural abnormalities of 13q frequently involve hetero- and homozygous deletions of a region in 13q14, distal to the retinoblastoma gene, which may be the site of a tumor suppressor gene. In contrast to a normal karyotype or structural changes of 13q, complex karyotypic abnormalities, high percentage of abnormal metaphases, trisomy 12 and structural changes involving the P53 tumor suppressor gene on 17p13 are adverse prognostic indicators. Cytogenetic and molecular findings provide important diagnostic, clinical, and prognostic information which can contribute to treatment decisions and follow-up of CLL patients.

A new subtype of pre-B acute lymphoblastic leukemia with t(5;12)(q31q33;p12), molecularly and cytogenetically distinct from t(5;12) in chronic myelomonocytic leukemia.

Translocation t(5;12)(q33;p13), resulting in an ETV6/PDGFRB gene fusion, is a recurrent chromosomal abnormality associated with chronic myelomonocytic leukemia (CMML). An analogous translocation was also found in four cell lines with features of pre-B acute lymphoblastic leukemia (ALL). Using fluorescence in situ hybridization (FISH) we show here that in three of these cell lines identical complex rearrangements occurred. However, the regions involved on 5q and 12p are different from the breakpoints in CMML, and the translocation is accompanied by seemingly identical cryptic deletions of both 5q and 12p chromosome sequences in all analyzed pre-B ALL cell lines. The similar cytogenetic, FISH, and immunophenotyping findings in the three cell lines suggest that the t(5;12)(q31q33;p12) defines a new entity of pre-B ALL.

Fusion of ETV6 to MDS1/EVI1 as a result of t(3;12)(q26;p13) in myeloproliferative disorders.

We identified a fusion between ETV6 on 12p13 and MDS1/EVI1 on 3q26 in a t(3;12)(q26;p13) found in two cases of myeloproliferative disorder. The resulting chimeric transcript consists of the first two exons of ETV6 fused to MDS1 sequences, which in turn is fused to the second exon of the EVI1 gene. It has recently been reported that MDS1 can be expressed in normal tissues both as a single gene and fused to EVI1. ETV6 does not contribute any known functional domain to the predicted fusion protein. Association with blast crisis and myelodysplastic syndrome-derived leukemia, bad prognosis, and relative complex karyotype are in agreement with observations made in other cases of t(3;12)(q26;p13). Furthermore, a comparison can be made with the formation of an AML1/MDS1/EVI1 fusion gene in translocations (3;21)(q26;q22).

del(7q) in chronic B-cell lymphoid malignancies.

Twelve patients with diagnosis of B-cell non-Hodgkin's lymphoma/leukemia and del[7q] were studied for their clinical, cytogenetic, and molecular characteristics. Eleven patients were classified as small cell lymphoma whereas one had a diffuse large cell lymphoma. Lymphoplasmacytic features were observed in six out of eleven small cell lymphomas. Morphologically and immunologically these small cell lymphomas could be classified as chronic lymphocytic leukemia (typical or atypical; 4 cases), marginal zone lymphoma (splenic lymphoma with villous lymphocytes; 1 case), mantle cell lymphoma (2 cases), or nonspecified, non-Hodgkin's lymphoma (4 cases). Eleven of twelve patients presented with peripheral blood and bone marrow involvement. Two of twelve cases showed del[7q] as the sole anomaly. Two different types of deletions were present: ten cases had del(7)(q21q31) and two cases had del(7)(q31q34). Cases that could be molecularly investigated did not show any involvement of BCL2, BCL3, or BCL6, and only one case had BCL1 rearrangement. The data indicate that del(7q) is associated with a subset of mature small B-cell lymphoproliferative disorders of which some but not all show lymphoplasmatic features.

Comparative study of antiphospholipid antibody detection in eleven Belgian laboratories.

Twenty-six plasma samples have been sent to 11 different Belgian laboratories in order to detect the presence of antiphospholipid antibodies, either by immunological methods and/or by coagulation tests. A good concordance between laboratories was observed for coagulation tests. Laboratories using detection tests and performing mixing procedures and neutralisation procedures displayed the highest sensitivity as compared with laboratories which did not perform one of these two latter procedures. The concordance between laboratories for the immunological methods was much worse as compared with coagulation tests. This may be attributable either to an intrinsic problem of the immunological tests or to a selection bias due the fact that the plasmas used in this study were selected in coagulation laboratories only where the chance to find a lupus anticoagulant positive/ELISA antiphospholipid negative sample is high.

Small B cell NHL and their leukemic counterpart: differences in subtyping and assessment of leukemic spread.

Three subtypes of small lymphocytic lymphoma were studied, namely B cell chronic lymphocytic leukemia (B-CLL), mantle cell lymphoma (MCL) and follicle center lymphoma (FCL). Agreement between tissue diagnosis, based on the proposal for a revised European-American classification of lymphoid neoplasms from the International Lymphoma Study Group, and the cytomorphological diagnosis on peripheral blood and/or bone marrow smears, using the proposals for the classification of chronic (mature) B and T lymphoid leukemias of the French-American-British Cooperative Group, was studied. Full agreement was found in 90% of the CLL and 82% of the FCL cases. In MCL cases, agreement was 65% including all cases classified as intermediate/mantle zone lymphoma according to FAB criteria. The incidence of bone marrow involvement detection in trephines compared to smears was equal in CLL (both 100%) and slightly higher in MCL (56 vs 48.5%); in FCL, however, trephine biopsies provided more reliable material (71 vs 35%).

Trisomy 3 in marginal zone B-cell lymphoma: a study based on cytogenetic analysis and fluorescence in situ hybridization.

Trisomy 3 represents the most frequent and consistent chromosomal abnormality characterizing the recently defined entity marginal zone B-cell lymphoma (MZBCL). By cytogenetic analysis and/or fluorescence in situ hybridization (FISH) on interphase nuclei we found in increased copy number of chromosome 3 in 22/36 (61%) successfully analysed cases, including 8/12 cases with extranodal MZBCL, 8/13 cases with nodal MZBCL, and 6/11 patients with splenic MZBCL. Sensitivity of interphase cytogenetics was somewhat higher than that of conventional cytogenetic investigation. Structural chromosomal changes involving at least one chromosome 3 were seen in 11/20 cases with an increased copy number of chromosome 3: +de(3)(p13) was demonstrated in three cases, and was the sole chromosomal abnormality in one of them; +i(3)(q10) was seen in two other patients; and rearrangements involving various breakpoints on the long arm of chromosome 3 were found in the remaining cases. FISH on metaphase spreads confirmed these structural abnormalities and additionally showed two unexpected translocations involving chromosome 3. We conclude that: (1) trisomy 3 occurs in a high proportion of extranodal, nodal and splenic MZBCL; (2) FISH on interphase nuclei is an additional and sensitive tool in detecting an increased copy number of chromosome 3 in MZBCL; (3) additional structural abnormalities involving the long arm of chromosome 3 are frequent but non-recurrent and are perhaps secondary changes; and (4) abnormalities such as +del(3)(pl3) and +i(3)(q10) suggest that genes located on the long arm of chromosome 3 are of particular importance in the pathogenesis of MZBCL.

Prognostic significance of bone marrow trephine and peripheral blood smears in 55 patients with mantle cell lymphoma.

Bone marrow trephine and peripheral blood smears taken at diagnosis of 55 cases of well-documented mantle cell lymphomas were reviewed in order to analyse the leukaemic involvement in this non-Hodgkin's lymphoma: its incidence, morphological characteristics and prognostic significance. A median survival of 36 months was found. The median age was 61 and the male to female ratio was 4:1. Morphologically 7 cases presented with a mantle zone pattern, all the others had a diffuse pattern. Involvement of the bone marrow was found in 58% and a trend for prolonged survival in patients with a negative trephine was seen. An absolute lymphocytosis above 10,000 mu l was found at diagnosis in 5 cases (10%) and had a statistically significant impact on survival. An additional 5 cases developed frank leukaemia during the course of the disease and died within 1 to 6 months of this evolution, suggesting that marked lymphocytosis is more a terminal event associated with an extremely poor prognosis than a presenting symptom. Finally we identified an additional parameter with statistically prognostic significance, namely, the presence of atypical cells in the peripheral blood even in the absence of an increased lymphocytosis.

Deletions of the long arm of chromosome 7 in myeloid disorders: loss of band 7q32 implies worst prognosis.

Clinical and cytogenetic data were analysed in 54 patients with acute non-lymphocytic leukaemias (ANLL) or MDS (myelodysplastic syndromes) and deletion of the long arm of chromosome 7 (7q-), in order to determine if there is a commonly deleted region in 7q and to establish possible correlations between karyotypic features, such as karyotype pattern, karyotype complexity, associated anomalies, and/or the type of deleted segments, and outcome of patients with these disorders. The median follow-up of our patients was 4 months (range 1-89), as was the median survival. In 30% of the cases there was a history of preceding MDS or previous chemotherapy. Clinical and cytogenetic remission was obtained in 7/36 patients treated with chemotherapy (CT). At the time of 7q- detection, three patients previously treated with CT for ANLL were in clinical remission. 5q- was the most recurrent associated abnormality. Complex karyotypes were observed in 68% of the cases. In univariate analysis, statistical differences in survival were observed according to diagnosis (therapy-related and secondary diseases had a worse prognosis than primary disorders), the chromosomal segments deleted (the loss of band 7q32 was of poor prognostic value), the karyotype complexity (patients with single anomalies did better than patients with complex anomalies) and the response to therapy (patients who achieved complete remission had a better survival probability). In multivariate analysis, the loss of band 7q32 was found to be significantly related to very poor prognosis. This finding suggests that band 7q32 may contain critical genes that should be explored at the molecular level.