Immunophenotypic analysis reveals heterogeneity and common biologic aspects in monoclonal B-cell lymphocytosis.

Monoclonal B-cell lymphocytosis (MBL) is the presence of small B-cell clones in the peripheral blood of healthy subjects. Most MBL have the characteristic phenotype of chronic lymphocyte leukemia (chronic lymphocytic leukemia (CLL)-like MBL), and depending on the number of monoclonal B-cells, may characterize a preclinical stage of the CLL. However, there are also MBL with an atypical (CD5(+) CD20(+/bright) CD23(dim/-) ) or a CD5(neg) phenotype, which remain largely unexplored. We performed an extended immunophenotypic, cytogenetic, and hematologic analysis in 75 CLL-like, 39 atypical, 50 CD5(neg) , and 7 biphenotypic MBL cases to detect differences or similarities among the MBL subsets. The phenotypic analysis showed expression variations in many surface markers and a wide spectrum of disease-specific phenotypes within each MBL subtype. Interphase fluorescent in situ hybridization analysis showed a different panel of aberrations according to the phenotype. Overall, del(13q14) and +12 were the most common abnormalities (39%), whereas del(11q13), del(17p13), and del(6q23) were detected only in 3, 1, and 0 cases, respectively. A comparison of MBL with overt chronic lymphoproliferations revealed common aspects in the preclinical state, regarding both the kind of cytogenetic aberrations detected and the lymphocyte composition. Our findings highlight not only the heterogeneity among MBL subsets but also indicate common biologic features which differentiate MBL from clinical disease.

Approach to Acute Myeloid Leukemia with Increased Eosinophils and Basophils.

There is remarkable morphologic and genetic heterogeneity in acute myeloid leukemia (AML). In a small percentage of cases of AML, increased eosinophils and/or basophils are present in the bone marrow and sometimes in the peripheral blood. This is often a puzzling diagnostic situation but also an important finding that requires special investigation. Unique chromosomal rearrangements have been correlated with an increased number of eosinophils and basophils in AML. The identification of the underlying genetic lesion that promotes eosinophilia and basophilia can dramatically change both the prognosis and the treatment of the patient. Thus, clinicians must be vigilant in searching for the cause of eosinophilia and basophilia in patients with AML, since the different causes may lead to different treatments and survival outcomes. In this article, we examine the significance of increased eosinophils and/or basophils in the context of AML, provide guidance that simplifies the differential diagnosis, and give prognostic and therapeutic information about specific subtypes of AML associated with eosinophilia and/or basophilia. Evidence supporting personalized (molecularly targeted) therapy for these patients is also presented.

The Prognostic Effect of CDKN2A/2B Gene Deletions in Pediatric Acute Lymphoblastic Leukemia (ALL): Independent Prognostic Significance in BFM-Based Protocols.

One of the most frequent genes affected in pediatric ALL is the CDKN2A/2B gene, acting as a secondary cooperating event and playing an important role in cell-cycle regulation and chemosensitivity. Despite its inclusion in combined CNA (copy-number alterations) classifiers, like the IKZF1plus entity and the UKALL CNA profile, the prognostic impact of the individual gene deletions outside the context of a combined CNA evaluation remains controversial. Addressing the CDKN2A/2B deletions' additive prognostic effect in current risk-stratification algorithms, we present a retrospective study of a Greek pediatric ALL cohort comprising 247 patients studied over a 24-year period (2000-2023). Herein, we provide insight regarding the correlation with disease features, MRD clearance, and independent prognostic significance for this ALL cohort treated with contemporary BFM-based treatment protocols. Within an extended follow-up time of 135 months, the presence of the CDKN2A/2B deletions (biallelic or monoallelic) was associated with inferior EFS rates (65.1% compared to 91.8% for the gene non-deleted subgroup, p < 0.001), with the relapse rate accounting for 22.2% and 5.9%, respectively (p < 0.001). The presence of the biallelic deletion was associated with the worst outcomes (EFS 57.2% vs. 89.6% in the case of any other status, monoallelic or non-deleted, p < 0.001). Survival differences were demonstrated for B-ALL cases (EFS 65.3% vs. 93.6% for the non-deleted B-ALL subgroup, p < 0.001), but the prognostic effect was not statistically significant within the T-ALL cohort (EFS 64.3 vs. 69.2, p = 0.947). The presence of the CDKN2A/2B deletions clearly correlated with inferior outcomes within all protocol-defined risk groups (standard risk (SR): EFS 66.7% vs. 100%, p < 0.001, intermediate risk (IR): EFS 77.1% vs. 97.9%, p < 0.001, high risk (HR): EFS 42.1% vs. 70.5% p < 0.001 for deleted vs non-deleted cases in each patient risk group); additionally, in this study, the presence of the deletion differentiated prognosis within both MRD-positive and -negative subgroups on days 15 and 33 of induction. In multivariate analysis, the presence of the CDKN2A/2B deletions was the most important prognostic factor for relapse and overall survival, yielding a hazard ratio of 5.2 (95% confidence interval: 2.59-10.41, p < 0.001) and 5.96 (95% confidence interval: 2.97-11.95, p < 0.001), respectively, designating the alteration's independent prognostic significance in the context of modern risk stratification. The results of our study demonstrate that the presence of the CDKN2A/2B deletions can further stratify all existing risk groups, identifying patient subgroups with different outcomes. The above biallelic deletions could be incorporated into future risk-stratification algorithms, refining MRD-based stratification. In the era of targeted therapies, future prospective controlled clinical trials will further explore the possible use of cyclin-dependent kinase inhibitors (CDKIs) in CDKN2A/2B-affected ALL pediatric subgroups.

Modern diagnostic approaches to cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma is a very aggressive tumor with poor survival. Therefore, early diagnosis and surgical resection are of paramount importance. Its diagnosis is difficult because access to the tumor is not easy. Biopsy is possible only for intrahepatic cholangiocarcinoma, which accounts for 10% of cases. Routine brush cytology from endoscopic retrograde cholangiopancreatography (ERCP) has a high specificity of 100% but unfortunately a low sensitivity of 30%. In this review we briefly describe new diagnostic techniques applicable to ERCP brush cytology specimens and targeting the genetic background of the disease, in particular fluorescence in situ hybridization (FISH) and digital image analysis (DIA). DATA SOURCES: The PubMed database up to 2011 was used for the retrieval of relevant articles. The search terms FISH, fluorescence in situ hybridization, DIA, digital image analysis and cholangiocarcinoma were used. Both original and review articles were used. RESULTS: FISH identifies cells with chromosomal abnormalities, mainly numerical aberrations, using a mixture of fluorescence-labeled probes. FISH offers a higher sensitivity than routine cytology, retaining a high level of specificity. The DIA criterion for malignancy is demonstration of aneuploidy. This technique increases the sensitivity to 40%, but the specificity remains low. Preliminary data from application to other tumors suggest that combination of FISH and DIA may be of further benefit. CONCLUSIONS: The new techniques offer a significantly enhanced diagnostic efficacy in the evaluation of ERCP brush specimens. Apart from contributing to a more timely diagnosis, their wider application to cholangiocarcinoma may also facilitate the genetic study of the disease and add to our understanding of oncogenesis at the molecular level, with the prospect of identifying targets for novel therapeutic interventions.

Chronic myeloid leukemia (CML) in children: classical and newer therapeutic approaches.

Chronic myeloid leukemia (CML) represents a rare myeloproliferative disease among children where allogeneic stem cell transplantation (SCT) remains the curative gold standard. However, the impressive early cytogenetic and molecular responses achieved by tyrosine kinase inhibitors [TKIs (imatinib, nilotinib, and dasatinib)] as first-line or even sole treatment in adults, has led to their increasing use also among children. Due to limited data regarding long-term results of TKIs and especially those of second generation in pediatric cohorts, we would like to add clinical information in this rare series of patients by reporting on four children with CML over a 10-year period, focusing on TKIs, dose escalations and clinical responses.

The Cytogenetic Profile of Primary and Secondary Plasma Cell Leukemia: Etiopathogenetic Perspectives, Prognostic Impact and Clinical Relevance to Newly Diagnosed Multiple Myeloma with Differential Circulating Clonal Plasma Cells.

Plasma cell leukemia (PCL) is a rare and aggressive plasma cell dyscrasia that may appear as de-novo leukemia (pPCL) or on the basis of a pre-existing multiple myeloma (MM), called secondary plasma cell leukemia (sPCL). In this prospective study, we have applied a broad panel of FISH probes in 965 newly diagnosed MM (NDMM) and 44 PCL cases of both types to reveal the particular cytogenetic differences among the three plasma cell dyscrasias. In order to evaluate the frequency and patterns of clonal evolution, the same FISH panel was applied both at diagnosis and at the time of first relapse for 81 relapsed MM patients and both at MM diagnosis and during sPCL transformation for the 19 sPCL cases described here. pPCL was characterized by frequent MYC translocations and t(11;14) with a 11q13 breakpoint centered on the MYEOV gene, not commonly seen in MM. sPCL had a higher number of FISH abnormalities and was strongly associated with the presence of del(17p13), either acquired at the initial MM stage or as a newly acquired lesion upon leukemogenesis in the context of the apparent clonal evolution observed in sPCL. In clinical terms, sPCL showed a shorter overall survival than pPCL with either standard or high-risk (t(4;14) and/or t(14;16) and/or del(17p13) and/or ≥3 concomitant aberrations) abnormalities (median 5 months vs. 21 and 11 months respectively, p < 0.001), suggesting a prognostic stratification based on cytogenetic background. These observations proved relevant in the NDMM setting, where higher levels of circulating plasma cells (CPCs) were strongly associated with high-risk cytogenetics (median frequency of CPCs: 0.11% of peripheral blood nucleated cells for high-risk vs. 0.007% for standard-risk NDMM, p < 0.0001). Most importantly, the combined evaluation of CPCs (higher or lower than a cut-off of 0.03%), together with patients' cytogenetic status, could be used for an improved prognostic stratification of NDMM patients.

Fish evaluation of additional cytogenetic aberrations and hyperdiploidy in childhood Burkitt lymphoma.

Beyond MYC rearrangement, Burkitt lymphoma (BL) often presents with additional aberrations. Biopsy touch imprints from 72 children with BL were tested with interphase fluorescence in-situ hybridization (i-FISH) for MYC, BCL2, BCL6, IGH, IGK and IGL rearrangements and copy-number aberrations involving 1q21/1p32, 7cen/7q31, 9cen/9p21, 13q14/13q34 and 17cen/17p13. Diploid status deviations were investigated with chromosome enumeration probes. MYC rearrangement was demonstrated in all cases. Additional aberrations included +1q (21/72:29.2%), +7q (14/72:19.4%), 13q- (14/72:19.4%), 9p-(6/72:8.3%) and hyperdiploidy (6/72:8.3%). Advanced clinical stage IV, +7q and 9p- were associated with shorter overall survival, with stage IV and +7q retaining prognostic significance on multivariate analysis. No relapse or death was reported among the hyperdiploid cases. This i-FISH investigation provides information on the genetic profile of BL and may prove valuable for patients with no karyotype analysis. Demonstration of hyperdiploidy could evolve research on clonal evolution pathways and probably identify a subgroup of children with favorable prognosis.

Copy Number Alteration Profile Provides Additional Prognostic Value for Acute Lymphoblastic Leukemia Patients Treated on BFM Protocols.

We present our data of a novel proposed CNA-profile risk-index, applied on a Greek ALLIC-BFM-treated cohort, aiming at further refining genomic risk-stratification. Eighty-five of 227 consecutively treated ALL patients were analyzed for the copy-number-status of eight genes (IKZF1/CDKN2A/2B/PAR1/BTG1/EBF1/PAX5/ETV6/RB1). Using the MLPA-assay, patients were stratified as: (1) Good-risk(GR)-CNA-profile (n = 51), with no deletion of IKZF1/CDKN2A/B/PAR1/BTG1/EBF1/PAX5/ETV6/RB1 or isolated deletions of ETV6/PAX5/BTG1 or ETV6 deletions with a single additional deletion of BTG1/PAX5/CDKN2A/B. (2) Poor-risk(PR)-CNA-profile (n = 34), with any deletion of ΙΚΖF1/PAR1/EBF1/RB1 or any other CΝΑ. With a median follow-up time of 49.9 months, EFS for GR-CNA-profile and PR-CNA-profile patients was 96.0% vs. 57.6% (p < 0.001). For IR-group and HR-group patients, EFS for the GR-CNA/PR-CNA subgroups was 100.0% vs. 60.0% (p < 0.001) and 88.2% vs. 55.6% (p = 0.047), respectively. Among FC-MRDd15 + patients (MRDd15 ≥ 10-4), EFS rates were 95.3% vs. 51.7% for GR-CNA/PR-CNA subjects (p < 0.001). Similarly, among FC-MRDd33 + patients (MRDd33 ≥ 10-4), EFS was 92.9% vs. 27.3% (p < 0.001) and for patients FC-MRDd33 - (MRDd33 < 10-4), EFS was 97.2% vs. 72.7% (p = 0.004), for GR-CNA/PR-CNA patients, respectively. In a multivariate analysis, the CNA-profile was the most important outcome predictor. In conclusion, the CNA-profile can establish a new genomic risk-index, identifying a distinct subgroup with increased relapse risk among the IR-group, as well as a subgroup of patients with superior prognosis among HR-patients. The CNA-profile is feasible in BFM-based protocols, further refining MRD-based risk-stratification.

Contribution of immunophenotype to the investigation and differential diagnosis of Burkitt lymphoma, double-hit high-grade B-cell lymphoma, and single-hit MYC-rearranged diffuse large B-cell lymphoma.

BACKGROUND: There are no immunophenotypic guidelines for the investigation of MYC-rearranged lymphomas. We aimed to identify simple immunophenotypic features that would help to differentiate between MYC-rearranged lymphomas and guide cytogenetic analysis. METHODS: We reviewed diagnostic samples from patients diagnosed with Burkitt lymphoma (BL), double-hit lymphoma (DHL), MYC-rearranged diffuse large B-cell lymphoma (MYC-DLBCL), and standard (non-MYC-rearranged) DLBCL over the last decade in our Institution. Using flow cytometry (with antibodies CD20, CD10, CD38, bcl-2, Ki-67, FMC-7, CD43, CD27, CD79b, CD23, and CD22) we determined antigen% expression and median-fluorescence intensity ratios (MFIR). The forward scatter (FS) and side scatter (SS) characteristics of tumor B-cells were compared with normal T-cells (B/T ratios) for patients with MYC-rearranged lymphomas. RESULTS: We identified 51 patients of whom 14 had BL, 10 had DHL (6 MYC+/BCL2+; 4 MYC+/BCL6+), 8 MYC-DLBCL, and 19 standard DLBCL. The significant differences (p <.05) were: higher CD38% in BL than standard DLBCL; higher CD10% in BL and DHL versus MYC-DLBCL and standard DLBCL; higher CD10MFIR in BL than MYC-DLBCL and standard DLBCL; higher Ki-67% in BL than DHL and MYC-DLBCL; higher bcl-2% in DHL than BL; higher FMC-7% in BL than MYC-DLBCL and standard DLBCL; and lower SS (B/T) ratio in DHL than MYC-DLBCL. CONCLUSIONS: The combination of CD38% > 90, CD10% > 80, CD10MFIR > 10, bcl-2% < 30, and Ki-67% > 70 was characteristic of BL. "Deviation" from these cut-offs should raise suspicion for DHL and, therefore, BCL2 and/or BCL6 FISH is required. We also found that a diagnosis of DHL rather than of MYC-DLBCL was significantly associated with CD10% > 60, Ki-67% > 50, and SS (B/T) <1.5.

Flow cytometric predictive scoring systems for common fusions ETV6/RUNX1, BCR/ABL1, TCF3/PBX1 and rearrangements of the KMT2A gene, proposed for the initial cytogenetic approach in cases of B-acute lymphoblastic leukemia.

INTRODUCTION: In B-acute lymphoblastic leukemia (B-ALL), the identification of cytogenetic prognostic factors is important for stratifying patients into risk groups and tailoring treatment accordingly. The purpose of this study was to propose flow cytometric (FCM) scoring systems (SSs) for predicting t(12;21)(p13;q22), t(9;22)(q34;q11), t(11q23), and t(1;19)(q23;p13.3) translocations. METHODS: We analyzed retrospectively the FCM immunophenotype of 377 patients with B-ALL with regard to the major cytogenetic findings revealed by interphase fluorescence in situ hybridization (i-FISH). Comparing descriptive data on the expression of each antigen and performing receiver operating characteristic (ROC) analysis, we identified the most reliable predictive markers for each translocation and sought to establish a specific SS for each translocation, based on specific antibody panels. RESULTS: CD27, CD9, CD66c, CD10, CD25, and CD34 were employed for the prediction of t(12;21), CD25, CD38, CD34, and CD66c for t(9;22), NG2, CD10, CD15, CD34, and CD20 for t(11q23), and CD34, cµ, CD123, and CD66c for t(1;19). The sensitivity and specificity, respectively, of each predictive score were 89.29% and 96.15% for t(12;21), 75.00% and 88.19% for t(9;22), 84.21% and 99.04% for t(11q23), and 85.71% and 92.71% for t(1;19). CONCLUSION: Four highly specific and significantly sensitive FCM-obtained SSs are proposed for the prediction of the four major translocations observed in patients with B-ALL. Prospective evaluation of the proposed SSs could lead to a better targeted cytogenetic investigation and therefore to more cost-effective laboratory practice.

"Bone marrow aspirate automated counts on hematology analyzers: formulating a scoring system based on hematology parameters, to discriminate reactive versus myelodysplastic syndrome-related bone marrows".

INTRODUCTION: Diagnosis of myelodysplastic syndromes (MDS) is usually challenging. In this context, we have attempted to employ data derived from automated analysis of bone marrow (BM) samples as an ancillary tool for the discrimination between reactive marrow and MDS. METHODS: A total of 101 BM anticoagulated samples referred for flow cytometry (FCM) analysis on the clinical suspicion of MDS had been previously counted in a Mindray BC-6800 hematology analyzer (testing set). Among them, 22/101 randomly selected BM samples (comparison set) had been also simultaneously counted by an Advia 2120 and a CELL-DYN Sapphire hematology analyzer. Selected parameters obtained by Mindray BC-6800 were retrospectively evaluated with ROC and regression analysis in an attempt to formulate a discriminative scoring system (SS) for MDS. This system was further evaluated in the comparison set. RESULTS: The diagnosis of MDS was established in 37/101 patients assessed ("MDS" group). Three patients were diagnosed with myelodysplastic/myeloproliferative neoplasm (MDS/MPN), while 61 revealed a "reactive" bone marrow ("RBM" group). Statistical analysis revealed significant differences in Hb, RDW-CV%, NRBC%, and RET% values between the "MDS" and the "RBM" group. Specific cutoff values were then indicated and employed for the formulation of a SS of high sensitivity (86.84%) and specificity (86.89%). The encouraging performance characteristics of the proposed SS were also confirmed in the BM comparison set. CONCLUSION: Automated BM counts on hematology analyzers contributed to the formulation of a SS for the screening discrimination between reactive and MDS BM fluids, which seems to be applicable and informative, regardless of the analyzer used.

Clinical presentation, diagnosis, and survival in cholangiocarcinoma: A prospective study.

BACKGROUND AND STUDY AIMS: The diagnosis of cholangiocarcinoma (CCA) is difficult. The present study aimed to assess the clinical features, diagnosis, and survival in CCA. PATIENTS AND METHODS: This is a prospective study on 46 patients with CCA who underwent endoscopic retrograde cholangiopancreatography (ERCP) or surgical resection and 20 controls with a clinical and ERCP suspicion for CCA in whom surgical biopsy and/or 4-year follow-up showed a benign biliary stricture. RESULTS: The median age at presentation was 71years (range 44-88). Thirty-four patients (73.9%) presented with painless jaundice. Median CA 19-9 value was 188IU/L (range 1-49,138), with a level of <100IU/L in 13 patients (28%). Total bilirubin was 11.9 (0.6-36.3)mg/dL. The tumour was intrahepatic in 3 (6.5%), hilar (Klatskin) in 25 (54.3%), and located in the lower third of the bile duct in 18 (39.1%) patients. The diagnosis was confirmed by positive cytology in 10 (21.7%), biopsy in 20 (43.5%), cholangioscopy in five (10.9%), and imaging and clinical grounds in 11 (23.9%) patients. Cytology was feasible in 36 patients; it was positive in 10 and "highly indicative" in two patients (33.3% sensitivity). Twenty-two patients (47.8%) were treated by surgical resection, and the rest were offered palliative biliary drainage. Mean estimated survival for the entire group of CCA patients was 21.5±3.3months. Survival was slightly longer in patients who underwent surgical resection than those who had palliative treatment; the estimated mean survival rates were 26.2±4.2 vs. 17.1±3.3months, respectively, but the difference was not statistically significant (p=0.115). CONCLUSION: The diagnosis of CCA is difficult and often delayed. The outcome is generally poor.

B-cell acute lymphoblastic leukemia with mature phenotype and MLL rearrangement: report of five new cases and review of the literature.

The association between mature-B phenotype and MLL abnormalities in acute lymphoblastic leukemia (ALL) is a very unusual finding; only 14 pediatric cases have been reported so far. We describe the clinical and biological characteristics and outcome of five pediatric cases of newly diagnosed B lineage ALL with MLL abnormalities and mature immunophenotype based on light chain restriction and surface Ig expression. Blasts showed variable expression of CD10/CD34/TdT. MLL abnormalities with no MYC involvement were detected in all patients by G-banding, FISH, and/or RT-PCR. Three patients were treated according to Interfant protocol, one to ALLIC-09, and one received B-NHL-BFM-2004. All patients achieved complete remission and three of them relapsed. Despite the small cohort size, it could be postulated that B lineage ALL with MLL abnormalities and mature phenotype is a distinct entity that differs both from the typical Pro B ALL observed in infants and mature B-ALL with high MYC expression.

Suboptimal erythropoietic response to hypoxemia in idiopathic pulmonary fibrosis.

BACKGROUND AND STUDY OBJECTIVES: Idiopathic pulmonary fibrosis (IPF) is a chronic inflammatory process characterized by severe derangement of gas exchange in the advanced stages of disease. However, erythrocytosis is infrequent in IPF. The aim of this study was to investigate the potential relation between the blunted erythropoietic response and the chronic inflammation. SUBJECTS: Nine patients (6 men and 3 women) with IPF and profound hypoxemia (PO(2) < 65 mm Hg) and 34 sex- and age-matched healthy volunteers participated in the study. METHODS: We evaluated the hematologic parameters, serum erythropoietin, tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-8 levels. We also studied the development of burst-forming unit-erythroid (BFU-E)-derived colonies in semisolid methylcellulose cultures in blood samples from all patients. RESULTS: Hemoglobin and serum erythropoietin levels were almost comparable between the two studied groups. On the contrary, serum TNF-alpha, IL-6, and IL-8 values were significantly higher in patients with IPF (p < 0.05, p < 0.01, and p < 0.001, respectively). IPF sera induced a significant growth inhibition of erythroid bursts arising from mononuclear cells of either patients or control subjects compared with heat-inactivated AB serum (p < 0.05 and p < 0.01, respectively). Moreover, there was an apparent increment in the number of BFU-E colonies when patients' mononuclear cells were cultured in comparison with those of healthy subjects (p < 0.05). CONCLUSIONS: Our findings suggest that in IPF there is an increased number of primitive erythroid progenitors, which fail to proliferate and differentiate in vivo, suggesting a kind of ineffective erythropoiesis. As a consequence, hemoglobin levels do not rise in proportion to the severity of hypoxemia. Cytokines released from alveolar macrophages seem to have not only local but also systemic effects, since the serum of these patients directly suppressed erythropoiesis; however, the suboptimal erythropoietic response to hypoxia cannot be entirely attributed to this suppression. It is possible that several other factors interfere, synergistically or additively.

Red cell macrocytosis in hypoxemic patients with chronic obstructive pulmonary disease.

Macrocytosis is a common finding in patients with chronic obstructive pulmonary disease (COPD). The cause for the elevation of mean corpuscular volume (MCV) in these patients remains elusive. In an attempt to determine the extent of macrocytosis in COPD patients and search for possible causative factors, we evaluated the hematologic parameters, F-cell percentage, blood gases and serum erythropoietin (Epo) Levels in 32 COPD clinically stable patients and 34 sex- and age-matched non-smoker healthy volunteers. An increased MCV was observed in almost half of the hypoxemic COPD cases (14/32 or 43.75%), while erythrocytosis developed to a lesser degree (37.5%). The erythropoietic response did not correlate with the severity of hypoxia. Moreover, no significant correlation was found between macrocytosis and hypoxemia or erythrocytosis and red cell size. In some cases the two phenomena occurred independently. The F-cell percentage was significantly elevated in the COPD group (P < 0.01) and was associated with MCV values (n = 32, r5 = 0.41, P < 0.05). This finding supports the hypothesis we put forward to explain the macrocytosis often observed in COPD, i.e., that the acute erythropoietic stress occurring repeatedly in these patients as a result of the frequent exacerbations may lead to waves of release of relatively immature, large red cells from the marrow, including an increased number of F-cells, reflecting the recruitment of normally dormant BFU-E (bursts forming units of erythrocyte precursors), which maintain the program for gamma-chain synthesis. The fact that erythrocytosis and macrocytosis, both being triggered by hypoxemia, do not occur consistently in all COPD patients indicates that many other factors may also intervene.

An extended fluorescence in situ hybridization approach for the cytogenetic study of cholangiocarcinoma on endoscopic retrograde cholangiopancreatography brushing cytology preparations.

The cytological diagnosis of cholangiocarcinoma has been significantly aided by applying a 4-probe fluorescence in situ hybridization system on endoscopic retrograde cholangiopancreatography brushing smears, aiming mainly at the detection of hyperdiploidy. However, this approach adds little to our understanding of the genetic background of the disease. With the prospect of obtaining additional data on chromosomal aberrations, we have extended the fluorescence in situ hybridization study, with the application of 4 independent 2-probe systems in 35 patients with documented cholangiocarcinoma. Fluorescence in situ hybridization assays were performed on endoscopic retrograde cholangiopancreatography brushing smears, with probes for the 7q31, 11q13 (CCND1), 17p53 (TP53), and 9p21 (INK4 locus) bands, together with the respective centromeric probe. Hyperdiploidy, involving at least 2 of the 4 chromosomes targeted, was found in 31 patients. 17p13 deletion was detected in 3, and 9p21 deletion, in 5 of the hyperdiploid cases, with the 2 aberrations concurrent in 1. CCND1 amplification was found in 1 case as the sole abnormality and in another together with hyperdiploidy, but in apparently unrelated clones. This work indicates that interphase fluorescence in situ hybridization is a practical and useful tool for the cytogenetic study of cholangiocarcinoma on endoscopic retrograde cholangiopancreatography brushing smears, which is often the only available tissue specimen of the tumor. Apart from hyperdiploidy, it provides additional data on the genetic profile of cholangiocarcinoma, especially regarding structural chromosomal aberrations and clonal diversity. This line of investigation may prove useful in the delineation of oncogenesis and the interpretation of the diverse clinical features of the disease.

High frequency of human leukocyte antigen class II DRB1*1602 haplotype in Greek patients with myelodysplastic syndrome and of DRB1*1501 in the low-risk subgroup.

Myelodysplastic syndromes (MDS) comprise a heterogenous group of clonal hematopoietic disorders in which the immune-mediated pathogenetic mechanisms are under investigation. Overrepresentation of human leukocyte antigen (HLA)-DR2 and its serologic split HLA-DR15 has been associated with low-risk MDS in certain ethnic groups and has been proposed as a predictive factor for a favorable response to immunomodulatory treatment. Because the HLA-DRB1*15 haplotype does not predominate in the Greek population, we investigated the frequency of HLA-DRB1 alleles among 114 patients of Greek origin suffering from various types of MDS: 36 refractory anemia (RA), 24 refractory anemia with ringed sideroblasts (RARS), 19 refractory anemia with excess of blasts (RAEB), 14 refractory anemia with excess of blasts in transformation (RAEB-t), 14 chronic myelomonocytic leukemia, and 7 hypoplastic MDS patients. HLA-DRB1 molecular typing was performed with polymerase chain reaction-sequence specific oligonucleotides and results were compared with that from a previously reported control Greek population. HLA-DRB1*1602 was the only allele that was significantly overrepresented in Greek MDS patients as a whole, whereas HLA-DRB1*1501 allele frequency was significantly higher in Greek patients with low-risk myelodysplasia. Our results suggest the possible value of HLA-DR15 and HLA-DR16 as determinants for immunomodulatory interventions, at least for Greek patients with low-risk MDS.

Duplication of Philadelphia chromosome and trisomy of chromosome 21 in a pediatric patient with acute lymphoblastic leukemia.

The evaluation of molecular and cytogenetic characteristics using novel techniques has significantly contributed into the insight of leukemia. In this study, immunoglobulin heavy chain gene rearrangements (V(H)D(H)J(H) region) were analyzed by polymerase chain reaction (PCR). Point mutations of the D835/I836 in the activation loop (AL) domain of the second tyrosine kinase domain of the fms-related tyrosine kinase 3 (FLT3) gene and NRAS (neuroblastoma cell line) point mutations were also analyzed by PCR. Furthermore, sequence analysis of the V(H)D(H)J(H) region was performed, as well as, chromosomal aberrations were identified by interphase fluorescence in situ hybridization (iFISH) in a 12.5-year-old boy with acute lymphoblastic leukemia. Positive MRD was found in bone marrow samples obtained at various time points during and after treatment completion prior to relapse. Molecular analysis of the FLT3 gene mutations revealed an acquired a G → T mutation at codon 835, which resulted to substitution of aspartate 835 for tyrosine (D835Y). Cytogenetic analysis with iFISH showed t(9;22) (q34;q11.2), with minor-BCR/ABL1 fusion gene in the majority of nuclei, while a subclone with duplication of the Philadelphia chromosome was observed. Triple signals of AML1 were detected in 80% of nuclei, which were compatible with trisomy of chromosome 21. BCR/ABL1 fusion gene, duplication of Philadelphia chromosome and persistence of MRD constitute inferior prognostic factors, while hyperdiploidy, trisomy of chromosome 21 and FLT3-AL mutations are related to better prognosis. The study of cytogenetic and molecular characteristics is essential in order to decide on the optimal treatment protocol in childhood leukemia.

Sequential monitoring of minimal residual disease in acute lymphoblastic leukemia: 7-year experience in a pediatric hematology/oncology unit.

We evaluated minimal residual disease (MRD) in 91 children with acute lymphoblastic leukemia (ALL) by PCR amplification of clonal rearrangements, immunoglobulin (IgH; VDJ rearrangement, CDR3 region) and T-cell receptor (TCRdelta). Sequential monitoring of MRD was performed at different time points during and after chemotherapy and was correlated to patient outcome. In total, 792 bone marrow samples were assessed for MRD at the end of induction, and during and after treatment completion. MRD positivity at the end of induction was detected in 12% of patients and was associated with high incidence of relapse, 54.55% (p = 0.0002), at 5 years. On the other hand, 88% of patients were MRD-negative at the end of induction and the relapse rate at 5 years was very low, 5%. The frequency of MRD decreased to 16% in the first 6 months of chemotherapy; however, the incidence of relapse in MRD-positive patients remained high, 42.8%. After treatment completion (24-36 months from diagnosis), 32% patients were MRD-positive and the relapse rate was 36.5% (p = 0.0009). Our results indicated that monitoring of MRD constituted an essential prognostic marker, and detection of MRD particularly at the end of induction and after treatment completion was strongly predictive for patient outcome.