International standardized approach for flow cytometric residual disease monitoring in chronic lymphocytic leukaemia.

The eradication of minimal residual disease (MRD) in chronic lymphocytic leukaemia (CLL) predicts for improved outcome. However, the wide variety of MRD techniques makes it difficult to interpret and compare different clinical trials. Our aim was to develop a standardized flow cytometric CLL-MRD assay and compare it to real-time quantitative allele-specific oligonucleotide (RQ-ASO) Immunoglobulin heavy chain gene (IgH) polymerase chain reaction (PCR). Analysis of 728 paired blood and marrow samples demonstrated high concordance (87%) for patients off-therapy. Blood analysis was equally or more sensitive than marrow in 92% of samples but marrow analysis was necessary to detect MRD within 3 months of alemtuzumab therapy. Assessment of 50 CLL-specific antibody combinations identified three (CD5/CD19 with CD20/CD38, CD81/CD22 and CD79b/CD43) with low inter-laboratory variation and false-detection rates. Experienced operators demonstrated an accuracy of 95.7% (specificity 98.8%, sensitivity 91.1%) in 141 samples with 0.01-0.1% CLL. There was close correlation and 95% concordance with RQ-ASO IgH-PCR for detection of CLL above 0.01%. The proposed flow cytometry approach is applicable to all sample types and therapeutic regimes, and sufficiently rapid and sensitive to guide therapy to an MRD-negativity in real time. These techniques may be used as a tool for assessing response and comparing the efficacy of different therapeutic approaches.

A study of disseminated intravascular coagulation in acute leukemia reveals markedly elevated D-dimer levels are a sensitive indicator of acute promyelocytic leukemia.

INTRODUCTION: While the presence of disseminated intravascular coagulation (DIC) has been implicated in worse clinical outcome in acute leukemia, the relationship between different subtypes of acute leukemia and the clinicopathologic features of DIC has not been systematically well studied. METHODS: In this study, we retrospectively reviewed 149 cases of newly diagnosed acute leukemia and assessed the utility of evaluating red blood cell morphologic features, and coagulation parameters in determining the presence of DIC as well as differentiating subtypes of acute leukemia. RESULTS: Review of our cohort demonstrates a novel finding, that elevated D-dimer concentrations ≥19 000 ng/mL fibrinogen equivalent units (FEU) are a sensitive diagnostic indicator of acute promyelocytic leukemia (APL) with moderate specificity, sensitivity 96%, specificity 92% in acute leukemia subtyping. Similar to other studies, APL showed an increased incidence of DIC (P < 0.01) compared to other subtypes of acute leukemia. Surprisingly, the presence of schistocytes on the peripheral blood smear was not a statistically significant indicator of DIC, sensitivity of 36% and specificity of 89%. Finally, the presence of DIC was not a significant indicator of poorer prognosis amongst all patients with AML. CONCLUSION: Overall we identify elevated D-dimer concentrations ≥19 000 ng/mL FEU are a sensitive indicator of acute promyelocytic leukemia (APL), with a sensitivity of 96% and specificity of 92% in the subtyping of acute leukemias, and that the presence of schistocytes in peripheral blood smears is not a diagnostically sensitive screening test for DIC with a sensitivity of 36%.

Spontaneously relapsing clonal, mucosal cytotoxic T-cell lymphoproliferative disorder: case report and review of the literature.

Primary T-cell lymphoma of the gastrointestinal tract is a rare and usually aggressive disorder that may be associated with celiac disease. The authors describe a unique case of a clonal proliferation of CD8+ T cells involving the oral mucosa, ileum, and colon of a 35-year-old man that has regressed spontaneously and recurred numerous times over a 9-year period without treatment. The patient's symptoms were limited to occasional rectal bleeding and recurring painful oral ulcers. Within the intestine, these collections of small T cells induced minimal architectural distortions and did not show extensive epitheliotrophism. Polymerase chain reaction and sequencing analyses revealed that the identical T-cell clone has been present for more than 9 years and in different mucosal locations in this patient. This may represent a unique T-cell lymphoproliferative process akin to a mucosal counterpart of lymphomatoid papulosis of the skin.

Aggressive cutaneous NK and NK-like T-cell lymphomas: clinicopathologic, immunohistochemical, and molecular analyses of 12 cases.

Natural killer (NK) and NK-like T-cell lymphomas are rare hematolymphoid malignancies that predominate in the upper aerodigestive system. They also involve other extranodal sites, including the skin. Primary cutaneous manifestations of NK and NK-like T-cell lymphomas are uncommon, and the clinicopathologic features are poorly understood. We have studied 12 patients of varied ethnic backgrounds with CD56-positive lymphomas in the skin. Six patients subsequently progressed to disseminated disease. These lymphomas showed the following immunophenotype: CD56+, CD43+, TCRb-, CD3-/+, CD20-, CD30-/+, CD4-, and CD8-. Two cases exhibited T-cell receptor gene rearrangements supporting a T-cell origin for these lymphomas, whereas the remaining 10 cases were likely derived from NK cells. Our results show inconsistent association of these lymphomas with Epstein-Barr virus (EBV), the multidrug resistance phenotype, and expression of P53. In addition, we found a previously unreported correlation between lymphomas harboring EBV mRNA and the expression of the multidrug resistance phenotype. These lymphomas were aggressive and were associated with rapid clinical progression, treatment failure, multiple relapses, and an average survival of 15 months from the time of diagnosis. Our results indicate the importance of recognizing this disease as a distinct subset of aggressive cutaneous lymphomas that may be diagnosed on the basis of morphology, immunophenotype, and gene rearrangement studies.

Blastic/blastoid transformation of follicular lymphoma: immunohistologic and molecular analyses of five cases.

Progression of follicular lymphoma to a higher-grade malignancy frequently heralds a poor prognosis. Clinical transformation is variably accompanied by a spectrum of histologic changes characterized by alteration in growth and cytology. Although several cytogenetic events and potential oncogenes have been documented in this progression, the underlying molecular mechanisms are largely unknown. We present five patients with an unusual histologic transformation of follicular lymphoma manifested by blastic/blastoid morphology. This transformation is histologically distinct from other types of transformation of follicular lymphoma. All five cases exhibited the t(14;18) translocation and expressed the BCL-2 protein. In addition, two of the five patients showed increased levels of the p53 protein within neoplastic cells implicating a possible role for this oncogene in blastic/blastoid transformation. The lack of BCL-1 and myeloid antigens by immunohistochemistry and flow cytometry studies served to distinguish blastic/blastoid transformation of follicular lymphoma from its morphologic mimics. This distinction is clinically important because lymphoblastic and myeloid leukemias require significantly different therapeutic modalities and show better prognosis. Moreover, the lack of Epstein-Barr virus-specific mRNA suggests that this virus is unlikely to participate in blastic/blastoid transformation of follicular lymphoma.

Novel factor V C2-domain mutation (R2074H) in two families with factor V deficiency and bleeding.

The molecular basis of Factor V deficiency has been defined in few patients only. We report a homozygous nucleotide change (G6395A) in two Tunisian probands with Factor V deficiency and bleeding episodes. This substitution results in the replacement of an arginine (R) by a histidine (H) in amino acid position 2074, located in the Factor V C2-domain. Mutations in this protein domain have not previously been described. Several lines of evidence support that this sequence variant is indeed disease causing: 1) Crystal structures of Factor V and molecular C2-domain modeling studies of H2074 suggest that the conserved R2074 is required for correct folding; 2) Structure-function studies of selective Factor V mutants (R2074A) demonstrate the importance of R2074 for structural stability of the Factor V C2-domain and for cofactor activity (1); 3) In Factor VIII, point mutations in codon 2209, which corresponds to position 2074 in Factor V, cause hemophilia A.

Familial coagulation factor V deficiency caused by a novel 4 base pair insertion in the factor V gene: factor V Stanford.

An index patient with pseudohomozygosity for factor V Leiden was identified. Each of his two children inherited a different paternal factor V allele; a daughter was heterozygous for factor V Leiden, with 100% factor V activity, and a son was heterozygous for factor V deficiency, with 50% factor V activity. Genomic DNA was obtained from family members, and the 25 factor V exons and flanking intronic regions were sequenced in the proband and confirmed in the children. Within exon 13 of factor V, a 4 base insertion was found at NT 2856 in the proband and son. but not the daughter. This mutation, here designated factor V Stanford, results in a frameshift with loss of a thrombin activation site (R1545V) and premature termination of translation at amino acid 1560.

Sensitivity and specificity of the APC resistance assay in detection of individuals with factor V Leiden.

Resistance to activated protein C (APC) is the most common cause of familial thrombophilia. The partial thromboplastin time (PTT)-based test for resistance to APC has been widely employed as a screening test for this disorder. However, the utility of this test for screening is not well characterized. More than 90% of patients with resistance to APC have the G1691A mutation in factor V (factor V Leiden). The authors studied the ability of a commercial APC resistance assay to correctly identify the factor V Leiden genotype in 130 individuals. At the recommended assay cut-off value of 2, the sensitivity of the APC resistance assay was 50%, with a specificity of 98%. Increasing the cut-off value increased the sensitivity but decreased the specificity of the test. Receiver operating characteristic (ROC) curve analysis indicated that the test was of intermediate utility. There was considerable overlap in APC ratios in the range of 2 to 3 between subjects with a normal factor V genotype and heterozygotes for factor V Leiden. The authors conclude that the APC resistance assay in its present form is not a useful screening test for factor V Leiden heterozygotes. Until the performance of this assay is improved, patients should have molecular diagnostic testing performed to determine their factor V Leiden status.

Heparin-induced platelet aggregation vs platelet factor 4 enzyme-linked immunosorbent assay in the diagnosis of heparin-induced thrombocytopenia-thrombosis.

Thrombosis occurs in an unpredictable subset of patients with heparin-induced thrombocytopenia (HIT). The diagnosis of HIT requires clinical suspicion and laboratory confirmation. Although the "gold-standard" diagnostic test is considered to be the serotonin release assay (SRA), most laboratories use heparin-induced platelet aggregation (HIPA), which is highly specific but reported to be less sensitive than the SRA. Recently, the heparin-platelet factor 4 (PF4) enzyme-linked immunosorbent assay (ELISA) has been reported to have comparable sensitivity to the SRA. We compared the HIPA and PF4 ELISA in serum samples from 146 patients examined for HIT and assessed whether either test predicted thrombotic risk. Results for 81 patients were positive for HIPA, PF4 ELISA, or both. Of these, 91% were HIPA-positive, while only 60% were PF4 ELISA-positive. Clinical information was available on 63 patients, 17 of whom had thrombotic events (10 venous, 6 arterial, and 1 both). Neither the HIPA nor the PF4 ELISA predicted thrombotic risk, but the HIPA proved to be a more sensitive test for laboratory confirmation.

Aggressive natural killer-like T-cell malignancy with leukemic presentation following solid organ transplantation.

NK-like T-cell malignancies are part of a spectrum of lymphoproliferative diseases that complicate immunosuppression associated with solid organ transplantation. We describe 2 patients with long-standing immunosuppression following solid organ transplantation. Both patients had systemic symptoms that included fever, myalgia, and weight loss. Organ involvement and lymphadenopathy were not initially observed. Unique to these 2 cases are the initial leukemic symptoms, which led to further characterization and identification of NK-like T-cell malignancies. Both patients exhibited an anomalous T/NK phenotype, CD56 positivity, and atypical blastic architecture of the large granular lymphocytes. Clonal rearrangement of T-cell receptor genes was detected in both patients. In 1 patient, a cytogenetic abnormality involving 8q24 was demonstrated. The disease course in both patients was aggressive, with involvement of multiple sites and rapid demise. This study emphasizes the importance of including NK-like T-cell malignancies in the differential diagnosis of lymphoproliferative disorders associated with immunosuppression and recognizing that an aggressive clinical course may follow leukemic presentation of disease.

Peripheral T-cell lymphoma complicated by a proliferation of large B cells.

We studied 14 cases that showed a morphologic appearance of peripheral T-cell lymphoma and contained substantial numbers of CD20+ large B cells. In all but 2 cases, the CD20+ large cells showed a mix of kappa and lambda light chain expression. Two cases showed a focal predominance of kappa expression. In situ hybridization using the EBER1 probe for detection of Epstein-Barr virus (EBV) RNA was performed on every case. EBV RNA was present in 10 cases. Of 8 cases with EBV RNA stained by immunohistochemistry for the latent membrane protein of EBV, 6 were positive. Double-labeling immunohistochemistry and in situ hybridization confirmed that EBV was present in the large B cells. Polymerase chain reaction (PCR) analysis showed a clonal rearrangement of the T-cell receptor (TCR)-gamma chain gene in 12 of 13 cases tested. One additional case showed a clonal rearrangement of the TCR-beta chain gene by Southern blot hybridization. PCR analysis showed a clonal immunoglobulin gene rearrangement in 5 cases, a suggestion of a clonal rearrangement in 1, an oligoclonal pattern in 4, and a polyclonal pattern in 4. The finding of large B and T cells may result in a misdiagnosis of a reactive process or of T-cell-rich B-cell lymphoma. The presence of EBV in some cases could cause further confusion with the reactive T- and B-immunoblastic proliferation of infectious mononucleosis.

Cephacetrile in the treatment of female pelvic inflammatory disease.

The efficacy and tolerability of cephacetrile administered by the intravenous or intramuscular route was evaluated in an open-label study of 55 hospitalized females diagnosed as having mild to severe pelvic inflammatory disease. Rapid improvement was observed in most patients. All patients were classified as clinically cured or significantly improved. Pain was absent or mild in 86% of the patients who received cephacetrile by intramuscular injection. Seventy-two percent of the patients given the drug intravenously experienced no localized pain. Phlebitis occurred in 6 of the 53 patients (11%) who received intravenous cephacetrile and thrombosis was present in 3 (6%). Rash and puritus was reported in 1 patient. Results of this study suggest that cephacetrile is an important and useful drug in the treatment of pelvic inflammatory disease.

Effect of adrenal suppression in gonadal gysgenesis.

Plasma steroids, estrogen, progesterone, and testosterone, and plasma gonadotropins FSH and LH were determined before and after adrenal suppression in an 18-year-old patient with gonadal dysgenesis (XX/XO). Total plasma estrogen was initially low (9.2 plus or minus 2.4 pg/ml) and did not change after adrenal suppression. Plasma progesterone (380 plus or minus 18 pg/ml) and testosterone (236 plus or minus pg/ml) were in the range found in the normal follicular phase and fell to 50 plus or minus 17 pg/ml and 112 plus or minus pg/ml, respectively, after adrenal suppression. Mean levels of FSH and LH were unchanged after adrenal suppression. These results suggest that the adrenal may make a significant contribution to plasma levels of progesterone and testosterone in patients with gonadal dysgenesis and may also contribute to the plasma levels of these steroids during the follicular phase of the mensturual cycle.

A microplate allele-specific oligonucleotide hybridization assay for detection of factor V Leiden.

Factor V Leiden is the most common genetic risk factor for thrombosis. Currently, the determination of factor V Leiden genotype is limited to laboratories with expertise in molecular methods to develop "home brew" assays using polymerase chain reaction (PCR) to amplify genomic DNA, followed by analysis of Mnl I restriction fragments. These methods are not standardized, are labor intensive, and have low throughput. We describe a method for determination of factor V Leiden genotype using allele-specific oligonucleotide capture probes coated onto 96 well plates, requiring only a thermal cycler and a microplate spectrophotometer to perform. With an automated strip washer and plate reader, genotypes could be determined in 80 min from completion of PCR. Within-run and between-run coefficients of variation for the assay were < 10%. In all 160 cases studied, the microplate assay correctly identified the factor V genotype. The microplate oligonucleotide hybridization assay is a simple and reliable system for determination of factor V Leiden genotype. The assay offers an automatable, high-throughput alternative to current testing methodologies.

Sensitive detection of clonal immunoglobulin rearrangements in frozen and paraffin embedded tissues by polymerase chain reaction heteroduplex analysis.

Molecular detection of a clonal population of B or T cells through analysis of rearranged antigen receptor genes is an essential adjunct to the morphologic, flow cytometric, and immunohistochemical evaluation of tissue specimens for the presence of leukemia or lymphoma. Combining polymerase chain reaction (PCR) with heteroduplex annealing and polyacrylamide gel electrophoresis (PAGE) has been used to detect clonal T-cell receptor rearrangements, particularly in skin biopsy specimens. The authors have developed a similar PCR heteroduplex assay for detection of clonal VDJ immunoglobulin gene rearrangements using two sets of primers based on relatively conserved consensus regions in the J(H) and framework I and 2 regions of the immunoglobulin heavy chain V region gene. This method is able to detect a clonal rearrangement when the clone comprises as little as 1% of the population in a polyclonal B-cell background. It may be used on fresh, frozen, or paraffin-embedded tissue and detects a clonal population in a majority of lymphoma subtypes. Compared with conventional PCR analysis, this method requires only a short additional cycle of denaturation and slow renaturation before PAGE. Interpretation is simplified as the clonal PCR product migrates away from the polyclonal background products.

A polymorphism in the BCL-6 gene is associated with follicle center lymphoma.

Follicle center lymphoma (FCL) accounts for approximately 40% of all non-Hodgkin's lymphomas (NHL). The genetic-environmental interactions involved in the etiology and pathogenesis of this disease are unknown. In our previous study a single nucleotide polymorphism (SNP) (397C) in the regulatory untranslated first intron region of the BCL-6 gene was found in four of the eight FCL patients but in none of the 10 healthy controls. To further evaluate the potential association between the 397C allele of the BCL-6 gene and FCL, we performed a case-control study. Genomic DNA was isolated from 85 FCL patients, from 98 control cases without a previous history of malignancy, treated at Stanford University Medical Center for non-malignant disorders and from 90 samples from the DNA Polymorphism Discovery Resource. The 397G and the 397C polymorphic alleles were identified by a PCR-RFLP method. To evaluate the possible effect of this polymorphism on gene expression, BCL-6 mRNA levels in nine FCL tumors with the 397G-G genotype and in nine FCL tumors with the 397G-C genotype were measured by quantitative real-time RT-PCR. The 397C polymorphic allele was found in 32 FCL cases (37.6%), in 20 controls (20.4%) and in 17 (18.9%) samples from the DNA Polymorphism Discovery Resource. The prevalence of the 397G-C and 397C-C genotypes was significantly higher in FCL cases than in control group (p = 0.01). No difference in BCL-6 gene expression was observed between FCL cases with 397G-G and 397G-C genotypes. The present study demonstrates a possible association between the 397C allele of the BCL-6 proto-oncogene and FCL. The similar levels of BCL-6 mRNA expression in 397G-G and in 397G-C FCL cases suggests that any possible oncogenic effect of the polymorphic allele would not simply be related to a direct effect on BCL-6 gene expression and suggests the existence of other FCL susceptibility genes that are in linkage disequilibrium with the 397C allele of the BCL-6 gene.

Recurrent thrombosis due to compound heterozygosity for factor V Leiden and factor V deficiency.

A point mutation in the factor V gene (factor V Leiden) is the most common cause of familial thrombophilia. Patients with factor V Leiden have an increased risk of thrombosis, particularly those homozygous for the mutation. However, the phenotype in individuals with the mutation is variable, suggesting that other factors influence thrombotic risk. We describe for the first time a family in which two independent defects in factor V co-exist: heterozygosity for factor V Leiden and factor V deficiency. Compound heterozygosity for these two defects results in a phenotype similar to a homozygous factor V Leiden state with profound resistance to APC and recurrent thrombosis.

Use of the polymerase chain reaction in the evaluation of cutaneous T-cell infiltrates.

Histologic evaluation of suspected cutaneous T-cell neoplasia is challenging. There is significant overlap with features of benign condition, and neoplastic cells often occur in a reactive background. Recently, molecular techniques using paraffin-embedded tissue have been applied to the diagnosis of cutaneous T-cell infiltrates. These methods are useful for determining whether a clonal population of T-cells is present. The advantages and limitation of molecular diagnostic methods in the diagnosis of cutaneous T-cell infiltrates are discussed.

High sensitivity and specificity of a new functional flow cytometry assay for clinically significant heparin-induced thrombocytopenia antibodies.

INTRODUCTION: Heparin-induced thrombocytopenia (HIT) is a life-threatening condition, in which the anticoagulant heparin, platelet factor 4 (PF4), and platelet-activating antibodies form complexes with prothrombotic properties. Laboratory tests to support clinical diagnosis are subdivided into functional, platelet activation assays, which lack standardization, or immunological assays, which have moderate specificity toward HIT. In this study, clinical performance of HITAlert, a novel in vitro diagnostic (IVD) registered platelet activation assay, was tested in a large cohort of HIT-suspected patients and compared with immunological assays. METHODS: From 346 HIT-suspected patients (single center), clinical data including 4T pretest probability results, citrated platelet-poor plasmas, and sera were collected, allowing direct comparison of clinical observations with HITAlert results. HITAlert performance was compared with PF4 IgG ELISA (246 patients, three centers) and PF4 PaGIA (298 patients, single center). RESULTS: HITAlert showed high sensitivity (88.2%) and specificity (99.1%) when compared with clinical diagnosis. Agreement of HITAlert with PF4 ELISA- and PF4 PaGIA-positive patients is low (52.7 and 23.2%, respectively), while agreement with PF4 IgG ELISA- and PF4 PaGIA-negative patients is very high (98.1 and 99.1%, respectively). CONCLUSION: HITAlert performance is excellent when compared with clinical HIT diagnosis, making it a suitable assay for rapid testing of platelet activation due to anticoagulant therapy.

Minimal residual disease quantification using consensus primers and high-throughput IGH sequencing predicts post-transplant relapse in chronic lymphocytic leukemia.

Quantification of minimal residual disease (MRD) following allogeneic hematopoietic cell transplantation (allo-HCT) predicts post-transplant relapse in patients with chronic lymphocytic leukemia (CLL). We utilized an MRD-quantification method that amplifies immunoglobulin heavy chain (IGH) loci using consensus V and J segment primers followed by high-throughput sequencing (HTS), enabling quantification with a detection limit of one CLL cell per million mononuclear cells. Using this IGH-HTS approach, we analyzed MRD patterns in over 400 samples from 40 CLL patients who underwent reduced-intensity allo-HCT. Nine patients relapsed within 12 months post-HCT. Of the 31 patients in remission at 12 months post-HCT, disease-free survival was 86% in patients with MRD <10(-4) and 20% in those with MRD ≥10(-4) (relapse hazard ratio (HR) 9.0; 95% confidence interval (CI) 2.5-32; P<0.0001), with median follow-up of 36 months. Additionally, MRD predicted relapse at other time points, including 9, 18 and 24 months post-HCT. MRD doubling time <12 months with disease burden ≥10(-5) was associated with relapse within 12 months of MRD assessment in 50% of patients, and within 24 months in 90% of patients. This IGH-HTS method may facilitate routine MRD quantification in clinical trials.