- Head of ImmunophenotypingRoyal Marsden Hospital, Sutton, Surrey, United Kindgomedit
Terminal deoxynucleotidyl transferase (TdT) has long been considered a diagnostic marker for acute lymphoblastic leukemia. Reports of TdT-positive cells in acute myeloid leukemia have lately questioned its diagnostic value. TDT has been... more
Terminal deoxynucleotidyl transferase (TdT) has long been considered a diagnostic marker for acute lymphoblastic leukemia. Reports of TdT-positive cells in acute myeloid leukemia have lately questioned its diagnostic value. TDT has been detected mainly by microscopy methods: immunofluorescence and immunocytochemistry. The aim of this study was to reevaluate the diagnostic importance of TdT in acute leukemia by using flow cytometry with a method that allows quantitative analysis. Fifty-eight cases of acute leukemia were studied and TdT expression was quantified using calibrated fluorescent beads. The highest TdT values were found in B lineage acute lymphoblastic leukemia (ALL) while acute myeloid leukemia (AML) had the lowest values, even in cases with a high percentage of TdT-positive cells. Biphenotypic leukemia had intermediate values between B-lineage and T-lineage acute leukemia. The difference between these groups was statistically significant (P < 0.0001). The TdT assay by flow cytometry was more precise than immunocytochemistry because it recognizes quantitative differences between ALL and AML. It is also valuable in better defining the maturation stages in pre-B ALL and T-ALL. We conclude that quantitative flow cytometry of TdT re-establishes the diagnostic value of this enzyme and has potential applications for the study of minimal residual disease.
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A 2-year-old girl of Pakistani ethnic origin had suffered from recurrent epileptic seizures from the third day of life and subsequently had shown developmental delay. While on holiday in Pakistan, she developed fever, coryzal symptoms and... more
A 2-year-old girl of Pakistani ethnic origin had suffered from recurrent epileptic seizures from the third day of life and subsequently had shown developmental delay. While on holiday in Pakistan, she developed fever, coryzal symptoms and cough and was noted to have a raised white cell count (WBC). She was treated with an oral cephalosporin. Her fever initially settled but 1 week later she was admitted to hospital following a grand mal seizure. Her cough had persisted and her lymphocyte count had risen dramatically. Acute leukemia was suspected and the family were advised to return to the United Kingdom (UK) immediately for further tests including a bone marrow examination. On returning to the UK, she was unwell with severe paroxysms of coughing accompanied by vomiting, fever and tachypnea. Her chest was clear on auscultation and she has a few small palpable cervical lymph nodes. There was no hepatomegaly or splenomegaly. FBC showed: WBC 98.36 10/l, lymphocyte count 72.56 10/l, hemoglobin concentration 12.1 g/dl and platelet count of 6276 10/l. The clinical features were considered typical of pertussis and of note, because of her neurological problems, she had not been immunized. However a nasal swab did not grow Bordetella pertussis and a hematology referral was therefore made for possible bone marrow examination to exclude a malignant cause of the lymphocytosis. The peripheral blood film (Figs. 1 and 2) showed mainly small mature lymphocytes but there were many smear cells and some large lymphoid cells with basophilic cytoplasm and nucleoli. Further diagnostic procedures were performed. FIGURE 1 Peripheral blood film showing three normal lymphocytes and one large lymphoid cells with an immature chromatin pattern and peripheral cytoplasmic basophilia. MGG6 100.
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beta and gamma T cell receptor (TCR) gene configuration was studied in 12 patients with large granular lymphocyte T cell leukemia (LGL- leukemia). Both genes were found rearranged in ten cases. In the remaining two patients TCR beta was... more
beta and gamma T cell receptor (TCR) gene configuration was studied in 12 patients with large granular lymphocyte T cell leukemia (LGL- leukemia). Both genes were found rearranged in ten cases. In the remaining two patients TCR beta was found in germline configuration. In one of them rearrangement of T cell-rearranging gene gamma (TRG gamma) and a gamma mRNA were demonstrated. We suggest that in this patient the leukemic T cells arose from one of the rare T cells bearing a gamma- delta rather than an alpha-beta TCR heterodimeric molecule. In the other patient several discrete TRG gamma rearrangements were detected. Because her leukemic cells were shown to be monoclonal on the grounds of their karyotype, we suggest that her leukemia originated before any rearrangement had taken place. The combined use of TCR beta and TRG gamma probes provides new information on the origin and clonal expansion of lymphoid cells in LGL-leukemia.
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We have investigated the role of immunophenotyping in distinguishing between leukemic B-cell lymphoproliferative disorders. Circulating cells from 666 cases were analyzed with a panel of markers by flow cytometry. The diseases included:... more
We have investigated the role of immunophenotyping in distinguishing between leukemic B-cell lymphoproliferative disorders. Circulating cells from 666 cases were analyzed with a panel of markers by flow cytometry. The diseases included: chronic lymphocytic leukemia (CLL), 400; prolymphocytic leukemia, 22; hairy cell leukemia (HCL), 40; HCL variant, 15; splenic lymphoma with villous lymphocytes, 100; follicular lymphoma, 26; lymphoplasmacytic lymphoma, 25; mantle-cell lymphoma, 20; and large cell lymphoma, 18. On the basis of the most common marker profile in CLL, CD5+, CD23+, FMC7- and weak expression (+/-) of surface immunoglobulin (SmIg) and CD22, we devised a scoring system that gives for each of these five markers a value of 1 or 0 according to whether it is typical or atypical for CLL. Scores range from 5 (typical of CLL) to 0 (atypical for CLL). Application of the scoring system to all the cases showed that 87% of CLL scored 5 and 4 and only 0.4% scored 0 or 1, whereas 89% of ...
Research Interests: Pathology, Leukemia, Medicine, Lymphoma, Humans, and 15 moreChronic Lymphocytic Leukemia, Pubmed, Lectins, Differential Diagnosis, CD, Clinical Sciences, Diffuse Large B-Cell Lymphoma, Immunophenotyping, Chi Square Distribution, B Lymphocytes, MANTLE CELL LYMPHOMA, Cell Adhesion Molecules, Follicular lymphoma, Lymphoproliferative disorders, and Diffuse large B cell lymphoma
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We have studied the immunological and cytogenetic features of 26 patients with acute leukaemia classified as biphenotypic according to a scoring system based on the number and lineage specificity of antigens expressed on the blast cells.... more
We have studied the immunological and cytogenetic features of 26 patients with acute leukaemia classified as biphenotypic according to a scoring system based on the number and lineage specificity of antigens expressed on the blast cells. The series included 19 ...
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One of the most intriguing features of chronic lymphocytic leukemia (CLL) is its clinical heterogeneity. The mutational status of immunoglobulin heavy chain variable region (IgVH) genes is one of the most powerful predictors of overall... more
One of the most intriguing features of chronic lymphocytic leukemia (CLL) is its clinical heterogeneity. The mutational status of immunoglobulin heavy chain variable region (IgVH) genes is one of the most powerful predictors of overall survival (OS) and progression-free survival (PFS). The expression of the tyrosine kinase ZAP-70 in CLL cells has been proposed as a surrogate marker for the mutational status of IgVH genes. Recent reports suggest that ZAP-70 over-expression is an independent prognostic factor for shorter OS and PFS. There is little information about correlation between ZAP-70 expression and other clinical and biological features in CLL. The aims of this study are 1) to evaluate prospectively ZAP-70 expression in a series of CLL patients; 2) to correlate this with clinical stage, lymphocyte morphology, CD38 expression and chromosomal abnormalities detected by FISH and 3) to analyze the independent prognostic value of ZAP-70 in predicting time to first treatment (treatm...
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Concurrent activation of BCL2 and MYC usually occurs in B cell non-Hodgkin lymphoma (B-NHL) by translocation of both oncogenes to both immunoglobulin heavy chain (IGH) alleles: this abrogates immunoglobulin synthesis. We have studied... more
Concurrent activation of BCL2 and MYC usually occurs in B cell non-Hodgkin lymphoma (B-NHL) by translocation of both oncogenes to both immunoglobulin heavy chain (IGH) alleles: this abrogates immunoglobulin synthesis. We have studied three B-NHL cell lines (DoHH2, VAL and ROS 50) and show that concurrent activation of BCL2 and MYC may follow translocation of both oncogenes to the same IGH allele. Conventional cytogenetics of DoHH2 suggested the presence of a t(14;18)(q32;q21) translocation. However, fluorescent in situ hybridization (FISH) studies using whole chromosome paints, alpha satellite probes and flow-sorted chromosomes as probes revealed an unexpected complexity of rearrangements involving chromosomes 8, 14 and 18, namely t(8;14;18)(q24;q32;q21). DNA blot and previous PCR analysis confirmed the juxtaposition of BCL2 major breakpoint region (mbr) with IGJH6, but also demonstrated a rearrangement within the first exon of MYC. The centromeric (5') MYC rearranged fragment c...
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We have analyzed the expression of the zeta chain of the T cell receptor/CD3 complex and the co-stimulatory molecule CD28 by dual colour immunofluorescence on T lymphocytes from patients with B cell chronic lymphocytic leukemia (CLL).... more
We have analyzed the expression of the zeta chain of the T cell receptor/CD3 complex and the co-stimulatory molecule CD28 by dual colour immunofluorescence on T lymphocytes from patients with B cell chronic lymphocytic leukemia (CLL). Zeta chain was significantly reduced on CD3-positive lymphocytes from 33 patients compared with normal controls (P<0.0001). The values were lower in stages B and C than in stage A. In five patients tested in partial remission the values were normal. CD28, investigated in CD3, CD4 and CD8 positive T cells from 18 CLL patients appeared to be reduced in the three subsets but more marked in CD8-positive lymphocytes. The loss of zeta chain and CD28 in a proportion of circulating T lymphocytes from CLL may underlie some of the known functional abnormalities of these cells and the immunodeficiency associated with the disease.
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5076Background: Cancer-secreted cytokines drive inflammation, supporting tumour growth and increasing the NLR. A high NLR associates with poor prognosis and treatment resistance (TXR). MDSCs are ar...
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Hairy cell leukemia (HCL) is a rare B-cell disorder, with a variant form, which differs from the former in clinical behaviour, morphology and immunophenotype. The resemblance of these malignancies to splenic marginal zone lymphoma (SMZL),... more
Hairy cell leukemia (HCL) is a rare B-cell disorder, with a variant form, which differs from the former in clinical behaviour, morphology and immunophenotype. The resemblance of these malignancies to splenic marginal zone lymphoma (SMZL), has led to the suggestion that they may share a common clonal progenitor that homes within the marginal zone of the splenic germinal centres. The immunophenotype and recent global gene expression results offer some support for this view, with a similar profile seen in HCL, and a subset of memory B-cells that are localized to the marginal zone. However, studies of the mutational status of the Ig genes are controversial. The majority of HCL cases are mutated, whereas up to 30% of SMZL cases are unmutated. Further investigation to understand these differences and to identify the cell of origin in these disorders is needed. In addition, there are no data on gene expression or VH mutation in HCL-variant (HCL-v), an entity with a poorer outcome and for w...
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Research Interests: Oncology, Flow Cytometry, Survival Analysis, Adolescent, Medicine, and 15 moreMultivariate Analysis, Acute Myeloid Leukemia, Humans, Internal Medicine, Recurrence, Clinical Sciences, Aged, Middle Aged, Bone Marrow Transplantation, Adult, Retrospective Studies, Prognosis, MINIMAL RESIDUAL DISEASE, Predictive value of tests, and Hematopoietic Stem Cell Transplantation
The clinical significance of detecting minimal residual disease (MRD) in B-lineage acute lymphoblastic leukaemia (ALL) was evaluated by quantitative flow cytometry using a combination of TdT with CD10 and CD19. 53 patients with B-cell... more
The clinical significance of detecting minimal residual disease (MRD) in B-lineage acute lymphoblastic leukaemia (ALL) was evaluated by quantitative flow cytometry using a combination of TdT with CD10 and CD19. 53 patients with B-cell precursor ALL were followed during and after completion of treatment (median follow-up 23 months). Nine patients relapsed and MRD had been detected in six of them, 5-15 weeks before relapse despite morphological complete remission. 43 patients remain in clinical remission and in none of these was MRD detected. Disease-free survival based on the detection of MRD by flow cytometry showed a statistically significant difference between both groups (P&lt;0.0001). The absence of MRD correlates with a low relapse rate, whereas the presence of MRD predicted early relapse. This study has shown that flow cytometry can improve the morphologic assessment of bone marrow (BM) remission status in B-lineage ALL. The finding of &lt; 5% blasts in BM aspirates did not correlate with &#39;true&#39; remission in a proportion of cases as residual leukaemic blasts were detected by flow cytometry in nine samples from six patients. On the other hand, the presence of &gt; 5% blasts assessed by morphology was not necessarily a feature of relapse in five patients as these cells were shown to have a phenotype identical to normal TdT-negative B-cell precursors. Quantitative flow cytometry was more informative than conventional morphology to assess remission status and showed a strong correlation with clinical outcome. This methodology is useful to define MRD in the majority of patients with B-lineage ALL and should be tested in prospective clinical trials.
Research Interests: Flow Cytometry, British, Treatment Outcome, Medicine, Humans, and 15 moreChild, Internal Medicine, Female, Male, Acute Lymphoblastic Leukaemia, Bone marrow, Recurrence, Bone Marrow Transplantation, Adult, Immunophenotyping, Clinical Significance, MINIMAL RESIDUAL DISEASE, Child preschool, Disease Free Survival, and Cardiovascular medicine and haematology
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An adequate bone marrow sample is essential for a rapid diagnosis of acute leukaemia by multicolour flow cytometry enabling the assignment of lineage and choice of therapy. Diagnosis is sometimes delayed due to difficulties in of... more
An adequate bone marrow sample is essential for a rapid diagnosis of acute leukaemia by multicolour flow cytometry enabling the assignment of lineage and choice of therapy. Diagnosis is sometimes delayed due to difficulties in of obtaining a bone marrow aspirate due to a 9dry tap9. In this study we evaluated immunophenotyping of mechanically disaggregated unfixed bone marrow trephine biopsies from 62 paediatric and adult patients at diagnosis (mostly acute leukemia). We compared the results to immunophenotyping of blood and aspirate samples and the trephine biopsy histology. In 26 of 62 cases no disease was present in the blood and the aspirate was a 9dry tap9. In 24 of 26 cases a diagnosis was reached by immunophenotyping from the disaggregated bone marrow biopsy. Results were concordant with histopathology in all 24 cases (22 acute lymphoblastic leukemia(ALL) cases, 2 acute myeloid leukemia (AML) cases). In 2 cases insufficient cells were obtained from the disaggregated biopsy to ...
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Despite advances in chronic lymphocytic leukaemia (CLL) treatment, globally chemotherapy remains a central treatment modality, with chemotherapy trials representing an invaluable resource to explore disease-related/genetic features... more
Despite advances in chronic lymphocytic leukaemia (CLL) treatment, globally chemotherapy remains a central treatment modality, with chemotherapy trials representing an invaluable resource to explore disease-related/genetic features contributing to long-term outcomes. In 499 LRF CLL4 cases, a trial with >12 years follow-up, we employed targeted resequencing of 22 genes, identifying 623 mutations. After background mutation rate correction, 11/22 genes were recurrently mutated at frequencies between 3.6% (NFKBIE) and 24% (SF3B1). Mutations beyond Sanger resolution (<12% VAF) were observed in all genes, with KRAS mutations principally composed of these low VAF variants. Firstly, employing orthogonal approaches to confirm <12% VAF TP53 mutations, we assessed the clinical impact of TP53 clonal architecture. Whilst ≥ 12% VAF TP53mut cases were associated with reduced PFS and OS, we could not demonstrate a difference between <12% VAF TP53 mutations and either wild type or ≥12% V...
Research Interests: Oncology, Leukemia, Medicine, Mutation, Gene, and 3 moreClinical Sciences, Sanger sequencing, and KRAS
BACKGROUND: T-cell prolymphocytic leukemia (T-PLL) is a rare, aggressive, post thymic lymphoid malignancy, with an incidence of approximately 0.1/100,000 people. T-PLL accounts for ~2% of all mature lymphocytic leukaemias in adults >30... more
BACKGROUND: T-cell prolymphocytic leukemia (T-PLL) is a rare, aggressive, post thymic lymphoid malignancy, with an incidence of approximately 0.1/100,000 people. T-PLL accounts for ~2% of all mature lymphocytic leukaemias in adults >30 years. Median overall survival (OS) ~20 months and long term remissions are infrequent. Intravenous alemtuzumab, a monoclonal antibody directed against CD52 remains the most effective treatment in T-PLL. At the Royal Marsden Hospital (RMH) we have been using alemtuzumab for T-PLL since the early 1990s. We describe our experience over the last 3 decades. METHODS: We included 174 T-PLL patients that were diagnosed or treated at RMH between 1989-2019. Immunophenotyping data was available through our hematological malignancy diagnostic service (HMDS) from 1998 onwards. Kaplan-Meier analysis was used for OS and disease free interval (DFI) RESULTS: There were 174 patients in total. Mean age at diagnosis was 61 years old (range 32-88) and M: F ratio was ~...
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Cells from the great majority of patients with chronic lymphocytic leukemia (CLL) express CD23. A recent histologic study has shown that CD23 is expressed more strongly in the proliferating centers of the lymph nodes, where the large... more
Cells from the great majority of patients with chronic lymphocytic leukemia (CLL) express CD23. A recent histologic study has shown that CD23 is expressed more strongly in the proliferating centers of the lymph nodes, where the large prolymphocytoid cells are located. The aim of our study was to quantify the expression of CD23 and CD21 in small and prolymphocytoid cells from patients with CLL and B-cell lymphomas, and correlate this expression with clinical parameters. Using quantitative flow cyto-metry we analyzed the antigen density of CD23 and CD21 in: 1) 101 cases of chronic lymphocytic leukemia, 84 typical, 14 with increased prolymphocytes (CLL/PL) and 3 atypical, 2) 15 cases of CD23 positive B-cell lymphoma with circulating lymphoma cells and 3) 8 normal subjects. The results were correlated with morphology and clinical staging. Cells from CLL and CLL/PL have a significantly higher number of CD23 molecules than normal and lymphoma B-cells (p<0.001 and p<0.001, respective...
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We studied the expression of the immunoglobulin-associated membrane protein B29 in 499 cases of chronic B cell diseases using the monoclonal antibody SN8 (CD79b). SN8 was positive in 5% (17/330) of chronic lymphocytic leukemia (CLL) and... more
We studied the expression of the immunoglobulin-associated membrane protein B29 in 499 cases of chronic B cell diseases using the monoclonal antibody SN8 (CD79b). SN8 was positive in 5% (17/330) of chronic lymphocytic leukemia (CLL) and 100% (15/15) of B prolymphocytic leukemia. The expression of B29 in other B cell disorders was, as a rule, significantly higher than in CLL. Two thirds of non-Hodgkin's lymphomas in leukemic phase were SN8 positive, including lymphoplasmacytic (45%), follicular (83%), mantle cell (92%) and splenic lymphoma with villous lymphocytes (74%) while only 25% of hairy cell leukemias were SN8 positive. Within CLL, 2.3% of typical cases were SN8+ while 16% of cases with atypical morphology and an increased number of prolymphocytes were SN8+. Our results suggest a useful role for SN8 in the immunophenotypic differentiation of B cell disorders as a marker for non-CLL diseases. The analysis of B29 expression may throw light into the structure of the B cell an...
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ZAP-70, CD38 and IGHV mutations have all been reported to have prognostic impact in chronic lymphocytic leukemia (CLL), both individually and in paired combinations. We aimed to determine whether the combination of all three factors... more
ZAP-70, CD38 and IGHV mutations have all been reported to have prognostic impact in chronic lymphocytic leukemia (CLL), both individually and in paired combinations. We aimed to determine whether the combination of all three factors provided more refined prognostic information concerning the treatment-free interval (TFI) from diagnosis. ZAP-70, CD38 and IGHV mutations were evaluated in 142 patients. Combining all three factors, the ZAP-70-/CD38-/Mutated group showed the longest median TFI (62 months, n = 37), ZAP-70+/CD38+/Unmutated cases the shortest (11 months, n = 37) and cases discordant for > or = 1 factor, an intermediate TFI (27 months, n = 68) (p = 0.006). Analysis of discordant cases revealed values that were otherwise masked when measuring single prognostic factors. The presence or absence of cytogenetic abnormalities did not explain the variability among discordant cases. Simultaneous analysis of ZAP-70, CD38 and IGHV mutations in CLL provides more discriminatory predi...
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The European Myeloma Network (EMN) organized two flow cytometry workshops. The first aimed to identify specific indications for flow cytometry in patients with monoclonal gammopathies, and consensus technical approaches through a... more
The European Myeloma Network (EMN) organized two flow cytometry workshops. The first aimed to identify specific indications for flow cytometry in patients with monoclonal gammopathies, and consensus technical approaches through a questionnaire-based review of current practice in participating laboratories. The second aimed to resolve outstanding technical issues and develop a consensus approach to analysis of plasma cells. The primary clinical applications identified were: differential diagnosis of neoplastic plasma cell disorders from reactive plasmacytosis; identifying risk of progression in patients with MGUS and detecting minimal residual disease. A range of technical recommendations were identified, including: 1) CD38, CD138 and CD45 should all be included in at least one tube for plasma cell identification and enumeration. The primary gate should be based on CD38 vs. CD138 expression; 2) after treatment, clonality assessment is only likely to be informative when combined with ...
Research Interests: Flow Cytometry, Medicine, Humans, Multiple Myeloma, Monoclonal Antibodies, and 13 moreDifferential Diagnosis, CD, Myeloma, Prognosis, Immunophenotyping, Somatic Cell Count, MINIMAL RESIDUAL DISEASE, Clinical Application, Cell count, Plasma cells, cytometry, Chromosome aberrations, and Cardiovascular medicine and haematology
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CD123 is an antibody that identifies the a chain of the human interleukin-3 receptor and is expressed in a variety of normal hematopoietic cells, acute leukemia and hairy cell leukemia (HCL). The aim of the study was to investigate the... more
CD123 is an antibody that identifies the a chain of the human interleukin-3 receptor and is expressed in a variety of normal hematopoietic cells, acute leukemia and hairy cell leukemia (HCL). The aim of the study was to investigate the diagnostic value of CD123 expression in B-cell disorders with circulating hairy and villous lymphocytes. We investigated the diagnostic value of CD123 expression in neoplastic cells from 59 patients with B-cell disorders with circulating hairy or villous lymphocytes: HCL (n=24), the variant form of HCL (n=11) and splenic lymphoma with villous lymphocytes (SLVL) (n=24). Cells from 12 patients with chronic lymphocytic leukemia were used as controls. Immunophenotypic analysis was performed by flow cytometry on 77 samples from peripheral blood (n=48), bone marrow (n=25) and spleen cell suspensions (n=4). Our findings show that cells from 95% of typical HCL express CD123 with strong to moderate intensity while this molecule is absent in circulating cells f...
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BACKGROUND Zeta-chain associated protein (ZAP)-70 has been proposed as a surrogate marker for immunoglobulin heavy-chain variable region (IgVH) mutation in chronic lymphocytic leukemia (CLL), but it is still not clear whether it is an... more
BACKGROUND Zeta-chain associated protein (ZAP)-70 has been proposed as a surrogate marker for immunoglobulin heavy-chain variable region (IgVH) mutation in chronic lymphocytic leukemia (CLL), but it is still not clear whether it is an independent prognostic factor. METHODS The authors evaluated ZAP-70 expression by flow cytometry in 201 untreated patients and correlated ZAP-70 levels with CD38 expression, genetic abnormalities detected by fluorescence in situ hybridization (FISH), and the time from diagnosis to first treatment. RESULTS Fifty-seven patients (28%) were positive for ZAP-70 (> or = 20%). Positive ZAP-70 status was associated with advanced disease stage, atypical morphology, CD38-positive status, trisomy 12, del(6q), or no detectable abnormalities; negative ZAP-70 status was correlated with del(13q) as a sole abnormality. The treatment-free interval (TFI) was 17.7 months for ZAP-70-positive patients and 44.6 months for ZAP-70-negative patients (P < 0.001). Multivar...
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4817 T-cell acute lymphoblastic leukemia (T-ALL) patients have increased risk for treatment resistance and early relapse. The precise bone marrow evaluation for the presence of minimal residual disease (MRD) is essential for guiding... more
4817 T-cell acute lymphoblastic leukemia (T-ALL) patients have increased risk for treatment resistance and early relapse. The precise bone marrow evaluation for the presence of minimal residual disease (MRD) is essential for guiding treatment options. This requires techniques more sensitive than the level of sensitivity of light microscopic technique such as multicolour flow cytometry (FCM). Immunophenotypic alterations called leukemia associated immunophenotypic patterns (LAIP) (i.e.aberrant myeloid markers) and ectopic phenotypic expression (i.e. appearance of immature phenotypes such as TdT, CD1a and CD3 outside their normal site in the thymus) are of benefit to track the residual leukemic cells in T-ALL. A retrospective data analysis of MRD was done comprising T-ALL patients diagnosed and followed-up at the Institute of Cancer Research/Royal Marsden Hospital by means of 3-colour flow cytometry (3C FCM).The aim was to answer a question whether the 3C FCM can reliably split patien...
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In vitro Acute Myeloid Leukemia (AML) models are currently based on cell lines or, if primary cells are used, require co-culture or supplementation with abnormally high concentrations of exogenous cytokines (EC). These conditions... more
In vitro Acute Myeloid Leukemia (AML) models are currently based on cell lines or, if primary cells are used, require co-culture or supplementation with abnormally high concentrations of exogenous cytokines (EC). These conditions introduce bias to the culture and fail to recapitulate the 3D environment integral to leukemic growth in bone marrow (BM). We have previously shown that human AML cell lines can be grown in 3D-polyurethane scaffolds (3D-PU) best when collagen-coated for 8 weeks in the absence of EC. This same cytokine-free platform supported proliferation and clonogenic capacity of human cord blood mononuclear cells (MNCs) for up to 6 weeks in static conditions (SC), with more rapid growth when cultured in a novel perfused 3D hollow-fibre bioreactor (PHFB), which utilizes permeable membranes embedded within the coated 3D-PU for the selective mass transport of nutrients/metabolites. Herein, we evaluated the potential for human primary de novo Mixed Phenotype Acute Leukemia (...
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T-cell prolymphocytic leukemia (T-PLL) is a rare T-cell disorder characterized by an aggressive clinical course and short survival. Immunophenotypically, the leukemic cells show heterogeneous CD4 and CD8 expression; most cases are... more
T-cell prolymphocytic leukemia (T-PLL) is a rare T-cell disorder characterized by an aggressive clinical course and short survival. Immunophenotypically, the leukemic cells show heterogeneous CD4 and CD8 expression; most cases are CD4+CD8− but, CD4+CD8+ and CD4−CD8+ cases are recognized. CD4+CD8+ phenotype is conventionally associated with cortical thymocytes, however, double positive T-prolymphocytes, are TdT −, CD1a − and CD3+ suggesting a post-thymic origin. Phenotypic changes in malignant cells while recognized in acute leukemias, are rare in mature T-cell disorders. In T-PLL, there is only 1 report describing 2 cases of phenotypic change (Tuset et al, Leukemia and Lymphoma 2001.42:1379-83). We retrospectively investigate the frequency of this phenomenon amongst 20 patients with T-PLL for whom sequential immunophenotyping of leukemic cells was available. This included the 2 cases previously reported. Diagnosis of T-PLL was based on clinical features, morphology, immunophenotype ...
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The prognostic value of ZAP-70, CD38 expression and IgVH somatic hypermutation(SHM) in CLL has been well documented. We investigated whether the proposed model of combining ZAP-70 and CD38 levels to identify patients likely to progress... more
The prognostic value of ZAP-70, CD38 expression and IgVH somatic hypermutation(SHM) in CLL has been well documented. We investigated whether the proposed model of combining ZAP-70 and CD38 levels to identify patients likely to progress (Del Giudice et al 2005, Schroers et al 2005) remained valid when mutational status was considered and studied which combinations of these 3 parameters provided the most valuable prognostic information. ZAP-70 and CD38 were evaluated by flow cytometry and IgVH SHM was analysed by direct sequencing with 98% cut off. All 3 parameters were studied in 115 untreated CLL patients, 90% of which were advanced stage:(stage A stable 10%, stage A progressive 30%, stage B 37%, and stage C 23%). ZAP-70 and IgVH SHM showed 68% concordance, CD38 and IgVH SHM concordance of 69% and 75% of patients, using cut offs of ≥30% and ≥7%, respectively. The impact on time to first treatment /treatment free interval (TFI) for these parameters can be seen in Table 1. Treatment F...
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4896 Current in vitro models for the study of AML rely on cell lines or, if primary cells are used, require abnormally high concentrations of exogenous cytokines and/or stromal cells in a co-culture system. These conditions introduce bias... more
4896 Current in vitro models for the study of AML rely on cell lines or, if primary cells are used, require abnormally high concentrations of exogenous cytokines and/or stromal cells in a co-culture system. These conditions introduce bias and artifact by selecting for culture-responsive cells and do not account for the 3D leukemic growth observed in the bone marrow. We have previously shown that human AML cell lines can be cultivated in 3D synthetic polymeric scaffolds coated with collagen and fibronectin for 8 weeks. We have also shown that human cord blood mononuclear cells can be cultured in the same 3D in vitro system with good viability (>90%), proliferation and clonogenic capacity in the absence of exogenous cytokines over a period of 4–6 weeks. Herein, we evaluate the potential for human primary AML cells to survive in a cytokine-free environment on 3D polyurethane (PU) scaffolds rendered bioactive by collagen type I over 35 days. After informed consent, peripheral blood (...
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2601 Poster Board II-577 Minimal residual Disease (MRD) monitoring is known to be of importance in guiding management in patients with acute myeloid leukaemia (AML) treated with chemotherapy. Many subtypes of AML do not, however, have a... more
2601 Poster Board II-577 Minimal residual Disease (MRD) monitoring is known to be of importance in guiding management in patients with acute myeloid leukaemia (AML) treated with chemotherapy. Many subtypes of AML do not, however, have a molecular marker available for MRD monitoring and in this setting the use of multiparametric flow cytometry (MFC) has been evaluated. Few studies have investigated the impact of MRD in the setting of allogeneic transplantation for AML. We speculate that, as with chemotherapy, the outcome in MRD positive patients will be inferior to MRD negative patients due to an increase in disease relapse. In order to investigate this hypothesis we studied 74 patients who underwent allogeneic transplantation for AML in a single centre from 2004–2008. The median age was 47.1 years (range: 20.8–70.1). The overall survival at 2 years was 48% with a median follow-up of 1.96 years. Conditioning was myeloablative in 37 patients and reduced intensity in 37 patients. 46 pa...
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A middle aged Northern European man presented with a submental lymph node and was found on imaging to have generalized lymphadenopathy. There was bone marrow infiltration by small apparently lymphoid cells, many with cytoplasmic tails;... more
A middle aged Northern European man presented with a submental lymph node and was found on imaging to have generalized lymphadenopathy. There was bone marrow infiltration by small apparently lymphoid cells, many with cytoplasmic tails; these cells were CD56(+) and weakly CD4(+). A provisional diagnosis was confirmed by further immunophenotyping.
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Deletions of 13q14.3 are well known in several malignancies and are thought to be associated with tumour suppressor function. The RB-1 gene is a tumour suppressor gene, but other loci including D13S319 and D13S25 telomeric to this within... more
Deletions of 13q14.3 are well known in several malignancies and are thought to be associated with tumour suppressor function. The RB-1 gene is a tumour suppressor gene, but other loci including D13S319 and D13S25 telomeric to this within 13q14.3 are deleted in B-cell chronic lymphocytic leukaemia (B-CLL), multiple myeloma and non-Hodgkin&#39;s lymphoma, with varying clinical significance. The fluorescence in situ hybridization screening of 22 patients with T-prolymphocytic leukaemia (T-PLL) for deletions of 13q14.3 revealed loss of D13S25 in 17 cases (mean 40% range 13-98%), with 11 patients having at least a 20% deletion. Mapping the deletions for the RB-1, D13S319,and D13S25 loci revealed D13S25 as the most frequently deleted marker. However, patients with only the D13S25 deletion had low percentages of cells with the deletion (12-13%), suggesting that loss of D13S25 on its own may not provide sufficient growth advantage. The use of the YAC 954c12, which maps immediately adjacent to D13S25, defined the telomeric border of the deletion in some of the cases. Inv(14)(q11q32) and t(14;14)(q11;q32) are characteristic of T-PLL, but are also observed in premalignant T-cell clones in patients with ataxia telangiectasia. Transition to overt leukaemia may result from loss of suppressor function. Thus, 13q14.3 deletions could contribute to the development of overt leukaemia in T-PLL, but the involvement of more than one gene in the region cannot be excluded.
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ABSTRACT