Demonstration That Mast Cells, T Cells, and B Cells Bearing the Activating Kit Mutation D816V Occur in Clusters within the Marrow of Patients with Mastocytosis
2004; Elsevier BV; Volume: 6; Issue: 4 Linguagem: Inglês
10.1016/s1525-1578(10)60529-6
ISSN1943-7811
AutoresMarcia L. Taylor, Devinder Sehgal, Mark Raffeld, Harold Obiakor, Cem Akin, Rose G. Mage, Dean D. Metcalfe,
Tópico(s)melanin and skin pigmentation
ResumoMastocytosis is characterized by focal heterotypic clusters of mast cells and lymphocytes in the bone marrow and by a somatically acquired activating Kit mutation, D816V. The relationship of the occurrence of this mutation to the heterotypic clusters of mast cells and lymphocytes in bone marrow is unknown. We hypothesized that these two unique features of mastocytosis were related. To explore this hypothesis, laser capture microdissected mast cells, B cells, and T cells, from both lesional and non-lesional areas of bone marrow biopsy tissues from patients with mastocytosis, were examined for the D816V mutation in their DNA, using HinfI restriction digestion of nested PCR products amplified from extracts of dissected cells. The D816V mutation was detected in mast cells, B cells, and T cells from lesional but not non-lesional areas of bone marrow tissues. B cells obtained from lesional areas of tissue were also assessed for clonality and were found to at least represent an oligoclonal population. Thus, mast cells and lymphocytes within focal aggregates in the bone marrow of those with mastocytosis are more frequently positive for the codon 816 activating mutation. Further, the B cell population is oligoclonal, suggesting that clonal proliferation is unlikely to be the basis of clustering. Mastocytosis is characterized by focal heterotypic clusters of mast cells and lymphocytes in the bone marrow and by a somatically acquired activating Kit mutation, D816V. The relationship of the occurrence of this mutation to the heterotypic clusters of mast cells and lymphocytes in bone marrow is unknown. We hypothesized that these two unique features of mastocytosis were related. To explore this hypothesis, laser capture microdissected mast cells, B cells, and T cells, from both lesional and non-lesional areas of bone marrow biopsy tissues from patients with mastocytosis, were examined for the D816V mutation in their DNA, using HinfI restriction digestion of nested PCR products amplified from extracts of dissected cells. The D816V mutation was detected in mast cells, B cells, and T cells from lesional but not non-lesional areas of bone marrow tissues. B cells obtained from lesional areas of tissue were also assessed for clonality and were found to at least represent an oligoclonal population. Thus, mast cells and lymphocytes within focal aggregates in the bone marrow of those with mastocytosis are more frequently positive for the codon 816 activating mutation. Further, the B cell population is oligoclonal, suggesting that clonal proliferation is unlikely to be the basis of clustering. Focal aggregates of mast cells, and T and B lymphocytes are frequently observed within the bone marrow of patients with systemic mastocytosis.1Akin C Jaffe ES Raffeld M Kirshenbaum AS Daley T Noel P Metcalfe DD An immunohistochemical study of the bone marrow lesions of systemic mastocytosis: expression of stem cell factor by lesional mast cells.Am J Clin Pathol. 2002; 118: 242-247Crossref PubMed Scopus (25) Google Scholar Such heterotypic aggregates are considered a major histopathological feature of this disease, and the occurrence of these lesions is one of the diagnostic criteria for mastocytosis. Mast cells within these aggregates are tryptase positive, and contain stem cell factor.1Akin C Jaffe ES Raffeld M Kirshenbaum AS Daley T Noel P Metcalfe DD An immunohistochemical study of the bone marrow lesions of systemic mastocytosis: expression of stem cell factor by lesional mast cells.Am J Clin Pathol. 2002; 118: 242-247Crossref PubMed Scopus (25) Google Scholar The basis for the formation of these aggregates is unknown.To further characterize these heterotypic collections of mast cells and lymphocytes and to explore their etiology, we elected to determine whether the cells within these aggregates were more likely to exhibit the codon 816 activating mutation in Kit,2Furitsu T Tsujimura T Tono T Ikeda H Kitayama H Koshimizu U Sugahara H Butterfield JH Ashman LK Kanayama Y Matsuzawa Y Kitamura Y Kanakura Y Identification of mutations in the coding sequence of the proto-oncogene c-kit in a human mast cell leukemia cell line causing ligand-independent activation of c-kit product.J Clin Invest. 1993; 92: 1736-1744Crossref PubMed Scopus (732) Google Scholar, 3Nagata H Worobec AS Oh CK Chowdhury BA Tannenbaum S Suzuki Y Metcalfe DD Identification of a point mutation in the catalytic domain of the protooncogene c-kit in peripheral blood mononuclear cells of patients who have mastocytosis with an associated hematologic disorder.Proc Natl Acad Sci USA. 1995; 92: 10560-10564Crossref PubMed Scopus (792) Google Scholar, 4Longley Jr, BJ Metcalfe DD Tharp M Wang X Tyrrell L Lu S Heitjan D Ma Y Activating and dominant inactivating c-KIT catalytic domain mutations in distinct clinical forms of human mastocytosis.Proc Natl Acad Sci USA. 1999; 96: 1609-1614Crossref PubMed Scopus (494) Google Scholar which is now documented to occur in multiple hematopoietic lineages in most adult patients with mastocytosis.5Akin C Kirshenbaum AS Semere T Worobec AS Scott LM Metcalfe DD Analysis of the surface expression of c-kit and occurrence of the c-kit Asp816Val activating mutation in T cells, B cells, and myelomonocytic cells in patients with mastocytosis.Exp Hematol. 2000; 28: 140-147Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 6Yavuz AS Lipsky PE Yavuz S Metcalfe DD Akin C Evidence for the involvement of a hematopoietic progenitor cell in systemic mastocytosis from single-cell analysis of mutations in the c-kit gene.Blood. 2002; 100: 661-665Crossref PubMed Scopus (147) Google Scholar Not only are cells expressing mutated Kit believed to have enhanced proliferation on exposure to stem cell factor, the ligand of Kit,5Akin C Kirshenbaum AS Semere T Worobec AS Scott LM Metcalfe DD Analysis of the surface expression of c-kit and occurrence of the c-kit Asp816Val activating mutation in T cells, B cells, and myelomonocytic cells in patients with mastocytosis.Exp Hematol. 2000; 28: 140-147Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 7Longley BJ Tyrrell L Lu SZ Ma YS Langley K Ding TG Duffy T Jacobs P Tang LH Modlin I Somatic c-KIT activating mutation in urticaria pigmentosa and aggressive mastocytosis: establishment of clonality in a human mast cell neoplasm.Nat Genet. 1996; 12: 312-314Crossref PubMed Scopus (553) Google Scholar, 8Hashimoto K Tsujimura T Moriyama Y Yamatodani A Kimura M Tohya K Morimoto M Kitayama H Kanakura Y Kitamura Y Transforming and differentiation-inducing potential of constitutively activated c-kit mutant genes in the IC-2 murine interleukin-3-dependent mast cell line.Am J Pathol. 1996; 148: 189-200PubMed Google Scholar, 9Kitayama H Kanakura Y Furitsu T Tsujimura T Oritani K Ikeda H Sugahara H Mitsui H Kanayama Y Kitamura Y Matsuzawa Y Constitutively activating mutations of c-kit receptor tyrosine kinase confer factor-independent growth and tumorigenicity of factor-dependent hematopoietic cell lines.Blood. 1995; 85: 790-798Crossref PubMed Google Scholar, 10Kitayama H Tsujimura T Matsumura I Oritani K Ikeda H Ishikawa J Okabe M Suzuki M Yamamura K Matsuzawa Y Kitamura Y Kanakura Y Neoplastic transformation of normal hematopoietic cells by constitutively activating mutations of c-kit receptor tyrosine kinase.Blood. 1996; 88: 995-1004PubMed Google Scholar but also to exhibit enhanced chemotaxis to stem cell factor.11Taylor ML Dastych J Sehgal D Sundstrom M Nilsson G Akin C Mage RG Metcalfe DD The Kit-activating mutation D816V enhances stem cell factor-dependent chemotaxis.Blood. 2001; 98: 1195-1199Crossref PubMed Scopus (43) Google Scholar We thus hypothesized that mast cells and lymphocytes within these aggregates would be more likely to express the codon 816 mutation, either collecting in focal areas in response to a stimulus such as stem cell factor, or because of clonal proliferation. To further examine clonality, we sought to determine whether the population of B cells within a heterotypic aggregate was monoclonal or oligoclonal.As will be shown, mast cells, and T and B lymphocytes obtained by laser capture microdissection of these lesions consistently demonstrated the codon 816 mutation. Mast cells and lymphocytes collected from non-lesional areas did not exhibit a codon 816 mutation within the limit of detection. Further, the clonal characteristics of B cells obtained from a lesional area of tissue were found to be at least oligoclonal. These data are most consistent with the hypothesis that these collections of cells occur by a mechanism other than clonal proliferation.Materials and MethodsReagents and AntibodiesAmpliTaq DNA polymerase and dNTPs were obtained from Perkin Elmer (San Francisco, CA); TaqStart antibody from Clontech (Palo Alto, CA); HinfI restriction enzyme from New England Biolabs (Beverly, MA); and proteinase K from Roche Diagnostics (Indianapolis, IN).Patient CharacteristicsThe clinical profile of the patient population is summarized in Table 1. A total of 10 patients (five females and five males, age range 23 to 75) comprising various categories of mastocytosis were studied. Five of the patients had indolent systemic mastocytosis with normocellular focal increases of mast cells. Three of the patients were diagnosed with smoldering systemic mastocytosis with hypercellular focal distribution of mast cells. Two patients had associated hematological non-mast cell disease, more specifically, myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML). The patient with MDS had a normocellular focal bone marrow involvement while the CMML patient had a hypercellular focal involvement. In all these cases, mast cells were accompanied by lymphoid aggregates. All patients exhibited urticaria pigmentosa and had tryptase levels in plasma that ranged from 26 to >200 ng/ml. The study was reviewed and approved by the Institutional Review Board of the National Institute of Allergy and Infectious Diseases (National Institutes of Health) and informed consent was obtained from all patients.Table 1Characteristics of Patients with MastocytosisType of patient*All positive for urticaria pigmentosa.PatientAgeGenderBone marrow biopsyTryptase (ng/ml)Indolent systemic mastocytosis139Fnormocellular, focal26268Mnormocellular, focal71353Fnormocellular, focal35452Mnormocellular, focal>200561Fhypercellular, focal198Smoldering systemic mastocytosis671Mhypercellular, focal192723Fhypercellular, focal>200845Mhypercellular, focal>200Mastocytosis with associated/hematologic non-mast cell disease975Mnormocellular, focal, MDSND†ND equals not determined.1059Fhypercellular, CMML110* All positive for urticaria pigmentosa.† ND equals not determined. Open table in a new tab Processing and Immunohistochemistry of Bone Marrow Biopsies from Patients with Mastocytosis and Normal Human AppendixTrephine core bone marrow biopsies were obtained from the posterior iliac crest. Biopsy tissues were decalcified in 0.07% (w/v) EDTA, placed in B5 fixative overnight and then embedded in paraffin. Eight-μm-thick sections were cut on charged slides and immunophenotyping was performed with antibodies against tryptase (Dako Corp., Carpinteria, CA; 1:50 dilution), CD20 (L26, Dako, 1:200 dilution) and CD3 (Dako, 1:100 dilution) using an automated immunostainer (Ventana Medical Systems, Inc, Tucson, AZ) according to the manufacturer's paraffin-embedded tissue staining protocols. Heat-induced antigen retrieval was performed with a microwave pressure cooker as described.12Quintanilla-Martinez L Thieblemont C Fend F Kumar S Pinyol M Campo E Jaffe ES Raffeld M Mantle cell lymphomas lack expression of p27Kip1, a cyclin-dependent kinase inhibitor.Am J Pathol. 1998; 153: 175-182Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar All primary antibodies were applied to the sections for 32 minutes.Frozen normal human appendix tissue from a 4-year-old child preserved in OCT was obtained from Childrens National Medical Center, Washington, D.C. Eight-μm-thick sections were cut on charged slides and immunophenotyping was performed with antibodies against CD79a (Pharmingen, San Diego, CA, 1:50 dilution). The primary antibody was applied for 30 minutes followed by incubation with the secondary antibody, goat-anti-mouse IgG biotin (Southern Biotechnology, Birmingham, AL, 1:50 dilution) for 20 minutes.Laser Capture MicrodissectionLaser capture microdissection (LCM) of cells from paraffin-embedded bone marrow biopsy tissue sections was performed using a PixCell laser capture microscope with an infrared diode laser (Arcturus Engineering, Santa Clara, CA). Briefly, the stained, dehydrated tissue section was overlaid with a transfer polymer film mounted on an optically transparent cap. Once the cells of interest (400 to 500 cells) were located, low energy laser pulses were applied leading to focal expansion, melting of the polymer, and resultant fusion of the microdissected tissue to the cap. The captured cells on the cap were digested with 40 μl of 0.5 mg/ml DNase-free proteinase K in 1X PCR buffer for 3 hours at 50°C. Following digestion, the extract was centrifuged at 2000 rpm for 2 minutes. The proteinase K enzyme was heat-inactivated and the entire extract was used directly for PCR.Detection of the D816V Mutation in Bone Marrow BiopsiesThe D816V mutation was amplified by nested polymerase chain reaction (PCR). The primers Kit−1-F (TCCTCCAACCTAATAGTGTATTCACAG) and Kit−1-R (TTTGCAGGACTGTCAAGCAGAGAATG) were used for the first round and Kit−2-F (TATCCTCCTTACTCATGGTCGG) Kit−2-R (AGA-GAATGGGTACTCACGTTTCC) for the second round of amplification as described.11Taylor ML Dastych J Sehgal D Sundstrom M Nilsson G Akin C Mage RG Metcalfe DD The Kit-activating mutation D816V enhances stem cell factor-dependent chemotaxis.Blood. 2001; 98: 1195-1199Crossref PubMed Scopus (43) Google Scholar The PCR was performed in a final volume of 50 μl containing 50 mmol/L KCl, 10 mmol/L-Tris-HCl, 200 μmol/L of each dNTP, 5 units AmpliTaq DNA polymerase and 0.5 μmol/L of each primer. One μl of the PCR product from the first round was used as the template for the second round of PCR. The rest of the components in the reaction mixture were the same as described above for the first round. The same temperature conditions were used for the first and second round touchdown PCR. There was an initial 95°C 1-minute denaturation step, followed by denaturation at 94°C for 1 minute and an extension at 72°C for 2 minutes; for the first cycle, annealing was at 68°C for 30 seconds which dropped down to 58°C at the end of the fifth cycle at the rate of 2°C per cycle. The annealing temperature for the remaining 23 cycles was 58°C. The PCR ended with a 5-minute extension at 72°C. The PCR was performed on a PTC-100 programmable thermal cycler with a hot bonnet (MJ Research, Inc., Watertown, MA).To detect the D816V mutation which creates an additional HinfI site, the second round PCR product was digested with the restriction enzyme HinfI for 2 hours at 37°C. The digested product was electrophoresed on a 20% TBE polyacrylamide gel (Novex, San Diego, CA). Predicted fragment sizes for the homozygous wild-type c-kit sequence were 68 and 43 bp and for the heterozygous mutated sequence were 68, 54, 43, and 14 bp. As the mutation is found on only one of the two alleles, cells with mutations also have the wild-type fragment of 68 and 43 bp generated from the wild-type copy of the c-kit gene. Control cell lines, HMC-1.1 (homozygous for the absence of the Kit D816V mutation), and HMC-1.2 (heterozygous for the Kit D816V mutation) were analyzed in parallel.Determination of the Sensitivity of Detection of the D816V MutationTo determine the sensitivity of detection of the D816V mutation in the assay, increasing numbers of mutant HMC1.2 cells (0,5,10,50,100) were mixed with wild-type HMC1.1 cells (100,95,90,50,0). The cells were then digested with proteinase K, followed by nested PCR and HinfI restriction digestion to detect the D816V mutation as described above. The level of detection was quantified by gel fluorescence imaging.11Taylor ML Dastych J Sehgal D Sundstrom M Nilsson G Akin C Mage RG Metcalfe DD The Kit-activating mutation D816V enhances stem cell factor-dependent chemotaxis.Blood. 2001; 98: 1195-1199Crossref PubMed Scopus (43) Google ScholarAmplification, Cloning, and Sequencing of Rearranged Immunoglobulin from B Cells in Bone Marrow and Control Human Appendix SectionsThe complementarity determining-region 3 (CDR3) of the rearranged immunoglobulin heavy chains in CD20-stained B cells from a lesional area of bone marrow from a patient with indolent mastocytosis and in CD79a-stained control normal appendix was amplified using previously published primers directed at conserved sequences present in all JH region genes (JH) and the framework region 3 (FR3).13Trainor KJ Brisco MJ Story CJ Morley AA Monoclonality in B-lymphoproliferative disorders detected at the DNA level.Blood. 1990; 75: 2220-2222PubMed Google Scholar, 14Ramasamy I Brisco M Morley A Improved PCR method for detecting monoclonal immunoglobulin heavy chain rearrangement in B cell neoplasms.J Clin Pathol. 1992; 45: 770-775Crossref PubMed Scopus (249) Google Scholar Hotstart PCRs were performed using TaqStart antibody. Forty cycles of amplification consisting of an initial denaturation at 95°C for 2 minutes, followed by denaturation at 94°C for 1 minute, elongation at 72°C for 1 minute, and annealing at 56°C for 30 seconds were performed with DNA from microdissected tissue (the total 40 μl of crude extract from laser capture microdissected cells was added as template). The PCR product was electrophoresed on a 20% TBE polyacrylamide gel (Novex).The purified PCR products were cloned into the pCR4-TOPO vector (TOPO TA Cloning Kit, lnvitrogen, Carlsbad, CA) and transformed into One Shot TOP 10 chemically competent E. coli (Invitrogen) according to the manufacturer's instructions. Plasmid DNA recovered from white (recombinant) colonies was digested with EcoRI to confirm the presence of the insert. The digested product was electrophoresed on a 20% TBE polyacrylamide gel (Novex). The cloned inserts were sequenced using T3 primers (5′-ATTAACCCTCACTAAAGGGA-3′) and the BigDye Terminator Cycle Sequencing Kit (PE Applied Biosystems, Foster City, CA); and run on the ABI Prism 310 Genetic Analyzer (PE Applied Biosystems). Sequence analysis was performed by MacVector Software version 7.0 (Accelrys, San Diego, CA). HCDR3 lengths were defined as the number of amino acids after the generally conserved VH amino acid Arg 94 and before the conserved JH amino acid Trp 103. The DH segment used was identified as having a stretch of at least seven nucleotides of uninterrupted identity with a germline gene sequence or eight or more identical nucleotides interrupted by one mismatch.ResultsLaser Capture MicrodissectionTo verify that laser capture microdissection could be applied to the study of mastocytosis lesions, mast cells, lymphocytes, B cells, and T cells were obtained by LCM from dehydrated and immunohistochemically stained bone marrow biopsy sections from patients with mastocytosis and examined (Figure 1). The upper panel in Figure 1 shows a representative picture of mast cells obtained from both lesional and non-lesional areas of tryptase-stained bone marrow biopsy tissue from a patient with mastocytosis. An extensive accumulation of tryptase-stained mast cells is shown in the lesional areas of the biopsy tissue compared to sparse numbers seen in the non-lesional area. Lymphocytes were usually found clustered adjacent to the mast cell infiltrates in lesional areas of tryptase-stained bone marrow biopsy tissues from patients with mastocytosis (Figure 1, middle panel). To confirm the results obtained in lymphocytes, tissues were immunostained with either anti-CD 20 or anti-CD3 mAb. As can be seen in the lower panel of Figure 1, both B cell and T cell aggregates were found in the lesional areas of bone marrow biopsy tissues with B cells generally found in greater numbers than T cells.Detection of the D816V MutationTo assess the role of the activating Kit mutation D816V in the pathology of mastocytosis, DNA obtained from discrete populations of cells from both lesional and non-lesional areas of bone marrow tissues by LCM were examined for the mutation. As can be seen in Figure 2A, the D816V mutation was detected in mast cells, lymphocytes, B cells, and T cells from lesional areas of bone marrow biopsy sections in all four patients with indolent mastocytosis. In contrast, the D816V mutation was not detected in non-lesional bone marrow cells in these patients. Similarly, the D816V mutation was detected in mast cells, lymphocytes, B cells, and T cells in the lesional but not in the non-lesional areas of bone marrow biopsies from patients with smoldering mastocytosis (Figure 2B) and in two patients with mastocytosis with associated hematological non-mast cell disease (Figure 2C).Figure 2Detection of the D816V mutation in mast cells, lymphocytes, B cells, and T cells in patients with different categories of mastocytosis. A: Indolent mastocytosis. B: Smoldering mastocytosis. C: Mastocytosis with associated hematological non-mast cell disease. Analysis of T cells and B cells in lesional areas only. A non-lesional area is defined as an area where there are a sparse number of mast cells. L, denotes lesional area; N, denotes non-lesional area.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Sensitivity of Detection of the D816V MutationTo determine the minimum number of mutant cells that can be detected by nested PCR, increasing numbers of mutant HMC 1.2 cells (0,5,10,50,100) were mixed with wild-type HMC 1.1 cells (100,95,90,50,0). As shown in Figure 3, the D816V mutation was detectable when at least 10 HMC 1.2 cells/per 100 total cells were present as revealed by three bands at 68, 54, and 43 bp (the 14-bp band is not shown in this figure). These results were also quantified by gel fluorescence (Figure 3). There was a fluorescence of 0.01 with 5 cells (band was not visible), 0.1 with 10 cells, 0.5 with 50 cells, and 0.55 with 100 cells. Therefore, as a general interpretation, the absence of detection of the D816V mutation in cells obtained from non-lesional marrow means that the mutational frequency is less than 1 in 10 cells.Figure 3Sensitivity of detection of the D816V mutation by nested PCR. Results of HinfI restriction digestion of mixtures of mutant HMC1.2 (0,5,10,50,100) and wild-type HMC1.1 cells (100,95,90,50,0) with the corresponding gel fluorescence graph. Data are representative of three experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Molecular Analysis of Clonality of B CellsTo explain how lymphocytes cluster in bone marrow tissue, assessment of IgH VDJ gene rearrangements in B cell clones obtained from lesional areas of a bone marrow biopsy from a patient with mastocytosis was performed. Approximately 400 to 500 B cells obtained by LCM from a lesional area of a bone marrow biopsy from one patient with indolent mastocytosis were examined (Figure 4A). Similar numbers of cells were obtained from normal human appendix. Analyses of the IgH VDJ gene rearrangements in B cells from this patient and from normal human appendix were then performed. According to PCR-based criteria established from a previous study,15Racanelli V Sansonno D Piccoli C D'Amore FP Tucci FA Dammacco F Molecular characterization of B cell clonal expansions in the liver of chronically hepatitis C virus-infected patients.J Immunol. 2001; 167: 21-29Crossref PubMed Scopus (99) Google Scholar the occurrence of one or two distinct bands is indicative of B cell monoclonality. However, B cell monoclonality was not observed in this patient. Oligoclonality, defined as three or more bands, was demonstrated both in B cells from the patient with mastocytosis and in normal human appendix (Figure 4B). These results were further confirmed by sequencing. Forty-five sequences from this patient with varying HCDR3 lengths fell into five groups. Twenty-four sequences obtained from normal human appendix fell into four groups (Table 2). D and J gene usage in the cells from the patient with mastocytosis and in cells from normal human appendix was comparable except that within cells from groups 1 and 5 from the patient with mastocytosis, the rarely expressed JH1 gene was rearranged. Group 3 identified in normal human appendix used the infrequently expressed JH2 gene. The IgH VDJ sequences found in B cells from the patient with mastocytosis are shown in Figure 4C.Figure 4Molecular analysis of clonality of B cells from bone marrow biopsies from a patient with mastocytosis. A: B cells obtained from lesional areas of a bone marrow biopsy from a patient with mastocytosis and from normal human appendix by LCM. B: Starting PCR product of B cells (400 to 500 cells) as well as cloned CDR3 obtained from microdissected B cells from a patient with mastocytosis and from normal human appendix. C: Corresponding IgH CDR3 VDJ sequences from the patient. The nucleotide and deduced amino acid sequences are shown with the segments encoded by D genes (regular font) and n nucleotides (bold) indicated. GenBank Accession Numbers for these and control human appendix cell sequences are given in Table 2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 2Summary of IgH VDJ and HCDR3 Sequencing Results from a Patient with Mastocytosis and Normal Human AppendixGroupNo. clonedHCDR3 length*Length of heavy chain third complementarity determining region (HCDR3) in amino acids.D-geneJ-geneNo. basepairs D-gene differs from germlineAccession Number†The sequences were submitted to GenBank and assigned the Accession Numbers shown.Patient bone marrow Group 1 (AC100)2817D2–8J10/24AY177274 Group 2 (AC103)914D3–10J65/28AY177275 Group 3 (AC151)39D6–19J42/15AY177277 Group 4 (AC148)37D3–16J43/14AY177278 Group 5 (AC146)212D3–22J10/27AY177276Human appendix Group 1 (H3-001)410D2–15J43/15AY177279 Group 2 (H3-002)114D3–16J42/18AY177280 Group 3 (H3-008)614D1–26J20/10AY177281 Group 4 (H3-003)138Unknown Only 6 bp longJ6AY177282* Length of heavy chain third complementarity determining region (HCDR3) in amino acids.† The sequences were submitted to GenBank and assigned the Accession Numbers shown. Open table in a new tab DiscussionBone marrow biopsies in mastocytosis are characterized by focal or diffuse accumulations of mast cells in paratrabecular and perivascular regions often associated with lymphoid aggregates.16Parker RI Hematologic aspects of mastocytosis: I: Bone marrow pathology in adult and pediatric systemic mast cell disease.J Invest Dermatol. 1991; 96: 47S-51SAbstract Full Text PDF Google Scholar, 17Mekori YA Lymphoid tissues and the immune system in mastocytosis.Hematol Oncol Clin North Am. 2000; 14: 569-577Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar However, neither a comprehensive assessment of the activating Kit mutation D816V in mast cells and lymphocytes in bone marrow from various categories of these patients, nor the explanation for the associated lymphocyte clustering found in lesions of tissues in this disease has previously been determined. This study did not examine other cell types not consistently found in these lesions including adjacent myeloid cells.In the present study, the D816V mutation was detected in mast cells, B cells, and T cell specifically in the lesional but not in the non-lesional areas of bone marrow biopsies from different categories of patients with mastocytosis. Our mutation sensitivity study indicates that mutation detection requires that at least 10% of the cell population contain the D816V mutation (Figure 3). While we cannot rule out the possibility that there may be a few mast cells present in the microdissected lymphoid aggregates, it is highly unlikely that these approach 10% of the total cells because adjacent tryptase staining of microdissected slides indicates few, if any, mast cells present in the selected areas. Thus, as may be seen in the upper panel in Figure 1, there is an extensive infiltration of tryptase-positive granulated mast cells clearly stained and with no evidence of other cell types in the lesional area of tryptase-stained bone marrow biopsies. This cluster of mast cells surrounds a lymphocyte population that was subsequently microdissected. We believe it is clear from this tryptase-stained slide that the lymphocyte cluster contained a negligible number of mast cells (at most less than 10, nowhere near the 50 cells required to detect the mutation). Also of note is that mast cells are mainly located in the periphery, which makes it easier to avoid microdissecting these cells with other cells. On closer examination, we believe one can also observe that most if not all of these mast cells were, in fact, not included in the microdissected sample as they remain in the tissue. Application of this technique has as a primary advantage, the ability to directly observe cell types for microdissection. It is our conclusion that between direct observation and the actual dissected materials provided for visualization that we avoided significant mast cell contamination in the lymphoid population that was microdissected and that the r
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