Heavy Chain Gene Rearrangement Studies Research Articles

Primary hepatic extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue

Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), is one of the specific type of low-grade B-cell lymphoma not infrequently found worldwide. It typically involves mucosal sites such as stomach and conjunctiva; however, primary hepatic MALT lymphoma has been extremely rarely reported. We describe a case of hepatic MALT lymphoma in a 70-year-old male patient who underwent left hepatectomy due to the incidentally detected liver masses at a medical checkup. The resected specimen revealed multinodular masses consisting of small-to-intermediate-sized lymphoid cells with serpentine pattern and focal lymphoepithelial lesions. The tumor cells were diffusely positive for CD20 and Bcl-2 but negative for CD3, CD10, CD5, CD23, CD43, and cyclinD1. The Ki-67 labeling index was 10% and immunoglobulin heavy chain gene rearrangement study confirmed monoclonal proliferation. In this paper, we discuss several unique clinicopathologic characteristics which will be helpful to the differential diagnosis of hepatic MALT lymphoma.

Immunoglobulin heavy chain gene rearrangement study in difficult cases of B-cell lymphoproliferative disorder

To evaluate the ancillary diagnostic value of IgH gene rearrangements in those B-cell lymphoproliferative disorder cases whom are difficult in making a final diagnosis. IgH gene clonal rearrangements were retrospectively analyzed in a total of 77 diagnostically difficult B-cell lympho-proliferative patients. Standardized BIOMED-2 system IgH gene clonality assay kit targeting FR1, FR2, FR3 was used, followed by heteroduplex-polyacrylamide gel electrophoresis (PAGE) and silver nitrate staining. The final diagnoses of the 77 cases were: 12 cases of reactive lymphoid hyperplasia, 20 cases of atypical lymphoid hyperplasia or suspicious lymphoma, and 45 cases of B-cell lymphoma. Detection rates of at least one positive reaction were 2/12, 11/20 (55%), 36/45 (80%) in the three groups, respectively. In B-cell lymphomas, the clonality detection rate of FR1, FR2 and FR3 was 60% (27/45), 60% (27/45) and 56% (25/45), respectively. The type distribution were: 20 marginal zone lymphomas, including 18 extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue, 7 diffuse large B-cell lymphomas, 7 follicular lymphomas, 1 mantle-cell lymphoma, 1 Burkitt's lymphoma, 4 plasma cell neoplasms and 5 unclassified B-cell lymphomas. Rearrangements of FR1, FR2 or FR3 were not detected in 9 (20%) of the B cell lymphoma cases, nevertheless, one of them had developed liver lesion later, and was confirmed finally to be B cell lymphoma. Fourteen patients of reactive lymphoid hyperplasia with positive IgH gene clonal rearrangements, and atypical lymphoid hyperplasia had follow-up history available. Four of them were diagnosed as lymphoid malignancies upon further biopsy, and in three of them, clonal IgH gene rearrangements were detected. B-cell lymphoproliferative disorder requiring a detection of clonal IgH gene rearrangement for making a final diagnosis. Combined detections of three IgH FR1, FR2 and FR3 rearrangements provide important ancillary diagnostic value in confirming suspected B-cell lympho-proliferative disorders. It is important to take an additional biopsy or to follow-up those patients who that have a detectable IgH gene clonal rearrangement but without apparent morphological evidence of lymphoma. For cases with a negative IgH gene rearrangements, it might be necessary to perform clonality analysis for other forms of gene rearrangements including IgH or IgK and IgL in order to further improve the detection sensitivity.

Clinicopathologic features of peripheral T-cell lymphoma, unspecified with follicular pattern

To study the clinicopathologic features of peripheral T-cell lymphoma, unspecified (PTL-U) with follicular pattern. The clinical data, hematoxylin and eosin-stained sections of lymph node biopsies and follow-up data of 18 cases of PTL-U associated with follicular growth pattern were reviewed and studied. Eight cases of reactive lymphoid hyperplasia were used as controls. Semi-quantitative observation by retiform micrometer rule was carried out. Immunohistochemical study was also performed in all cases. T-cell receptor and immunoglobulin heavy chain gene rearrangement studies were conducted by polymerase chain reaction-based method. The median age of the patients was 53 years. The male-to-female ratio was 1.57:1 in lymphoma group. All of the lymphoma patients presented with superficial lymphadenopathy, with (8/18) or without B symptoms. Histologically, the lymphoma was characterized by follicles of various sizes and shapes. The T zones were expanded by medium-sized lymphoma cells which contained clear cytoplasm and irregular nuclei. Mitotic figures were commonly identified. Immunohistochemical study confirmed that the lymphoma cells were of T-lineage. The proliferative index, as highlighted by Ki-67, was higher [average = (38.24 +/- 13.42)%/mm2] than that in the control group. T-cell receptor gene rearrangement was demonstrated in 71.4% (10/14) of the lymphoma cases. A definitive diagnosis of PTL-U with follicular pattern can be made on the basis of morphologic examination, immunohistochemical assessment and clinical features. Cases with atypical features can further be delineated by molecular analysis. Long-term follow up of these patients is prudent.

Epstein-Barr virus positive B-cell lymphoproliferative disorder/polymorphous B-cell lymphoma of the urinary bladder: A case report with review of literature

We report an unusual case of a localized Epstein-Barr virus (EBV)-positive B cell lymphoproliferative disorder (LPD)/polymorphous B cell lymphoma of the urinary bladder in a 67 years old female patient. She had no known predisposing immunodeficiencies and presented with recent onset of hematuria. The CT and cystoscopic examination revealed a localized 2.5 cm polypoid or plaque-like mucosal mass on the right posterior and lateral wall of the bladder. The biopsy sample showed a diffuse and densely polymorphous atypical lymphoid infiltrate admixed with numerous small lymphocytes, histiocytes and occasional plasma cells and neutrophils. The large atypical cells were CD20+, CD79a+, CD30+, CD43+ and they were strongly positive for EBV by in situ hybridization using anti-EBER-1 probe. Polymerase chain reaction (PCR) for immunoglobulin heavy chain gene rearrangement study showed a clonal gene rearrangement. The findings indicated EBV+LPD of the bladder. Primary lymphoma of bladder is rare and primary EBV+LPD of the bladder has not been previously described. Potential misdiagnosis of poorly differentiated urothelial carcinoma can occur and accurate diagnosis depends on comprehensive immunohistochemical and molecular workups.

Potential curability of cladribine in selected patients with hairy cell leukemia

18010 Background: Hairy cell leukemia (HCL) is an uncommon, chronic B-cell lymphoproliferative disorder associated with pancytopenia, splenomegaly, and recurrent infections. Cladribine as a single seven-day continuous intravenous infusion achieves an overall response rate of 98%, and complete response rate of 91%. Follow-up studies have documented minimal residual disease (MRD) in at least 50% of complete responders and continued clinical relapses, with 108 months median follow-up (range, 86–172). This suggests that all complete responders are at continuous risk for relapse. With twenty-year follow-up now available in some patients, we sought to determine if any patients were without MRD and potentially cured of HCL after a single seven-day course of cladribine. Methods: The Scripps Clinic cladribine phase II database of 379 patients was screened for patients who are alive, in a continuous hematologic remission, and received only a single seven-day course of cladribine. Patients agreed to undergo complete blood counts (CBC) and bone marrow aspiration and biopsy (BM). BM samples were analyzed for morphologic evidence of HCL and for MRD by flow cytometry to detect monoclonal B-cells with HCL phenotypic characteristics. Immunoglobulin heavy chain gene rearrangement studies were performed in some patients. Results: Of the 379 patients screened, we had accurate contact information on 75 patients; 20 patients met inclusion criteria and agreed to undergo CBC and BM. Patient characteristics: 17 males and 3 females; median age, 53 years (range, 39 to 75); median pretreatment duration, 2 years (range, 0.1 to 12.3); median duration following cladribine treatment, 17 years (range, 11 to 20). Prior to cladribine, 13 patients were untreated; 10 had undergone splenectomy, interferon, or chlorambucil. All patients had a normal CBC. Of these 20 patients, 9 patients had no evidence of MRD on BM; 7 patients had MRD; 4 patients had morphological evidence of HCL. Conclusions: Cladribine can potentially cure HCL in a subset of patients. Patients with MRD or even morphologically demonstrable disease can remain asymptomatic with normal blood counts up to twenty years after a single course of cladribine. No significant financial relationships to disclose.

A 3-way collision tumor of the upper respiratory tract: a composite of 2 immunophenotypically distinct mantle cell lymphomas and a plasmacytoma

Composite lymphoma (CL) is composed of 2 or more morphologically and immunophenotypically distinct lymphomas in a single anatomical site. Here we report a unique CL of the upper respiratory tract in an elderly male patient. Morphologically, the lymphoma was composed of 2 distinct and well-demarcated areas consisting of monotonous small to medium-sized lymphocytes and sheets of mature-appearing plasma cells. Immunophenotyping by both flow cytometry and immunohistochemistry revealed that the small to medium-sized lymphocytes were composed of 2 distinct subpopulations sharing a CD5(+)/CD19(+)/CD20(+)/CD22(+)/CD23(-)/FMC-7(+)/cyclin D1(+) immunophenotype but with different immunoglobulin (Ig) light and heavy chain expression, consistent with 2 immunophenotypically distinct mantle cell lymphomas (MCLs); the plasma cells were composed of CD38(bright +)/CD138(+)/IgG kappa-restricted plasma cells, consistent with a plasmacytoma. Fluorescence in situ hybridization showed the t(11;14) translocation present in the lymphocyte region but absent in the plasma cell area. Ig heavy chain gene rearrangement studies on manually dissected populations showed 2 distinct patterns for the MCL and plasmacytoma. To our knowledge, this is the first report of a 3-way CL consisting of 2 immunophenotypically distinct MCLs and a plasmacytoma.

Diffuse large B-cell lymphoma with expression of anaplastic lymphoma kinase protein: clinicopathologic and immunohistochemical study of 5 cases

To study the clinicopathologic features of diffuse large B-cell lymphoma (DLBCL) with expression of anaplastic lymphoma kinase (ALK) protein. Nine hundred and forty-five (945) cases of DLBCL (including 177 consultation cases) diagnosed according to the 2001 World Health Organization classification of tumors of hematopoietic and lymphoid tissues were enrolled into the study. Immunohistochemical study for anti-ALK-11 was performed using LSAB technique. The ALK-positive cases were further confirmed by immunohistochemical study using EnVision technique. Only ALK-positive cases by EnVision technique were further analyzed by immunostaining for antigens including CD20, CD3, CD30, EMA, granzyme-B, TIA-1 and PC. Immunoglobulin heavy chain gene rearrangement study was also performed and follow-up data collected. There were altogether 5 (4 males and 1 female) cases of DLBCL showing expression of ALK protein. The age of the patients ranged from 34 to 72 years. All were primary nodal DLBCL. One case belonged to clinical stage I, 2 in stage II and 2 in stage III. The duration of follow up ranged from 4 to 32 months. Three patients subsequently died and the longest survival was 32 months. Morphologic subtypes included centroblastic 2, anaplastic 1, immunoblastic with plasmacytoid differentiation 1 and plasmablastic 1. Immunohistochemically, 4 cases were CD20 positive (including 2 centroblastic, 1 anaplastic and 1 immunoblastic cases). The plasmablastic case expressed kappa light chain and was negative for CD20. Rearrangement of immunoglobulin heavy chain gene was demonstrated in all 5 cases studied. As for ALK protein staining, a mixed membranous and cytoplasmic (1 immunoblastic case), granular cytoplasmic (2 centroblastic and 1 anaplastic cases) and mixed nuclear and cytoplasmic (1 plasmablastic case) patterns were observed. Expression of ALK protein is a rare phenomenon in DLBCL and can be seen in centroblastic, anaplastic, immunoblastic and plasmablastic subtypes. It is often associated with aggressive clinical behavior and worse prognosis. A new pattern of ALK protein expression, mixed membranous and cytoplasmic, is reported.

T-cell/histiocyte-rich B-cell lymphoma: histology, immunophenotype and differential diagnosis

To study the histology, immunophenotype and differential diagnosis of T-cell/histiocyte-rich B-cell lymphoma (TCRBCL). A review of 245 cases of so-called Hodgkin lymphoma diagnosed during the period from 1980 to 2000 in 3 hospitals in Guangzhou, 8 cases were reclassified as TCRBCL, according to the 2001 World Health Organization classification of lymphoid neoplasms. An additional 8 cases of TCRBCL were retrieved from consultation files, as well as routine biopsy cases encountered between 2000 and 2004. Immunohistochemical studies were performed on paraffin-embedded tissue by SP technique in order to study the immunophenotype of the large neoplastic cells (CD20, CD79a, CD3, CD45RO, CD15, CD30, CD10, bcl-6 and EMA) and background non-neoplastic cells (CD3, CD8, CD20, CD45RO, CD79a, CD57, CD68, CD21, CD35, cyclin D1, TIA-1). In-situ hybridization for EBER 1/2 and immunoglobulin heavy chain gene rearrangement study were also performed in 4 and 4 cases respectively. Among the TCRBCL cases studied, there were 8 males and 8 females. The age of patients ranged from 10 to 68 years old (mean = 40.3 years old). All had lymphadenopathy and hepatosplenomegaly. On presentation, 3 cases belonged to stage II, 10 cases stage III and 3 cases stage IV. Histologically, scattered atypical large neoplastic cells were seen in a background of small lymphocytes and sometimes histiocytes. The large cells exhibited CD20+, CD79a+, EMA+, CD15- and CD30- phenotype. On the other hand, the background small lymphocytes were CD3 and CD45RO-positive. Most of these background T cells expressed CD8 and TIA-1, while they were mostly CD57-negative. The histiocytic cells were CD68-positive; and CD21 and CD35-positive follicular dendritic cell meshworks were absent. In-situ hybridization for EBER 1/2 showed negative nuclear signals. Immunoglobulin heavy chain gene rearrangement study revealed clonal pattern in all the 4 cases tested. TCRBCL is a rare subtype of lymphoma, with distinctive histology and immunophenotype. The above features are helpful in delineating this entity from Hodgkin lymphoma, reactive lymphoid hyperplasia and lymphomatoid granulomatosis.

Afectación cutánea específica en pacientes con mieloma múltiple. Estudio clínico-patológico, inmunohistoquímico y citogenético de 40 casos

Introduction Specific cutaneous involvement in patients with multiple myeloma is very rare. When it does occur, it is in patients with advanced stages of multiple myeloma and with a large tumorous mass. In this study, we analyzed 40 patients with specific cutaneous involvement from multiple myeloma, with no bony lesions underlying the skin lesions, and we reviewed the related literature. Objectives We were particularly interested in the clinical course of these patients, including survival once skin metastases had developed and the possible influence of the different treatments administered. Material and methods The study was completed by determining the histopathological pattern, the immunohistochemical profile, the type of immunoglobulin and immunoglobulin light chain produced by the neoplastic plasma cells that infiltrated the skin, as well as the cytogenetic characteristics of these cells. Results From a clinical standpoint, the skin lesions consisted of multiple nodules or plaques with erythematous or violaceous coloration, and variable location. Histopathologically, two patterns were seen: nodular and diffuse interstitial. The plasma cells showed cytological atypia, and in one case they displayed a spindle shape, giving the lesion a sarcomatoid appearance. Immunohistochemically, these neoplastic plasma cells were strongly positive for CD79a, CD138, and EMA, while the immunoexpression of CD38 and CD43 was less intense and constant. In 39 of the 40 patients, the type of monoclonal immunoglobulin produced by the neoplastic plasma cells in the skin lesions was determined: 18 patients had IgA myeloma (5 IgA κ and 13 IgA λ), 19 patients had IgG myeloma (15 IgG κ and 4 IgG λ) and 2 patients had IgD λ myeloma. The 22 cases in which immunoglobulin heavy chain gene rearrangement studies by PCR were carried out for the JH gene showed monoclonal rearrangement, while viral studies to try to identify genetic material of the HHV-8 virus and the Epstein- Barr virus gave negative results in all cases. These 22 cases studied using FISH showed the deletion of the rb-1 retinoblastoma gene in the neoplastic plasma cells that infiltrated the dermis. Despite aggressive chemotherapy, all of the patients died a few months after the skin lesions developed. Conclusions In our series, a perfect correlation was seen between the findings of serum electrophoresis and the immunohistochemistry of the skin lesions with regard to the type of immunoglobulin and the immunoglobulin light chain restriction produced by the neoplastic plasma cells. Patients with multiple myeloma have a very short survival period once specific skin lesions appear, regardless of the therapy administered. The deletion of the rb 1 gene may be a prognosis marker to identify those patients with especially aggressive forms of multiple myeloma.

Lymphocytic Mastitis and Diabetic Mastopathy: A Molecular, Immunophenotypic, and Clinicopathologic Evaluation of 11 Cases

Lymphocytic mastitis and diabetic mastopathy are uncommon fibroinflammatory breast diseases. The lesions seen in these entities are unique in that the associated lymphoid infiltrates are composed of predominantly B cells. In addition, B-cell lymphoepithelial lesions, a finding commonly associated with extranodal marginal zone B-cell/mucosa-associated lymphoid tissue (MALT) lymphomas, are also often present in lymphocytic mastitis and diabetic mastopathy. Although the clinical and immunomorphologic features are well characterized, the clonality of the B-cell infiltrate and the lymphomatous potential of lymphocytic mastitis and diabetic mastopathy have not been emphasized in the literature. We evaluated 11 cases of lymphocytic mastitis/diabetic mastopathy for immunoglobulin heavy chain gene rearrangement and correlated the findings with all available clinical data. A longstanding history of Type I diabetes mellitus was present in seven patients. One nondiabetic patient had Sjogren's syndrome, and two patients had no history of diabetes mellitus or other autoimmune disease. Clinical data were unavailable for one patient. B-cell–predominant lymphoid infiltrates were seen in all cases, and B-cell lymphoepithelial lesions were found in five. No evidence of a B-cell clone was found in any of the 11 cases by appropriately controlled immunoglobulin heavy chain gene rearrangement studies, and none of the patients developed lymphoma during follow-up intervals ranging from 2–126 months. These findings suggest that despite the presence of B-cell–predominant lymphoid infiltrates and lymphoepithelial lesions, lymphocytic mastitis and diabetic mastopathy do not appear to be associated with an increased risk for lymphoma.

Primary lymphoma of bilateral external auditory canals

We report a rare case of primary lymphoma originating in the bilateral external auditory canals. A 49-year-old man presented with cauliflower-like tumors on both of his external auditory canals. Immunohistopathologic examination and immunoglobulin heavy chain gene rearrangement studies led to a diagnosis of B-cell lymphoma. He had no other systemic involvement. After chemotherapy, total gross tumor removal by surgery was achieved, and no recurrence has been detected during 1 year of follow-up. To our knowledge, this is the third case of malignant lymphoma originating in the external auditory canal to be reported in the literature, and this is the first report that includes immunohistochemical findings of fresh tissue. (Am J Otolaryngol 2002;23:49-52. Copyright © 2002 by W.B. Saunders Company)

Differentiating Lymphoblastic Lymphoma and Ewing's Sarcoma: Lymphocyte Markers and Gene Rearrangement

We encountered a child with an intraosseous small round cell tumor that was negative for LCA, CD20 (L26), and CD3 and positive for vimentin, CD99 (MIC-2), and periodic acid-Schiff. The tumor exhibited rosette-like formations. This case was initially interpreted as Ewing's sarcoma (ES); however, additional studies revealed positivity for CD79a, CD43, and TdT expression, and an immunoglobulin heavy chain gene rearrangement (IgH-R) by polymerase chain reaction (PCR) established this to be a precursor B-lymphoblastic lymphoma. Because the differential diagnosis of ES and lymphoblastic lymphoma can be difficult and the differential diagnostic value of leukocyte antigens and immunoglobulin heavy chain gene rearrangement studies have not been fully evaluated, we conducted a more extensive investigation on 33 (21 soft tissue and 12 intraosseous) ES cases. Cases were retrieved from the files of the Department of Pathology at Georgetown University and from the Soft Tissue Registry of the Armed Forces Institute of Pathology. The cases were studied by light microscopy, immunohistochemistry, and PCR for IgH-R and T cell receptor gamma chain gene rearrangement (Tγ-R). There were 17 females and 16 males; the mean age was 29.3 years. Locations included the extremities (n = 17) and trunk (n = 16). All cases fit the ES spectrum by light microscopy and immunohistochemistry, as previously determined, and were negative for lymphoid markers (LCA, CD3, CD20, CD43, CD79a, and TdT), CD10 and CD34. CD99 was positive in 31/33 and bcl-2 was weakly positive in 13/33 cases. All 21 cases studied for gene rearrangements by PCR were negative for IgH-R and Tγ-R. Distinction of intraosseous lymphoblastic lymphoma from ES may be difficult because lymphomas may occasionally exhibit unexpected morphologic and immunophenotypic properties including LCA, CD3 and CD20 negativity and cytokeratin positivity. Additional analysis using CD79a, CD43, TdT, and PCR should be performed to avoid misdiagnosis. True ES is negative for lymphoid markers including CD79a, CD43, and TdT, as well as for IgH-R and Tγ-R.

Immunoglobulin Heavy-chain Gene Rearrangement Studies by Southern Blot Using DNA Extracted From Formalin-fixed, Paraffin-embedded Tissue

Immunoglobulin Heavy-chain Gene Rearrangement Studies by Southern Blot Using DNA Extracted From Formalin-fixed, Paraffin-embedded Tissue

A novel 8-bp insertion in codon 281 of p53 in a patient with acute lymphoblastic leukaemia and 2 separate leukaemic clones

We report a novel p53 insertion in a case of aggressive acute lymphoblastic leukaemia in a 16 year old male, in which 2 separate leukaemic clones were previously identified by T-cell receptor σ and immunoglobulin heavy chain gene rearrangement studies. Initial p53 mutation screening of blast cells from 29 patients with acute leukaemia by PCR-denaturing gradient gel electrophoresis showed that 2 had a silent codon 213 polymorphism and only the index case had a somatic mutation identified to be an 8 bp insertion in codon 281 (5′CCGGGGGG-3′). This insertion was associated with the second, more aggressive clone which underwent clonal evolution and became resistant to cytotoxic chemotherapy. With an allele-specific primer in PCR, we were able to demonstrate the presence of this clone as a minority at disease presentation, and in 2 of 3 collections of peripheral blood progenitor cells. © 1998 Wiley-Liss, Inc.

Thrombocytopenia following autologous bone marrow transplantation: Evidence for autoimmune aetiology and B cell clonal involvement

Thrombocytopenia outlasting anaemia and neutropenia is a well recognised sequel of autologous bone marrow transplantation (BMT) but the pathogenesis remains unclear. Autoimmune destruction of platelets has been suggested as a possible mechanism. We studied 5 patients who had undergone autologous BMT and were found to have persistent thrombocytopenia (< 150 x 10(9)/l) 6 months from transplantation with a normal haemoglobin level and granulocyte count. Apart from a mild reduction in the megakaryocyte numbers in one case, no other quantitative or qualitative defects of the megakaryocyte lineage were present to explain the peripheral thrombocytopenia. Two cases had positive anti-platelet autoantibodies. Immunoglobulin heavy chain gene rearrangement studies of peripheral blood and bone marrow mononuclear cells using the polymerase chain reaction showed evidence of clonal rearrangement in one of the two cases with positive anti-platelet autoantibodies. Our results support the previous reports that anti-platelet antibody-mediated destruction of platelets may play a role in the pathogenesis of post-autologous BMT thrombocytopenia. Furthermore, the demonstration of a clonal B cell expansion in one of the cases with anti-platelet antibodies suggests an aetiological link between clonal B cells, autoantibody production and thrombocytopenia.

Oligoclonal B‐cell leukemia characterized by spontaneous cell division and telomere association

Cytogenetic analysis of unstimulated cultures from a female patient with chronic B-cell leukemia (CLL) revealed three cytogenetically distinct clones, suggesting that the patient's leukemia was oligoclonal. Immunoglobulin heavy chain gene rearrangement studies revealed 1 germline and 4 rearranged bands, indicative of an oligoclonal leukemic population. Further evidence of oligoclonality was provided by X-linked RFLP studies. This is the first report of oligoclonality in CLL demonstrated by cytogenetic, immunoglobulin gene rearrangement, and X-chromosome inactivation studies. In addition to oligoclonality, the patient's leukemic cells exhibited telomere association, a Robertsonian translocation, and clonal evolution, suggesting an underlying genomic instability.

Complex karyotypic evolution in B-cell chronic lymphocytic leukemia

Cytogenetic analysis at diagnosis in a female patient with chronic B-cell leukemia showed a single abnormal clone with a 4p+ abnormality, 46,XX, −4, +der(4)t(4;?)(p16;?). Six additional clones evolved from this clone during the following 4 1 2 years and showed 3p+,4p−, and 11q− chromosomes in addition to the 4p+ abnormality. Immunoglobulin heavy chain gene rearrangement studies showed two rearranged bands and a faint germline band. Following splenectomy, a strong germline and faint rearranged bands were seen, suggesting that the majority of cells were normal, whereas cytogenetic studies showed that the karyotypically abnormal cells were still present. The combination of cytogenetic and Ig gene rearrangement studies provides detailed information regarding the number of circulating normal and leukemic cells.