Monoclonal Immunoglobulin Heavy Chain Research Articles

Immunohistochemical and gene rearrangement studies of central nervous system lymphomatoid granulomatosis

Lymphomatoid granulomatosis (LYG) is a rare multisystem disorder with characteristic angiocentric lymphoproliferative features, most frequently involving the lung, skin, and rarely the CNS. LYG has been classified into three subtypes based on the relative proportions of atypical and inflammatory infiltrating cells. Most systemic LYGs have been shown to be EBV-associated, T-cell rich, B-cell proliferative disorders. Here, we present four cases of LYG arising from the CNS and have analyzed them by immunohistochemistry to assess the phenotype of the infiltrate, and by PCR-SSCP (single-strand conformation polymorphism) analysis for immunoglobulin heavy chain (IgH) and T-cell receptor (TcR) gamma gene rearrangements. Three cases revealed perivascular infiltration of T-cell dominant lymphoid cells, two cases showed monoclonal TcRgamma gene rearrangement, while the remaining case had a B-cell immunophenotype and monoclonal IgH gene rearrangement with EBV genome expression. This is the first report of a gene rearrangement study on CNS-LYG. We confirm that some cases of CNS-LYG are derived from T-cell monoclonal lymphoproliferative disease, although this disease should be classified as a borderline malignancy and should be separated from overt malignant lymphoma of CNS.

Oral non-Hodgkin’s lymphoma: review of the literature and World Health Organization classification with reference to 40 cases

Forty cases of oral cavity non-Hodgkin's lymphoma (NHL) were evaluated for sex, age, location, clinical presentation, and World Health Organization (WHO) histological subtype. Fifty-three percent were female and the mean age was 71. The upper jaw (maxilla or palatal bone), mandible, palatal soft tissue, and vestibule and gingivae (maxillary or mandibular soft tissue involvement only) were, respectively, the most common locations. Swelling, ulceration, and radiographic destruction of bone were the most frequent signs. Most of the lymphomas were of B cell lineage (98%), and the majority of these B cell lymphomas (58%) were histologically subtyped as diffuse large B cell lymphoma, which is considered to have an aggressive clinical course. An immunohistochemical panel was used in the majority of cases to confirm the lineage and to help characterize the subtype. B and T cell specific markers were used to show lineage of the neoplastic cells. Additional markers were used to help confirm specific subtypes that characteristically show specific positivity to some of these antibodies. Molecular studies to detect monoclonal immunoglobulin heavy chain (IgH) gene rearrangements and Bcl-1 and Bcl-2 gene translocations were performed in cases in which the diagnosis was in question. The current WHO classification is also reviewed in detail.

Presence of Preleukemic Clones at Birth in the Majority of Children with B-Lineage Acute Lymphoblastic Leukemia.

The proof for the prenatal origin of childhood acute lymphoblastic leukemia (ALL) comes from the detection of concordant leukemia in monozygotic twins and the identification of translocation breakpoint genomic sequences at birth in a limited number of ALL patients with t(4;11) or t(12;21) chromosomal translocation. However, most patients with childhood ALL lack leukemia-specific fusion gene sequences. Therefore, we have used the rearranged immunoglobulin heavy chain (IgH) genes as a marker for the detection of preleukemic clones at birth. Guthrie card blood spots of 32 children with B-lineage ALL treated at our institution were available for this retrospective study. The ALL patients had a median age of 5 years (range, 15 months to 14 years) and had median presenting white blood cell (WBC) counts of 10150/μl (range, 800 to 103800/μl). In all patients a monoclonal IgH gene rearrangement was obtained from diagnostic bone marrow and sequenced. Clone-specific primers were designed using the specific D-N-J and N-D-N sequences. A two-stage polymerase chain reaction (PCR) using a semi-nested approach was developed to improve sensitivity and specificity of amplification. In all 32 patients, one leukemic cell could be detected in a background of 105 normal blood mononuclear cells. Nineteen of the 32 patients (59%) had detectable IgH gene rearrangements at birth using the sensitive semi-nested PCR. Sequencing of the PCR products obtained from Guthrie card blood spots revealed the identical sequences identified from diagnostic leukemic cells. The fetal characteristics of the leukemic cells were indicated by the small numbers of nucleotides inserted into the N region and the shortened D germ line segments. Interestingly, five of the six children (83%) with hyperdiploid ALL had detectable preleukemic clones at birth. Four of the five children (80%) with pro-B ALL, 13 of the 21 children (62%) with cALL and only two of the six children (33%) with pre-B ALL had preleukemic clones on their cards. We did not observe any differences in age at diagnosis or presenting WBC count between the 19 patients with preleukemic clones at birth and the 13 patients whose Guthrie cards were tested negative. Our results suggest that the majority of children with B-lineage ALL has preleukemic clones already at birth indicating a prenatal origin of leukemia. In addition, postnatal factors are important in leukemogenesis as well because of the long latency periods until clinical diagnosis of leukemia.

Mono/oligoclonal pattern of Kaposi Sarcoma‐associated herpesvirus (KSHV/HHV‐8) episomes in primary effusion lymphoma cells

Primary effusion lymphoma (PEL) is a rare lymphoma of B-cell origin, developed in serous cavities. PEL tumor cells are latently infected with Kaposi sarcoma-associated herpesvirus (KSHV) and in most cases co-infected with Epstein-Barr virus (EBV). In 15 primary PEL tumors including 10 EBV-positive cases, we analyzed the fused terminal repeat (TR) regions of KSHV episomes using pulsed-field gel electrophoresis and Southern blot. On the same genomic DNA samples, the cellular clonality was assessed by Southern blot and PCR detection of monoclonal immunoglobulin heavy chain (IGH) VDJ gene rearrangements, associated in the EBV-infected cases, with Southern blot analysis of the fused termini of EBV episomes. Monoclonal IGH gene rearrangements were detected in 13 tumors using Southern blot, in 11 cases using PCR, and in all cases considering both methods. EBV infection was monoclonal in all EBV-positive cases. However, only 5 PEL tumors were found to be monoclonally infected with KSHV. In the 10 other cases, we found a biclonal (2 bands; n = 4) or an oligoclonal pattern (3-6 bands; n = 6) of KSHV episomes. We hypothesized that the apparent discrepancy between viral and cellular clonalities in PEL might be due to several phenomena including complex mechanisms of genomic recircularization, insertion of duplicated sequences into the TR region and simultaneous infection of tumor cells with defective KSHV variants. KSHV infection of contaminating nontumoral cells, superinfection from lytically infected cells or viral integration events might also explain the oligoclonal pattern of KSHV infection. Several of these mechanisms, not mutually exclusive, might coexist in a single tumor.

Isolation and transplantation of highly purified autologous peripheral CD34+ progenitor cells: purging efficacy, hematopoietic reconstitution in non-Hodgkin's lymphoma (NHL): results of Japanese phase II study

The purging efficacy of positive selection of autologous CD34+ PBSC with a clinical scale method of magnetic-activated cell sorting system (CliniMACS) was investigated in 48 patients with non-Hodgkin's lymphoma (NHL). The median purity and recovery rate of the CD34+ cells post-selection were 93.3% (range 32.6-99.3) and 72.2% (range 20.5-309.8), respectively. The real-time PCR method to detect the patient-specific monoclonal immunoglobulin heavy chain gene rearrangement (minimal residual tumor; MRT) and CD19 and CD20 positivities were used for the detection of contaminating NHL cells before and after CD34+ selection. After selection, the median (range) depletion rate of MRT was 2.53 (1.52-4.78) log, and that of CD19+ cell and CD20+ cell was 2.46 (0.74-3.64) log and 2.32 (0.40-4.01) log, respectively. In 41 patients, high-dose chemotherapy was performed, followed by the transplantation of the isolated CD34+ cells. Rapid neutrophil recovery as well as platelet recovery was seen with a median time to reach 0.5 x 10(9)/l neutrophils of 10 days (range 8-13) and 20 x 10(9)/l platelets of 14 days (range 10-34), respectively. The present study demonstrated that CliniMACS is a highly effective positive selection method and a high purging efficacy could be obtained without compromising the hematopoietic reconstitution capacity of the graft in NHL patients undergoing high-dose chemotherapy.

Oligoclonal/Multiclonal Pattern of Kaposi Sarcoma-Associated Herpesvirus (KSHV/HHV-8) Episomes in Primary Effusion Lymphoma Cells.

Primary effusion lymphoma (PEL) is a rare non-Hodgkin lymphoma of B-cell lineage origin, developed in the serous body cavities and mostly observed in the course of HIV infection. PEL tumor cells are latently infected with Kaposi sarcoma-associated herpesvirus (KSHV) and in most cases, co-infected with Epstein-Barr virus (EBV). As a virological marker of clonality in PEL, we analyzed the fused terminal repeat regions (TR) of KSHV episomes using pulsed-field gel electrophoresis and Southern blot hybridization, in 15 primary PEL tumors including 10 EBV+ cases, and in 6 PEL cell lines. The cellular clonality was assessed on the same genomic DNA samples, by Southern blot and PCR detection of monoclonal immunoglobulin heavy chain (IgH) VDJ gene rearrangements, associated in the co-infected PEL with Southern blot analysis of the fused termini of EBV episomes. Monoclonal IgH gene rearrangements were detected in 87% (13/15) of primary PEL tumors using Southern blot, in 73% (11/15) using PCR analysis, and in all tumors considering both methods. All the co-infected PEL also displayed a monoclonal EBV infection. However, only 5 primary PEL tumors were found to be monoclonally infected with KSHV. In the 10 remaining cases, as well as in the KSHV+/EBV-negative BC-3 cell line, we found a biclonal (2 bands)(n=3) or an oligoclonal/multiclonal pattern (3–6 bands)(n=7) of KSHV episomes. KSHV infection of non-tumoral contaminating cells, superinfection mechanisms from lytically infected tumor cells, or viral integration events might explain such discordant findings between the viral and cellular clonality of PEL.

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

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.

Clonality analysis of B-cell lymphoproliferative disorders using PCR and melting curve analysis.

Detection of a monoclonal immunoglobulin heavy chain gene (IgH gene) rearrangement is commonly used to support the diagnosis of B-cell non-Hodgkin lymphoma. We investigated the application of melting curve analysis as a substitute for polyacrylamide gel electrophoresis (PAGE) in the detection of monoclonal IgH gene rearrangements after PCR. A total of 140 cases were selected for this study, including 63 B-cell malignancies with a previously documented monoclonal IgH gene rearrangement. These 140 specimens were tested using PCR with melting curve analysis, and the results obtained were compared with PAGE results to calculate the relative sensitivity and specificity of melting curve analysis. Melting curve analysis detected monoclonal rearrangements in 56 of 63 specimens (relative sensitivity 88.9%). No false positives were detected (relative specificity = 100%). False-negative results were obtained only when a weak monoclonal band was present on PAGE. These results show that a positive result on melting curve analysis is specific for a monoclonal IgH gene rearrangement. However, with a sensitivity of only 88.9%, the majority of negative results would require further evaluation of the amplicons using PAGE. The application of melting curve analysis in the detection of monoclonal IgH gene rearrangements in the clinical laboratory setting is discussed.

Contribution of IgH-PCR to the Evaluation of B-Cell Lymphoma Involvement in Paraffin-Embedded Bone Marrow Biopsy Specimens

We investigated whether the determination of clonality by polymerase chain reaction (PCR) analysis of immunoglobulin heavy chain (IgH) gene rearrangements could be helpful in the evaluation of B-cell lymphoma (BCL) involvement of bone marrow (BM) biopsy specimens. We evaluated 83 paraffin-embedded BM biopsy specimens from 26 patients with BCL. When BM biopsy specimens considered positive, “suspicious,” or negative by morphologic and immunohistochemical examination were evaluated by PCR, a monoclonal B-cell population was detected in 81% (39/48), 64% (9/14), and 11% (2/18), respectively. In most cases, a reproducible monoclonal IgH gene rearrangement was observed from BM and extramedullary sites. Nevertheless, in 4 cases, a different and independent monoclonal IgH rearrangement was observed during the disease course. PCR is efficient and complementary to morphologic and immunohistochemical examination for the evaluation of BCL involvement of BM biopsy specimens, especially when a reproducible rearrangement is found in 2 different samples.

A single primer pair immunoglobulin polymerase chain reaction assay as a useful tool in fine-needle aspiration biopsy differential diagnosis of lymphoid malignancies.

Different polymerase chain reaction (PCR) assays have proved to be useful tools for the detection of clonal immunoglobulin heavy chain (IgH) gene rearrangements in frozen or paraffin-embedded tissues. However, the usefulness of a single primer pair PCR assay on fine-needle biopsy aspirates has not been proven yet. The authors conducted a wide prospective study of 148 lymph nodes and extranodal lymphoid infiltrates obtained by fine-needle aspiration biopsy (FNAB). The power of a single primer pair PCR amplification of the hypervariable CDRIII region IgH genes was evaluated using a pair of consensus primers. The PCR cytologic results were compared with the final clinicopathologic diagnosis of each case assessed by combining cytologic and/or immunophenotypic data and histologic features or clinical follow-up. Among the 139 cases with an evaluable PCR result, 35 of 40 (87%) B-cell non-Hodgkin lymphomas were detected as a monoclonal band. Monoclonal IgH bands also were detected in two of the five (40%) T-cell lymphomas, two of the seven (29%) Hodgkin lymphomas, and 5 of the 87 (6%) reactive lymphoid disorders. These results are similar to those obtained by other authors using seminested PCR or combining different PCR tests in each sample obtained by FNAB or excisional biopsy. The results of the current study demonstrate the convenience of a single primer pair PCR amplification over seminested methods in terms of lower cost and workload. The existence of PCR false-negative and false-positive results for lymphoma makes it necessary to combine the information obtained by PCR with cytologic and/or immunophenotypic data to optimize the number of lymphoid malignancies correctly diagnosed by FNAB.

Specificity of polymerase chain reaction-based clonality analysis of immunoglobulin heavy chain gene rearrangement for the detection of bone marrow infiltrate in B-cell lymphoma-associated haemophagocytic syndrome.

As a wide range of disorders underlie haemophagocytic syndrome, a rapid distinction between benign polyclonal and malignant monoclonal lymphoid proliferations is critical. We investigated whether polymerase chain reaction (PCR) amplification of immunoglobulin heavy chain gene rearrangement could efficiently detect clonal B-cell populations in non-diagnostic marrow for B-cell lymphoma-associated haemophagocytic syndrome (B-LAHS). On amplifying two DNA samples per biopsy, no reproducible monoclonal PCR result was found in reactive haemophagocytic marrows. In contrast, four out of nine assessable B-LAHS patients with histomorphologically and immunohistochemically lymphoma-free bone marrow showed a reproducible monoclonal immunoglobulin heavy chain gene rearrangement. At the molecular level, two B-LAHS patients had lymphoma-free marrow as demonstrated by patient-specific PCR, suggesting that haemophagocytic marrow is not always associated with lymphoma involvement. PCR-based demonstration of clonal B-cell populations in marrow would add an extra dimension to B-LAHS diagnosis.

Rapid and Accurate Detection of Monoclonal Immunoglobulin Heavy Chain Gene Rearrangement by DNA Melting Curve Analysis in the LightCycler System

The detection of immunoglobulin heavy chain gene rearrangement (IgH-R) is a standard tool for distinguishing polyclonal from monoclonal B-cell populations. Current DNA-based polymerase chain reactions (PCR) strategies can diagnose monoclonal IgH-R either by measuring the length of the amplicon or by detecting gel mobility variations owing to sequence-dependent conformational changes. However, amplification and analysis remain sequential operations usually requiring manual transfer. We have developed a novel PCR strategy for detecting monoclonal IgH-R that monitors fluorescence of the specific double-stranded DNA binding dye SYBR Green I during melting curve analysis using the LightCycler System. We compared polyacrylamide gel electrophoresis (PAGE) versus melting curve analysis in 130 clinical DNA samples from formalin-fixed, paraffin-embedded (FFPE) tissues (mostly skin biopsies) of 128 patients. The identical FR3 primers were used to amplify the IgH variable region for both analytic techniques. We detected IgH-R in 24 DNA samples from FFPE tissue of 22 patients. Melting curve analysis, compared to PAGE, revealed no false negative and no false positive results, yielding both sensitivity and specificity equal to 100%. We also compared Southern blot analysis versus melting curve analysis in 23 clinical DNA samples from fresh-frozen lymph nodes of 23 patients. We detected IgH-R by melting curve analysis in 7 DNA samples from fresh-frozen lymph nodes. Melting curve analysis, compared to Southern blot analysis, revealed sensitivity equal to 58.3% (7 of 12) and specificity equal to 100% (11 of 11). We conclude that continuous fluorescence monitoring of PCR products with DNA melting curve analysis can rapidly and reproducibly distinguish polyclonal from monoclonal B-cell populations.

Primary follicular lymphoma of the testis in childhood: an entity with peculiar clinical and molecular characteristics

Background/Aims: Paediatric primary follicular lymphoma of the testis (PPFLT) is exceptional: the few reported cases seem to lack BCL-2 gene rearrangement and/or protein expression. The aim of this study was...

T-cell/histiocyte-rich large B-cell lymphoma is a disseminated aggressive neoplasm: differential diagnosis from Hodgkin's lymphoma.

An accurate diagnosis of T-cell/histiocyte-rich large B-cell lymphoma needs to take into consideration those forms of Hodgkin's lymphoma also characterized by a predominance of small lymphocytes and histiocytes, i.e. nodular lymphocyte predominance Hodgkin's lymphoma and lymphocyte-rich classical Hodgkin's lymphoma. We have studied the clinical, phenotypic and genetic features of a series of 12 cases of T-cell/histiocyte-rich large B-cell lymphoma along with 18 cases of Hodgkin's lymphoma for comparative purposes. Of the Hodgkin's lymphoma cases, there were 11 lymphocyte predominance type and seven classic type. T-cell/histiocyte-rich large B-cell lymphomas presented usually in advanced stages (III or IV in 11/12 cases), frequently with 'B' symptoms (6/9 cases), and followed a more aggressive course than Hodgkin's lymphoma (4/8 patients died due to the tumour in T-cell/histiocyte-rich large B-cell lymphoma versus 0/15 in Hodgkin's lymphoma). T-cell/histiocyte-rich large B-cell lymphoma cases showed diffuse effacement of the nodal architecture by a proliferation of scattered large atypical B-cells obscured by a background of small T-lymphocytes (more CD8+, TIA1+ than CD57+). Five cases showed also a prominent histiocytic component. The large B-cells expressed CD45 and often EMA (6/10 cases). On the other hand, CD 30, CD15 and latent infection by Epstein-Barr virus (EBV) were generally lacking. bc l6 and CD10 were, respectively, detected in 6/6 and 1/5 cases. Conventional polymerase chain reaction (PCR) showed monoclonal immunoglobulin heavy chain (IgH) gene rearrangements in all T-cell/histiocyte-rich large B-cell lymphomas studied (5/5), but did not detect any case with t(14;18) involving the major breakpoint region (0/4). The differential diagnosis of T-cell/histiocyte-rich large B-cell lymphoma from Hodgkin's lymphoma is facilitated by the integration of different immunophenotypic, molecular and clinical findings. T-cell/histiocyte-rich large B-cell lymphoma is a monoclonal neoplasm of bc l6+ B-cells with a phenotypic profile similar to lymphocyte predominance Hodgkin's lymphoma, suggesting a germinal centre origin and a possible relation to this disease. Therefore, in order to distinguish it from lymphocyte predominance Hodgkin's lymphoma, characterization of the reactive background, IgH gene rearrangement studies by conventional PCR and clinical features are more useful. In contrast, T-cell/histiocyte-rich large B-cell lymphoma can be distinguished from classical Hodgkin's lymphoma thanks to the presence of monoclonal IgH rearrangement and the CD 30-CD15-CD45+EMA+ immunophenotypic profile of the neoplastic cells in T-cell/histiocyte-rich large B-cell lymphoma.

Heavy chain disease.

The heavy chain diseases (HCDs) are rare B-cell malignancies that are distinguished by the production of a monoclonal immunoglobulin heavy chain (HC) without an associated light chain by the malignant B-cells. There are three types of HCD defined by the class of immunoglobulin (Ig) HC produced: IgA (alpha-HCD), IgG (gamma-HCD), and IgM (mu-HCD). Alpha-HCD is the most common and occurs most commonly as intestinal malabsorption in a young adult from a country bordering the Mediterranean Sea. Treatment consists of antibiotics and improved nutrition and hygiene. Surgery is occasionally required for patients with bulky masses at risk for bowel perforation. If there is no response to antibiotics or if aggressive non-Hodgkin's lymphoma (NHL) is diagnosed, the patient should be treated with chemotherapy. Gamma- and mu-HCD are rare and essentially are found in patients with a B-cell NHL that produces an abnormal Ig heavy chain. These patients occasionally may be diagnosed with a monoclonal gammopathy of undetermined significance (MGUS). Patients with MGUS with NHL should be administered chemotherapy. Screening the serum and urine of patients with lymphoplasmacytoid NHL would likely identify more patients with gamma- or mu-HCD.

In situ localization of follicular lymphoma: description and analysis by laser capture microdissection

AbstractFrom 1992 to 2000, we identified 23 lymph node biopsies with focal germinal centers (GCs) containing centrocytes staining strongly for bcl-2 protein, whereas most of the remaining lymph node showed bcl-2–negative follicular hyperplasia. We propose the designation in situ localization of follicular lymphoma (FL) for this phenomenon. In 2 additional cases, bcl-2+ follicles with features of in situ FL were identified in association with other low-grade B-cell lymphomas. To investigate the clonality of the bcl-2+follicles, we performed laser capture microdissection of bcl-2+ and bcl-2 follicles from the same lymph node in 5 cases, and analyzed them in parallel by polymerase chain reaction (PCR) amplification of immunoglobulin heavy chain (IgH) genes. In 4 of 5 cases the bcl-2+ follicles contained monoclonal IgH gene rearrangements, whereas the bcl-2− GCs exhibited a polyclonal ladder. A BCL2/JH gene rearrangement was detected in 6 of 14 (43%) evaluable cases. There were 5 patients with synchronous evidence of FL at another site. There were 13 patients who, without a prior diagnosis of FL, had clinical follow-up; one developed FL in an adjacent lymph node within one year, and 2 manifested FL at 13 and 72 months, respectively. There are 10 patients who have not yet shown other evidence of FL. These results suggest that at least close to half of these cases (8/18; 44%) represent homing to and early colonization of reactive GCs by FL. Other cases might represent FL at the earliest stage of development, or a preneoplastic event, requiring a second hit for neoplastic transformation. These findings provide insight into the pathophysiology of early FL, and illustrate the utility of immunohistochemistry for early diagnosis.

Immunoglobulin Heavy Chain Gene Analysis in Lymphomas: A Multi-Center Study Demonstrating the Heterogeneity of Performance of Polymerase Chain Reaction Assays

Determination of monoclonality through an evaluation of immunoglobulin heavy chain (IgH) gene rearrangements is a commonly performed and useful diagnostic assay. Many laboratories that perform this assay do so by the polymerase chain reaction (PCR). To evaluate current methods for performing IgH gene testing, 19 different Association of Molecular Pathology (AMP) member laboratories analyzed 29 blinded B cell and T cell lymphoid neoplasm samples of extracted DNA and formalin-fixed, paraffin-embedded (FFPE) tissue and were asked to complete a technical questionnaire. From this study, it is clear that Southern blot analysis remains the diagnostic gold standard, with a 100% diagnostic sensitivity and specificity. There was, however, remarkable heterogeneity in the performance of, and results obtained from, IgH PCR assays with diagnostic sensitivity ranging from over 90% to as low as 20%, when evaluating the same specimens. Many laboratories overestimate the diagnostic sensitivity of their IgH PCR assay, and there was a significant, and under appreciated, drop-off (from 61.3% to 41.8%) in detection in paired FFPE as compared with fresh/frozen tissues. Fixation has a dramatic impact on the inability to perform the test on FFPE (43.1%) versus DNA already extracted from fresh or frozen tissue (2.8%). A number of variables that affected the outcome of IgH PCR were identified. Strategies that improved the detection of monoclonal IgH rearrangements include: the addition of FRII to the FRIII upstream primer (increasing detection from 57.3% to 73.6%) and the use of the FR3A rather than the FR3 FRIII primer (increasing detection from 54.7% to 69.7%). Although numerous variables (from DNA extraction to PCR product detection) were evaluated, making it difficult to mandate alterations in laboratory practice, these findings ought to prompt diagnostic molecular pathology laboratories to reevaluate their claims of sensitivity, as well as their methodologies. Both pathologists and surgeons need to ensure that not all submitted material is fixed, if there is adequate sample. Importantly, there is a need for greater standardization to reduce the unacceptably high false negative rate of this crucial diagnostic assay.

Primary Lung Small B-Cell Lymphoma versus Lymphoid Hyperplasia: Evaluation of Diagnostic Criteria in 26 Cases.

Primary lung non-Hodgkin's lymphoma is a rare neoplasm mostly represented by low-grade B-cell lymphomas of mucosa-associated lymphoid tissue. Their diagnostic criteria are now well defined on surgical specimens, but pathologists may experience difficulties in distinguishing them on exiguous biopsies from benign lymphoid hyperplasia and other lymphomas. Therefore, we examined a series of 26 lung lymphoid lesions to further define the pathologic features of either lymphoma or lymphoid hyperplasia on small specimens. We observed 16 primary lung non-Hodgkin's lymphomas with a large predominance of low-grade mucosa-associated lymphoid tissue-type lymphomas (87.5%, n = 14). There were no autoimmune disorders, but three patients had a concomitant infectious disease (hepatitis C virus and Helicobacter pylori gastritis). One patient presented with a synchronous pulmonary adenocarcinoma. As well as the classical mucosa-associated lymphoid tissue cellular infiltrate, immunohistochemical characterization of the 14 mucosa-associated lymphoid tissue-type lymphomas revealed the CD20+/CD43+ centrocyte-like cell phenotype in 10 cases (71.5%). Although the lymphoepithelial lesions observed in all lymphomatous cases have been reported in lung lymphoid hyperplasia, the determination of B-cell CD20+/CD43+ phenotype of the intraepithelial lymphocytes highly increased the specificity of lymphoepithelial lesions. A monoclonal immunoglobulin heavy chain gene rearrangement was present in 71.4% of the mucosa-associated lymphoid tissue-type lymphoma specimens. Investigation of H. pylori by polymerase chain reaction detection was negative, even for the two cases associated with H. pylori gastritis.

Acute lymphoblastic leukaemia: correlation between morphological/immunohistochemical and molecular biological findings in bone marrow biopsy specimens

Background—Although numerous antibodies suitable for use on paraffin wax embedded sections are available for the subtyping of acute leukaemia (acute myelogenous leukaemia (AML) and acute lymphoblastic leukaemia (ALL)) in bone...

下腿に発生したｄｉｆｆｕｓｅ ｌａｒｇｅ Ｂ‐ｃｅｌｌ ｌｙｍｐｈｏｍａの１例

We report a case of primary cutaneous large B-cell lymphoma in a 63-year-old man who exhibited two firm nodules on his right lower leg for about one year.Histopathological examination revealed a dense infiltrate of atypical, large lymphocytes with mitotic figures from the entire dermis to the subcutaneous tissue. Bottom-heavy pattern was appparent. By immunohistochemical studies, atypical lymphocytes were found to be positive for CD20 and CD79a. A monoclonal immunoglobulin heavy chain gene rearrangement by Southern blot analysis was revealed in the involved tissue. On physical examination, no superficial lymphadenopathy was observed. CT scans and Ga scintigraphy showed no evidence of malignancy. According to the REAL classification, the present case corresponded to the entity, “diffuse large B-cell lymphoma” and EORTC classification classified it as large B-cell lymphoma of the leg. Local excision and local electron beam irradiation (total 40Gy) were administered. The patient has been free of disease for 1-year of follow up. [Skin Cancer (Japan) 2000; 266-270]