IgH基因单克隆重排检测及其在B细胞性非霍奇金淋巴瘤诊断中的应用

 目的：建立准确可靠、操作性强、适用于临床实际工作的免疫球蛋白重链（immunoglobulin heavy chain,IgH）基因单克隆重排检测方法,用于B细胞性非霍奇金淋巴瘤（B-cell non-Hodgkin lymphoma,B-NHL）的辅助诊断。方法：采用骨架区(framework region,FR）引物FR2、FR3和重链连接区 （joining region of heavy chain,J_H）引物LJH、VLJH组合、A管+B管模式、半巢式聚合酶链式反应(polymerase chain reaction,PCR)法对121例B-NHL、58例T细胞性非霍奇金淋巴瘤（T-cell non-Hodgkin lymphoma,T-NHL）和19例淋巴结反应性增生的石蜡组织进行IgH基因单克隆重排检测,分析IgH基因单克隆重排检出率在B-NHL组、T-NHL组和淋巴结反应性增生组中的差异,以及B-NHL中联合应用FR2和FR3与单独应用FR2、FR3之间IgH基因单克隆重排检出率的差异。结果：118例成功检测的B-NHL中,IgH基因单克隆重排检出率为81%（96/118）;54例成功检测的T-NHL中,IgH基因单克隆重排检出率为4%（2/54）;19例成功检测的淋巴结反应性增生中未检出IgH基因单克隆重排。B-NHL组与T-NHL组、淋巴结反应性增生组相比,IgH基因单克隆重排检出率差异具有显著性（P<0.05）。B-NHL中, FR2基因单克隆重排检出率为58%（68/118）,FR3基因单克隆重排检出率为55%（65/118）,联合应用FR2和FR3,IgH基因单克隆重排检出率为81%（96/118）,联合应用FR2和FR3与单独应用FR2、FR3的检出率有显著差异（P<0.05）。结论：采用FR2、FR3、LJH及VLJH引物组合、A管+B管模式和半巢式PCR法进行石蜡组织IgH基因单克隆重排检测,简单易行,结果准确可靠,阳性率较高,可用于临床B-NHL的辅助诊断。     

Isolated pleural PTLD after cardiac transplantation

Preamble: Epstein–Barr virus infection (EBV) and immunosuppression promote emergence of posttransplant lymphoproliferative disorders (PTLD) in patients undergoing organ transplantation. Objective: We report a case of PTLD confined to the pleura. Findings: The patient was a 62-year-old male who had undergone cardiac transplant in 1993 for ischemic heart disease. Seven years later, he presented with dyspnea and bilateral pleural effusions. The CT scan revealed left sided pleural base thickening. The cytology of the pleural fluid and fine needle aspirate of the pleura was both suggestive of PTLD. However, the tissue submitted for ancillary studies did not contain the diagnostic material. A clinical decision was made to withdraw immunosuppressive therapy and start rituximab. His clinical course was complicated by Pneumocystis carinii pneumonia and he died 4 months after the diagnosis of PTLD. Autopsy revealed bilateral pleural effusions with pleural nodules involving the visceral and parietal pleura of both lungs. Immunohistochemistry demonstrated B cell lineage with κ/λ ratio of 1. PCR studies done on the pleural nodules (postmortem specimen) revealed the presence of EBV DNA and absence of human herpes virus 8 (HHV8) DNA. In situ hybridization revealed positive staining for EBV RNA within the neoplasm. Conclusion: Pleural-based PTLD is rare. Cytology in conjunction with immunophenotyping and molecular studies can be useful for a definitive diagnosis. In our case, cytology sample was suggestive of PTLD. PCR studies performed on the antemortem specimen confirmed the presence of monoclonal IgH gene rearrangement, while the postmortem specimen revealed oligoclonal IgH gene rearrangement. The change from monoclonal to oligoclonal IgH gene rearrangement suggests reversion of monoclonal to polyclonal PTLD following rituximab and CHOP therapy. We also demonstrated EBV DNA and RNA in the tumor nodules, supporting EBV-induced PTLD.     

Molecular insight into the diagnosis of lymphoma

Malignant lymphoma may be very difficult to diagnose with routine histopathological methods because they may recapitulate benign architecture or contain benign infiltrates. The best method of diagnosis is to establish the clonal profile of the lymphocyte population, since a monoclonal proliferation is highly suggestive of neoplasia. By means of a PCR (polymerase chain reaction) method it is possible to detect the immunoglobulin heavy chain (IgH) gene rearrangement and therefore establish the lymphocyte clonality. PCR with primers complementary to relatively conserved regions called frameworks (FR1-FR3) laying among hyper variable regions (CDR1-CDR3) of IgH gene unable us to detect monoclonal versus polyclonal B-cell population. The length of the PCR product with these primers is unique if we deal with a monoclonal population. On the contrary, a polyclonal population gives PCR products of a different size. In this retrospective study we used a semi-nested PCR to analyse 37 paraffin-embedded specimens. All of them had been evaluated previously by pathohistological and immunophenotypic criteria. A number of polyclonal (PBL and tonsils from healthy donors) and monoclonal cells (PBL from CLL patients, Raji cell line) were analyzed as controls. Clonality was successfully determined in all specimens. Our results support the concept that molecular techniques such as PCR provide a helpful approach for detection of monoclonal immunoglobulin rearrangements in malignant lymphoma. This is especially true for border cases, but always in the combination with other pathohistological methods.     

Consensus primers for detecting monoclonal immunoglobulin heavy chain rearrangement in B cell lymphomas

AIMS: To demonstrate the usefulness of polymerase chain reaction (PCR) methodology with both the FR2A/LJH/VLJH and the FR1c/LJH/VLJH primer sets for detecting monoclonal immunoglobulin heavy chain (IgH) gene rearrangement in B cell non-Hodgkin lymphomas (B-NHLs). METHODS: Eighty three patients with B-NHL were enrolled in this study. DNA was isolated from paraffin wax embedded sections and amplified by PCR to determine the sequences of the rearranged IgH gene. Each PCR product was subcloned. Cycle sequences and sequence analyses were done to determine the clone specific IgH variable region (VH) sequences. RESULTS: Clonal IgH gene rearrangements were detected in 45 cases with FR2a/JH/VLJH and in 14 of the remaining cases with FR1c/JH/VLJH. Most of the cases detectable with FR2a/JH/VLJH were derived from VH3 and VH4 families. Five of six cases in the VH1 family and one in the VH7 family were amplified with the FR1c/JH/VLJH primer set only. CONCLUSION: The detection rate of IgH rearrangement in B-NHLs can be increased by using both FR2A/LJH/VLJH and FR1c/LJH/VLJH, and these two primer sets are suitable for routine PCR methodology. Moreover, each primer set appears to be closely related to VH family specificity.     

Improvements to B cell clonality analysis using PCR amplification of immunoglobulin light chain genes.

AIMS: Clonality analysis using polymerase chain reaction (PCR) amplification of the immunoglobulin heavy chain (IgH) gene is an important aid to the diagnosis of B cell lymphoproliferative diseases. However, the method has a relatively high false negative rate. In an attempt to improve detection rates simple PCR strategies for clonality analysis of B cell populations using amplification of Ig light chain genes have been developed. METHODS: Novel PCR protocols, designed to amplify Ig kappa and Ig lambda light chain genes, were evaluated using high molecular weight DNA samples from 28 selected cases of B cell lymphoma with known light chain expression and 12 reactive lymphoid specimens. Products were run on 10% polyacrylamide minigels using heteroduplex analysis. Conventional IgH PCR analysis was also performed. Twelve randomly selected formalin fixed, paraffin wax processed samples from cases submitted for molecular genetic analysis were also studied. RESULTS: Polyclonal products were seen in all reactive lymphoid samples. Using Ig kappa PCR, 24 of 28 lymphomas, including four of five IgH negative cases, displayed monoclonal patterns. Using Ig lambda PCR, eight of 12 Ig lambda expressing tumours, including two of five IgH negative cases, showed monoclonal patterns. Standard IgH PCR demonstrated monoclonality in 23 of 28 B cell lymphomas. The detection rate was improved to 27 of 28 lymphomas using heavy and light chain PCR. Efficient amplification was achieved using paraffin wax processed samples, seven of which showed monoclonality compared with eight using IgH PCR. CONCLUSIONS: Ig light chain PCR, used in conjunction with heavy chain analysis, enables improved detection of B cell monoclonality using routine histological specimens and can provide additional clone specific markers for the study of the biology of B cell tumours.     

Clonal analysis of a solitary follicular nodule of the thyroid with the polymerase chain reaction method.

Solitary follicular nodules of the thyroid occasionally create a diagnostic problem, especially in the differential diagnosis between adenoma and nodular hyperplasia To obtain confident histologic parameters of clonal lesions, we analyzed DNA samples prepared from paraffin-embedded archival tissue from 20 solitary follicular nodules of the thyroid for clonality with the polymerase chain reaction (PCR) method. On the base of X chromosome inactivation mosaicism, we tested restriction fragment-length polymorphism of the phosphoglycerate kinase (PGK) gene and a highly polymorphic short tandem repeat of the human androgen receptor (HUMARA) gene. Of 18 informative cases, 10 were monoclonal, 7 were polyclonal, and 1 showed microsatellite instability. All of the five completely encapsulated nodules were monoclonal. Four of the five unencapsulated nodules showed polyclonality. Of the seven partially encapsulated nodules, four were monoclonal, and the others were polyclonal. The former showed 50% or more of encapsulation degree, whereas the latter showed less than 50%. The capsule tended to be thicker in monoclonal nodules (mean, 0.33 mm) than in polyclonal nodules (mean, 0.13 mm). Other histologic features of the nodules and surrounding parenchymal changes had no significance with respect to predicting clonality. This study suggests that the degree of encapsulation and capsular thickness are morphologically important for predicting the clonality of the thyroid nodule.     

移植后淋巴组织增生性疾病的临床病理分析

目的探讨移植后淋巴组织增生性疾病（PTLD）的临床及病理特征,提高其诊断和治疗水平。方法对4例移植后淋巴组织增生性疾病行HE和免疫组织化学EnVision法染色、原位杂交及聚合酶链反应,复习其临床资料并随访。结果4例中3例是肾移植后,其中2例为多形性PTLD,1例为单形性PTLD;另1例是异体骨髓移植后PTLD的“早期”病变。2例EB病毒阳性。4例移植后所用免疫抑制剂均以环孢A类药为主,辅以激素。例1～4从移植到诊断PTLD的时间为42、7、129、2个月。例3多形性PTLD的临床分期为Ⅱ期（诊断PTLD后2个月死亡）;其余均为Ⅰ期。均存活,诊断PTLD后生存期为40（例1）、26（例2）、5（例4）个月。结论PTLD是发生在器官移植后,具有独特的形态和临床特征的淋巴组织增生性疾病,部分病例与EB病毒感染有关。其预后与临床分期相关,免疫抑制剂减量可能有效。     

肝移植术后淋巴组织增生性疾病的临床病理分析

目的探讨肝移植术后淋巴组织增生性疾病（PTLD）的临床病理特征。方法对3例肝移植术后PTLD行HE、免疫组织化学染色及TCR、IgH基因重排检测,同时复习其临床资料并随访。结果3例中,2例位于肝门部,1例位于肝动脉旁淋巴结。临床表现为发热和梗阻性黄疸。病理诊断例1为单形性T细胞PTLD的周围T细胞淋巴瘤,例2、例3为单形性B细胞PTLD的淋巴浆细胞样淋巴瘤和弥漫大B细胞淋巴瘤。基因重排检测例1 TCR阳性,例2、例3 IgH阳性。3例EBV免疫组化染色均阳性。行再次移植降低免疫抑制剂用量并配合抗病毒、抗CD20单抗和化疗等治疗,例1、例3分别于术后6个月和4个月死于淋巴瘤复发和肺感染,例2随访11个月无瘤生存。结论PTLD是肝移植术后严重的并发症之一,具有独特的形态和临床特征。病因可能与EB病毒感染和免疫抑制有关,需经病理组织学检查确诊。     

滤泡型淋巴瘤中t(14;18)染色体易位的分析及其临床意义

目的探讨滤泡型淋巴瘤（FL）的分子遗传学特征及其在病理诊断中的意义。方法收集55例FL石蜡标本,对照组小B细胞淋巴瘤28例和反应性滤泡增生（RFH）10例,应用套式PCR技术检测FL中,免疫球蛋白重链基因（IgH）的克隆性重排;应用标准PCR技术检测55例FL中t（14;18）易位,以10例RFH做对照;采用双色荧光原位杂交（FISH）技术检测20例淋巴结FL中t（14;18）易位,以4例RFH作为对照;并与PCR检测结果进行比较。结果（1）55例FL中,结内49例,结外6例。男性33例,女性22例,男女比为1．5：1。发病年龄36—79岁（中位年龄57岁）;FL分级：FL1—3分别为25例、19例和11例。（2）55例中50例（90％）检出β-肌动蛋白（actin）,该50例中FR3A阳性24例（48％）,FR2阳性25例（50％）,其中15例（30％）呈FR3A和FR2双阳性,共34例（68％）IgH基因重排。对照组小B细胞淋巴瘤28例中,25例检出β—actin,其中FR3A阳性18例（64％）,FR2阳性17例（61％）,共24例（86％）可检测出克隆性IgH基因重排。4例RFH均未检出IgH基因重排。（3）在44例结内FL中检出15例（34％）t（14;18）易位,其中14例在MBR,1例在mcr。（4）20例中,有16例（80％）可检出t（14;18）易位。结论（1）IgH克隆性重排在FL中的检测率比其他小B淋巴细胞低。（2）FISH检测石蜡包埋组织中t（14;18）易位有助于FL的诊断。FISH比PCR的敏感性更好,操作简便,可用于检测石蜡包埋组织中的分子遗传学改变。     

B淋巴细胞增生性疑难病例中IgH基因克隆性重排的分析

目的 探讨IgH基因克隆性重排对B淋巴细胞增生性疑难病变的辅助诊断价值.方法 检测77例B淋巴细胞增生性疑难病例中IgH基因的克隆性重排情况,均采用BIOMED-2系统IgH克隆性试剂盒中FR1、FR2、FR3三组家族引物进行PCR及聚丙烯酰胺凝胶电泳,硝酸银染色后观察,并对照最终病理诊断进行分析.结果 77例病变的最终病理诊断:B淋巴细胞反应性增生12例,不能排除B淋巴细胞不典型增生或淋巴瘤20例,B细胞性淋巴瘤45例.三组中FR1、FR2和FR3至少有一个为阳性的比值分别为2/12、11/20(55%)和36/45(80%).B细胞性淋巴瘤中,FR1、FR2和FR3的阳性率分别为60%(27/45)、60%(27/45)、56%(25/45),其类型有边缘区B细胞性淋巴瘤20例(其中黏膜相关淋巴组织型结外边缘区淋巴瘤18例,结内边缘区淋巴瘤2例),弥漫性大B细胞淋巴瘤7例,滤泡性淋巴瘤7例,套细胞性淋巴瘤1例,Burkitt淋巴瘤1例,浆细胞瘤4例,不能分型5例.FR1、FR2和FR3三者检测均为阴性但仍诊断为淋巴瘤9例(20%),其中1例后来出现肝脏B细胞淋巴瘤.对IgH基因重排阳性的B淋巴细胞反应性增生和不典型增生14例的随访结果,4例重新取活检后诊断为B细胞性淋巴瘤,其中3例IgH基因重排检测为阳性.结论 联合检测IgH基因FR1、FR2和FR3克隆性重排对B淋巴细胞增生性疑难病变诊断有重要的辅助价值;对形态改变和免疫表型诊断淋巴瘤依据不足而基因重排阳性者,重取活检或随访有一定价值;对阴性病例有必要补充IgH基因重排及IgK和IgL基因重排的检测以提高检测敏感性.     

The pathology of posttransplant lymphoproliferative disorders occurring in the setting of cyclosporine A-prednisone immunosuppression.

Posttransplant lymphoproliferative disorders (PTLDs) were diagnosed in 43 patients from the Pittsburgh-Denver series between June 1980 and March 1987. This constitutes a detection rate of 1.7%. Major categories of clinical presentation included a mononucleosislike syndrome, gastrointestinal/abdominal disease, and solid organ disease. The median time of onset in patients initially immunosuppressed with cyclosporine-A (CsA)-containing regimens was 4.4 months after transplant, regardless of tumor clonality. A strong association of PTLD with Epstein-Barr virus (EBV) was observed. A histologic spectrum of lesions from polymorphic to monomorphic was observed. Whereas polymorphic lesions could be either clonal or nonclonal, monomorphic lesions appeared to be clonal in composition. The presence of large atypical cells (atypical immunoblasts) or necrosis did not appreciably worsen the prognosis. Twelve patients had clonal, 13 had nonclonal, and five had both clonal and nonclonal tumors. Clonality was indeterminate in 13 cases. Most patients were treated with a regimen based on reduced immunosuppression and supportive surgery. Almost all nonclonal and about half of the clonal lesions respond to this conservative therapy, indicating that it is an appropriate first line of treatment. This behavior suggests that a spectrum of lesions ranging from infectious mononucleosis to malignant lymphoma constitutes the entity known as PTLD. Some monoclonal tumors can undergo regression, however, apparently in response to host immune control mechanisms. Because of its short latency and strong association with EBV, PTLD is an important model for the study of virus-associated tumor progression in humans.     

Increased sensitivity of B-cell clonality analysis in formalin-fixed and paraffin-embedded B-cell lymphoma samples using an enzyme blend with both 5′→3′ DNA polymerase and 3′→5′ exonuclease activity

Polymerase chain reaction (PCR)-based detection of immunoglobulin heavy chain (IgH) gene rearrangement for determination of B-cell clonality needs to be simple but optimally sensitive. Efficient IgH PCR analysis can be hampered by sequence variability in the template DNA, despite of the use of degenerative primers. To improve sensitivity of the B-cell clonality analysis in formalin-fixed and paraffin-embedded (FFPE) tissues, we have performed framework three-area (FR3)/joining gene (JH) IgH PCR utilizing an enzyme blend (rTth DNA Polymerase, XL) providing both 53 polymerase and 35 exonuclease activities. The DNA samples were extracted from FFPE biopsies of 43 mature B-cell lymphoma cases of so-called germinal center and post-germinal center origin, including 6 nodal follicular lymphomas (FL), 15 gastric mucosa-associated lymphoid tissue (MALT) lymphomas, and 22 gastric diffuse large B-cell lymphomas (DLBCL). Of the cases, 31 (17 DLBCL and 14 MALT lymphoma) represented small endoscopic biopsies. Serial dilutions of target DNA were applied to avoid inconsistent bands that may be seen when the input amount of template is too low, which can be the case when DNA is extracted from FFPE endoscopic gastric biopsies. Using conventional Taq polymerase, consistent monoclonal product was found in 53% (23/43) of the cases (FL: 67%; MALT lymphoma: 47%; DLBCL: 55%). The rTth polymerase showed reproducible monoclonal pattern in 72% (31/43) of the cases (FL: 67%; MALT lymphoma: 73%; DLBCL: 73%); the sensitivity is compatible with one that can be detected with conventional FR3/JH PCR in fresh/frozen tissues. In conclusion, the rTth DNA polymerase greatly improves sensitivity of FR3/JH PCR in FFPE biopsies of mature B-cell lymphomas, most probably by increasing the primer matches during PCR amplification.     

Posttransplantation lymphoproliferative disorders in pediatric patients undergoing liver transplantation

OBJECTIVES: To study the clinicopathologic and molecular genetic findings in posttransplantation lymphoproliferative disorders (PTLDs) following pediatric liver transplantation and to determine the applicability of a recently proposed consensus classification system. DESIGN: The clinical, pathologic, and molecular genetic findings of 11 PTLDs that occurred in 10 patients are presented. These 10 patients were derived from a group of 121 pediatric patients who underwent liver transplantation at the University of California, San Francisco. The PTLDs were classified using the proposed Society for Hematopathology scheme. Clonality was determined by immunohistochemical detection of monotypic immunoglobulin or by using polymerase chain reaction-based methods to detect monoclonal immunoglobulin heavy-chain gene rearrangements. Epstein-Barr virus (EBV) was detected by immunohistochemistry, in situ hybridization, or polymerase chain reaction. Epstein-Barr virus typing and the presence of LMP1 gene deletions were also analyzed by polymerase chain reaction. RESULTS: There were 3 early lesions, 4 polymorphic PTLDs, and 4 monomorphic PTLDs. Monoclonality was demonstrated in 8 of 9 cases assessed. Epstein-Barr virus was present in all cases; of 9 cases assessed by polymerase chain reaction, the virus was type A in 8 and type B in 1. No EBV LMP1 gene deletions were identified. The corresponding liver explants were negative for EBV in 8 cases and positive in 1 case. Greater than 3 foci of disease and monomorphic PTLD were associated with decreased actuarial survival (P <.05). CONCLUSIONS: The prognosis of pediatric patients with PTLD is favorable for early lesions and polymorphous PTLD, particularly in patients with localized disease. Multifocal disease and monomorphic PTLD are associated with an unfavorable prognosis.     

原发性睾丸恶性淋巴瘤克隆性IgH基因重排的检测

目的：探讨原发性睾丸淋巴瘤克隆性IgH基因重排的检测方法。方法：用半巢式PCR方法和聚丙烯凝胶（PAGE）－银染法检测14例睾丸恶性淋巴瘤的IgH基因重排。结果：与免疫组化对照，PCR检测的睾丸原发性B细胞淋巴瘤FR3A检出测率为85.71%，FR2的检出率为64.29%，而二者的综合检出率为100％，互补性较强。结论：半巢式PCR方法及PAGE－银染法对于原发性睾丸细胞淋巴瘤IgH基因的检出率有较高的灵敏度，FR3A和FR2引物的共同应用可以有效提高综合检出率，在目前情况下，应用PCR方法来鉴别恶性淋巴瘤，必须结合HE形态和免疫表型等综合判断。     

Cytologic features of post-transplant lymphoproliferative disorder

Post-transplant lymphoproliferative disorders (PTLD) are Epstein-Barr virus (EBV)–associated lymphoid proliferations which affect approximately 2% of organ allograft recipients. Although the histologic features of PTLD are well described, they have been described only rarely in cytologic specimens. The cytomorphologic features of PTLD in body fluid specimens, needle aspirations, and a gastric brushing specimen from seven patients with histologically confirmed PTLD were therefore reviewed. In the cytologic specimens, PTLD was characterized by a mostly polymorphous population of lymphoid cells containing many large transformed lymphocytes, occasional immunoblast-like atypical lymphocytes, necrosis, and, frequently, obvious plasmacytoid differentiation. The presence of EBV was documented in five of the seven cases in the corresponding tissue biopsies. The four patients with PTLD in a body fluid specimen all died within 3 months of detection of the PTLD in the body fluid. The three remaining patients are alive with resolution of PTLD (follow-up of 7, 8, and 14 months). The diagnosis of PTLD should be suggested when cytologic specimens from organ allograft recipients show a polymorphous atypical lymphoid proliferation, frequently with plasmacytoid differentiation and necrosis. Cytologic samples may provide the initial diagnosis of this potentially fatal disease and allow appropriate intervention. The presence of PTLD in a body fluid specimen is a poor prognostic indicator. Diagn. Cytopathol. 16:489–496, 1997. © 1997 Wiley-Liss, Inc.     

Clinical Characteristics of Monomorphic Post-transplant Lymphoproliferative Disorders

Post-transplant lymphoproliferative disorders (PTLD) are a heterogeneous group of lymphoproliferative disorders associated with immunosuppression and Epstein-Barr virus infection. PTLD is classified into three major categories: early lesions, polymorphic PTLD, and monomorphic PTLD. The majority of monomorphic PTLD cases are non-Hodgkin''s lymphoma of B-cell origin. This retrospective study was conducted to investigate the incidence, clinical manifestation, treatment, and outcomes of monomorphic PTLD among 5,817 recipients of solid organ or allogeneic hematopoietic stem cell transplantation from five institutions. Fourteen patients with monomorphic PTLD were identified (male:female 11:3; median age 42.6 yr, range 24-60). The overall incidence rate was 0.24%. The most common disease type was diffuse large B cell lymphoma (n=7). The median time between the transplant and diagnosis of PTLD was 85.8 months. However, all cases of PTLD after allogeneic hematopoietic stem cell transplantation occurred within 1 yr after transplantation. Ten of the 14 patients had EBV-positive tumor. Fourteen patients received combination systemic chemotherapy and four patients were treated with radiation therapy. Ten patients achieved a complete response (CR) and two patients a partial response (PR). The median follow-up period for surviving patients was 36.6 months. Nine patients remain alive (eight CR, one PR). Nine of 11 solid organ transplantations preserved graft function. The present study indicates a lower incidence rate and a longer median time before the development of PTLD than those of previous reports. Careful monitoring was needed after allogeneic hematopoietic stem cell transplantation for PTLD.     

Discordant bone marrow involvement in diffuse large B-cell lymphoma: comparative molecular analysis reveals a heterogeneous group of disorders

Discordant bone marrow (BM) involvement in patients with a diagnosis of large-cell non-Hodgkin's lymphoma (NHL) is characterized by marrow infiltrates predominantly composed of small lymphoid cell, cytologically compatible with low-grade NHL. Although this phenomenon is well described morphologically, molecular data concerning the relationship of the two lesions are lacking. The aim of the study was to investigate the clonal relationship of discordant lymphoma manifestations by using immunoglobulin heavy chain gene (IgH), as well as bcl-2 rearrangements, as molecular markers. IgH rearrangements were amplified by PCR with consensus primers directed against framework regions 3 or 2 (FR3 and FR2), followed by automated fragment length analysis and sequencing in selected cases. Rearrangements of the bcl-2 gene were identified with primers against the major breakpoint region. Small BM infiltrates were isolated by laser capture microdissection. In addition, immunohistochemistry was performed on paraffin sections using antibodies against CD3, CD10, CD20, bcl-2, bcl-6, p53, and the Ki67 antigen. Paraffin-embedded tissues of 21 cases diagnosed as diffuse large B-cell lymphoma (DLBCL) with discordant BM involvement and no previous history of low-grade B-cell NHL were analyzed. After review of immunohistochemical stains, 5 cases were excluded either as concordant BM infiltrates by large-cell lymphoma with abundant reactive T-cells (2 cases) or as benign, reactive lymphoid infiltrates (3 cases), as confirmed by a polyclonal pattern in the IgH analysis. Of the remaining 16 cases, a common clonal origin was confirmed in 8 cases by the presence of an identical clonal IgH rearrangement or bcl-2 rearrangement. In 4 cases, identification of distinct IgH or bcl-2 rearrangements gave evidence for the presence of two clonally unrelated neoplasms. The remaining 4 cases were not evaluable for technical reasons. Morphological, phenotypical, and molecular findings were compatible with a lymphoma of germinal center origin in the majority of cases. However, in 4 cases, flow cytometric analysis of the BM infiltrates revealed a B-cell chronic lymphocytic leukemia phenotype. Two of these cases were clonally related to the DLBCL and thus represented Richter's transformation. In summary, discordant BM infiltrates in DLBCL represent a heterogeneous group of disorders, encompassing cases with a clonally related, clinically occult small-cell component, as well as cases with two clonally distinct, unrelated B-cell neoplasms presenting synchronously at different locations.     

PCR-based clonality analysis of B-cell lymphomas in paraffin-embedded tissues: diagnostic value of immunoglobulin kappa and lambda light chain gene rearrangement investigation

Polymerase chain reaction (PCR)-based analysis, employed for detecting immunoglobulin heavy chain (IgH) gene rearrangements, has become a diagnostic tool widely used in the investigation of B-cell lymphomas, but the overall sensitivity of these methods does not exceed 80%, notably in germinal center (GC) and post-GC B-cell origin lymphomas. Many PCR strategies devised for detecting immunoglobulin light chain (IgL) gene rearrangements have been developed to enhance the clonality detection rates. However, the feasibility of these methods in routine clinical diagnosis using paraffin-embedded tissues has not yet been investigated sufficiently. We studied a large series of 108 cases of B-cell lymphomas, as well as 20 reactive lymphoid tissues using degenerate primers to amplify immunoglobulin kappa (Igkappa) and lambda (Iglambda) light chain genes. B-cell clonality was further investigated using semi-nested PCR for IgH gene rearrangements. B-cell clonality was detected in 74%, 56.5%, and 43.5% of cases using IgH, Igkappa, and Iglambda PCR, respectively. By combining these methods, the clonality detection rate increased to 93.5%. Only polyclonal patterns were noted in reactive lymphoid samples. We concluded that in addition to the established methods for IgH analysis, a PCR-based approach for IgL gene rearrangements analysis improves the clonality detection rate in over 90% of B-cell lymphoma cases using routine histological specimens with poor preservation of the genomic DNA.     

Polymerase chain reaction (PCR) detection of B cell clonality in Sjögren's syndrome patients: a diagnostic tool of clonal expansion

Sjögren's syndrome (SS) is an autoimmune disease characterized by clonal B cell attack of the exocrine glands and dysregulated expression of B cell‐activating factor (BAFF). Based upon the current data of increased rates of lymphoid malignancy, as non‐Hodgkin's lymphoma (NHL) is associated with SS, we propose the detection of clonal rearrangements of immunoglobulin heavy chain (IgH) gene in those patients as a predictor of malignant clonal expansion. To test our proposal, we examined the IgH clonal rearrangements in SS patients (60) and healthy control subjects (42) having chronic non‐specific sialadenitis, to determine the presence of clonal B cells in minor labial salivary glands (MSG) of SS patients. Clonal B cell expansion was assessed by two polymerase chain reaction (PCR) assays: (i) semi‐nested PCR, against sequences encoding framework regions FR3, FR2 and FR1c of the variable chain IgH gene in B cells present in the MSG infiltrate; and (ii) the PCR–enzyme‐linked immunosorbent assay (ELISA) technique, against the major and minor breakpoint regions of the Bcl‐2 oncogene coupled with a variable segment of the IgH to assess the Bcl‐2/JH translocation. When FR3, FR2 and FR1c primers were employed, we detected B cell monoclonality in 87% of the SS patients and 19% of the control subjects. The association between inflammation severity of the MSG pattern and the presence of B cell clonality was found to be statistically significant (P < 0·01). We concluded that the presence of B cell clonality in MSG can be used as a index of an altered microenvironment favouring the development of lymphoma in SS patients.