Non-mediastinal grey zone lymphomas and report from the workshop

Grey zone lymphomas represent borderline lesions between classical Hodgkin lymphoma (cHL) and other morphologically and immunophenotypically related diseases and entities like nodular lymphocyte predominant HL, T-cell rich B-cell lymphoma, ALK-negative anaplastic large cell lymphoma (ALCL), diffuse large B cell lymphoma (DLBCL) anaplastic variant and primary mediastinal LBCL. The sharp definition of morphological, immunophenotypical and molecular features of the "text-book cases" of each disease and the comparison with grey zone cases has reduced most of the latter cases. Two reports in this workshop dealt with the problematic non-mediastinal grey zone lymphomas, one with a cHL of T cell-type presenting in the skin as a ALK-negative ALCL and the other with the grey zone between cHL of B-cell type and ALK-negative ALCL.     

Molecular diagnosis of primary mediastinal B cell lymphoma identifies a clinically favorable subgroup of diffuse large B cell lymphoma related to Hodgkin lymphoma

Using current diagnostic criteria, primary mediastinal B cell lymphoma (PMBL) cannot be distinguished from other types of diffuse large B cell lymphoma (DLBCL) reliably. We used gene expression profiling to develop a more precise molecular diagnosis of PMBL. PMBL patients were considerably younger than other DLBCL patients, and their lymphomas frequently involved other thoracic structures but not extrathoracic sites typical of other DLBCLs. PMBL patients had a relatively favorable clinical outcome, with a 5-yr survival rate of 64% compared with 46% for other DLBCL patients. Gene expression profiling strongly supported a relationship between PMBL and Hodgkin lymphoma: over one third of the genes that were more highly expressed in PMBL than in other DLBCLs were also characteristically expressed in Hodgkin lymphoma cells. PDL2, which encodes a regulator of T cell activation, was the gene that best discriminated PMBL from other DLBCLs and was also highly expressed in Hodgkin lymphoma cells. The genomic loci for PDL2 and several neighboring genes were amplified in over half of the PMBLs and in Hodgkin lymphoma cell lines. The molecular diagnosis of PMBL should significantly aid in the development of therapies tailored to this clinically and pathogenetically distinctive subgroup of DLBCL.     

56例伴纵隔大包块淋巴瘤患者的临床特征及预后分析

目的:分析伴纵隔大包块淋巴瘤患者的发病情况、临床表现、治疗效果及预后。方法:回顾性分析2007年2月至2018年1月诊治的56例初诊伴有纵隔大包块患者的临床资料,包括临床特征、分型、分期、疗效及预后。结果:56例患者中,男性25例(45%),女性31例(55%),中位年龄31(17-72)岁。其中≤40岁患者48例(86%)。按病理分型,其中弥漫大B细胞淋巴瘤非特指型(DLBCL-NOS)9例(16.1%),原发纵隔大B细胞淋巴瘤(PMBCL)26例(46.4%),经典型霍奇金淋巴瘤(c HL)17例(30.4%),纵隔灰区淋巴瘤(MGZL)4例(7.1%)。所有患者均采用化疗或化学免疫治疗,中位随访时间54.5(11-149)个月。c HL组中联合受累野放疗(IFRT)10例,完全缓解(CR)10例(58.8%),部分缓解(PR)2例(11.8%),疾病进展(PD)5例(29.4%),治疗过程中有6例患者出现复发/难治而接受自体造血干细胞移植(auto-HSCT)。c HL组1年总生存率(OS)为94.1%,2年OS率为88.2%,5年OS率为67.2%。MGZL组中,联合IFRT 3例,auto-HSCT 1例,CR 2例(50%),PD 2例(50%)。MGZL组1年及2年OS率均为66.7%,5年OS率33.3%。PMBCL组中,联合IFRT 8例,auto-HSCT 7例,CR 22例(84.0%),PR 2例(8.0%),疾病稳定(SD)1例(4.0%),PD 1例(4.0%)。PMBCL组1年及2年OS率均为100%,5年OS率为95.7%。DLBCL-NOS组中,联合IFRT 3例,auto-HSCT 2例,CR 4例(44.5%),PR 2例(22.2%),疾病稳定(SD)1例(11.1%),PD 2例(22.2%)。DLBCL-NOS组1年OS率100%,2年及5年OS率均为77.8%。4组间OS率存在显著性差异(P<0.05),组间比较,PMBCL组生存期显著长于MGZL组(P<0.01)及c HL组(P<0.05),与DLBCL-NOS无显著性差异(P>0.05)。结论:纵隔肿物因其解剖部位独特,常表现为独特的临床特征。c HL、MGZL、PMBCL、DLBCL-NOS等几种原发或伴发纵隔大包块的B细胞起源的淋巴瘤,好发于年轻患者,其临床表现类似,但治疗效果差异显著。在现有治疗条件下,PMBCL的预后优于MGZL及c HL。     

蛋白和基因水平探索H/RS细胞的起源

目的：从蛋白、组织和单细胞的基因水平进一步探索H／RS细胞的来源及其克隆性。方法首先对33例经典型霍奇金淋巴瘤(cHL)标本进行B细胞特异性激活蛋白(BSAP)和CD20的检测，然后对33例cHL患的石蜡刮片组织和部分阳性病例的微切割细胞进行免疫球蛋白重链基因克隆性重排检测，并对同一病例的石蜡刮片组织和微切割细胞的扩增产物进行测序分析比较。结果33例中30例的H／RS细胞表达BSAP，10例表达CD20，BSAP和CD20的表达率差异有显性(P=0.000)，对照病例中反应性增生淋巴结的B淋巴细胞和B细胞淋巴瘤肿瘤细胞的BSAP和CD20表达均为100％，T细胞淋巴瘤的肿瘤细胞无表达；33例中的16例石蜡刮片组织IgH基因重排阳性，微切割的19管H／RS细胞有14管出现重排阳性，细胞数目不同的各管阳性率差异无显性(P=0．280)；同一病例的石蜡刮片组织和微切割细胞的PCR产物测序结果均为IgH可变区片段，但是碱基序列并不完全相同。结论：进一步支持cHL中大多数H／RS细胞为B细胞起源，且可能来源于其不同的分化阶段。     

Aberrant immunoglobulin class switch recombination and switch translocations in activated B cell-like diffuse large B cell lymphoma

To elucidate the mechanisms underlying chromosomal translocations in diffuse large B cell lymphoma (DLBCL), we investigated the nature and extent of immunoglobulin class switch recombination (CSR) in these tumors. We used Southern blotting to detect legitimate and illegitimate CSR events in tumor samples of the activated B cell-like (ABC), germinal center B cell-like (GCB), and primary mediastinal B cell lymphoma (PMBL) subgroups of DLBCL. The frequency of legitimate CSR was lower in ABC DLBCL than in GCB DLBCL and PMBL. In contrast, ABC DLBCL had a higher frequency of internal deletions within the switch mu (Smu) region compared with GCB DLBCL and PMBL. ABC DLBCLs also had frequent deletions within Sgamma and other illegitimate switch recombinations. Sequence analysis revealed ongoing Smu deletions within ABC DLBCL tumor clones, which were accompanied by ongoing duplications and activation-induced cytidine deaminase-dependent somatic mutations. Unexpectedly, short fragments derived from multiple chromosomes were interspersed within Smu in one case. These findings suggest that ABC DLBCLs have abnormalities in the regulation of CSR that could predispose to chromosomal translocations. Accordingly, aberrant switch recombination was responsible for translocations in ABC DLBCLs involving BCL6, MYC, and a novel translocation partner, SPIB.     

Clinicopathological features of malignant lymphoma in Japan: the Miyagi Study

The Miyagi Study is an epidemiological study of malignant lymphoma, including immunological and genetic analyses, constructed by a population-based registration system covering Miyagi prefecture, Japan. A total of 1,552 newly diagnosed cases in Miyagi between 2002 and 2008 were enrolled in this study; 75% were B-cell lymphomas, 19% were T-cell and natural killer-cell (T/NK-cell) lymphomas, and 5% were Hodgkin's lymphomas. The most frequent subtype of B-cell lymphoma is diffuse large B-cell lymphoma, followed by follicular lymphoma and extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (51%, 24% and 8%, respectively). Thus, follicular lymphoma accounts for 18.2% of newly diagnosed cases in Miyagi; unexpectedly, its frequency is similar to that reported in Western countries. The common subtypes of T/NK-cell lymphoma are peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma, and adult T-cell leukemia/lymphoma (30%, 15% and 14%, respectively). Most of the data are similar to those reported in Asian countries, except for follicular lymphoma. We also analyzed the CD20 expression in B-cell lymphomas by flow cytometry for the cell membrane expression and by immunohistochemistry for the cytoplasmic expression. The cell membrane expression of CD20 protein may determine the susceptibility of B-cell lymphomas to anti-CD20 antibody therapy. The lack of CD20 expression was confirmed by both methods in 4 cases of 585 newly diagnosed cases (0.7%) and in 5 of 67 recurrent cases (7.5%). Furthermore, 23 cases (6.5%) showed the discrepancy of CD20 expression between both methods. The Miyagi Study has revealed the latest epidemiological features of malignant lymphoma in Japan.     

Diminished expression of CD19 in B-cell lymphomas

BACKGROUND: CD19 is expressed on most B-cell lymphomas; however, the frequency and types of B-cell lymphomas with low-level expression of CD19 are not well characterized. METHODS: We reviewed flow cytometric histograms specifically for decreased CD19 expression on 349 cases analyzed by the Flow Cytometry Laboratory at University Hospitals of Cleveland (Cleveland, Ohio). Results of flow cytometry were correlated with the morphologic diagnosis. RESULTS: Of the cases reviewed, 125 (36%) showed a visible decrease in CD19 expression compared with normal B lymphocytes. Decreased CD19 expression was noted in 79% of follicular lymphomas (27 of 34), 36% of small lymphocytic lymphomas/chronic lymphocytic leukemias (82 of 228), 31% of mantle cell lymphomas (4 of 13), 24% of diffuse large B-cell lymphomas (8 of 33), and 13% of marginal zone B-cell lymphomas/lymphoplasmacytoid lymphomas (4 of 30) and was not observed in any Burkitt lymphoma (0 of 5) or hairy cell leukemia (0 of 6). Decreased CD19 expression was significantly more frequent in follicular lymphomas than in other lymphoma subtypes (P < 0.001). No significant difference was observed in the frequency of decreased CD19 expression based on histologic grade of follicular lymphoma. CONCLUSIONS: Diminished expression of CD19 expression occurs frequently in B-cell lymphomas, in particular follicular lymphoma, and may be helpful in identifying B-cell lymphoma cells in complex cell mixtures such as bone marrow specimens.     

The results of histological and immunohistological studies of primary biopsies in 400 patients with non-Hodgkin's lymphoma in the North-West region of Russia (according to WHO classification)

AIM: To determine relative frequency of non-Hodgkin's lymphoma (NHL) variants according to WHO classification categories in the North-West of Russia by the data of the local pathomorphological department. MATERIAL AND METHODS: Four hundred consecutive untreated patients with NHL were diagnosed according to the WHO classification between January 2000 and October 2003 in the Regional Bureau of Pathology, St-Petersburg. The patients' age, gender, location of the biopsied tumor focus were considered. The immunohistochemical study was performed by the paraffin section-immunoperoxidase method. Cases of plasma cell myeloma and bone marrow trephine biopsy diagnosed neoplasms were excluded, the rest series of 377 cases was compared to other regional world series. RESULTS: B-cell lymphomas accounted for 79.6% and T-cell type for 20.4% of 377 NHL cases. 33.2% cases were different histological variants of diffuse large B-cell lymphoma, 17.0% were B-cell small lymphocytic lymphomas, 11.0%--follicular lymphomas, 4.5%--mantle cell lymphomas, 2.1%--extranodal marginal zone B-cell lymphomas (MALT-type), 1.9%--primary B-cell mediastinal lymphomas. Peripheral T-cell lymphomas, unspecified and anaplastic large T/null cell lymphomas were diagnosed in 6.4 and 4.2% of 377 cases, respectively. Other lymphoma diagnoses comprised 19.7%. CONCLUSION: Diffuse large B-cell lymphomas and B-cell small lymphocytic lymphomas are prevalent among B-cell neoplasms. Follicular lymphomas and mantle cell lymphomas are less common in the North-West of Russia compared to Europe and USA. The relative frequency of extranodal marginal zone B-cell lymphomas (MALT-type) is lower and anaplastic large T/null cell lymphomas is higher than these in International Lymphoma Study Group, Taiwan and Japan series. This study provides evidence that the distribution pattern of non-Hodgkin's lymphoma subtypes in the North-West of Russia shows significant differences with those from the rest of the world.     

Loss of p73 promotes dissemination of Myc-induced B cell lymphomas in mice

Mice engineered to express c-Myc in B cells (Eμ-myc mice) develop lethal lymphomas in which the gene encoding the p53 tumor suppressor is frequently mutated. Whether the p53 homolog p73 also functions as a tumor suppressor in vivo remains controversial. Here we have shown that p73 loss does not substantially affect disease onset and mortality in Eμ-myc mice. However, it does alter the phenotype of the disease. Specifically, p73 loss decreased nodal disease and increased widespread extranodal dissemination. We further found that p53 acted as the dominant tumor suppressor during the onset of Eμ-myc–driven B cell lymphomagenesis, while p73 modulated tumor dissemination and extranodal growth. Immunophenotyping and expression profiling suggested that p73 loss allowed increased maturation of malignant B cells and deregulated genes involved in lymphocyte homing and dissemination of human lymphomas. Consistent with this, p73 expression was frequently downregulated in a large cohort of human mature aggressive B cell lymphomas, and both the incidence and degree of p73 downregulation in these tumors correlated with their extranodal dissemination status. These data indicate that p73 is a modifier of Myc-driven lymphomas in mice, favoring tumor dissemination, and suggest that p73 could be a biomarker for human B cell lymphoma dissemination, a notion that can now be tested in clinicopathologic correlation studies.     

Pathogenesis of Hodgkin's lymphoma

In Hodgkin's lymphoma (HL), the B cell origin of the tumour cells, the Hodgkin and Reed-Sternberg (HRS) cells, has been disclosed by molecular single cell analysis about 10 yr ago. This finding formed the basis for various studies aimed to better understand the pathogenesis of this peculiar malignancy and the pathophysiology of the HRS cells. Work of our groups in this regard was focussed recently on two main topics, namely the study of differential gene expression in HRS cells and the pathogenesis of composite lymphomas. Composite lymphomas are combinations of HL and B cell non-Hodgkin lymphomas, that turned out to be often clonally related. By molecular analysis of several composite lymphomas for potential transforming events, we identified examples of both shared as well as distinct transforming events. Comparing gene expression profiles of HL-derived cell lines with the corresponding profiles from other B cell lymphomas and normal B cell subsets revealed a global down-regulation of the B cell-specific gene expression signature in HRS cells. Moreover, we identifed aberrant expression and activity of multiple receptor tyrosine kinases in HRS cells of classical and to a lesser extend lymphocyte predominant HL, which appears to be a unique feature of HL, and may offer novel strategies for treatment.     

伴有明显淋巴滤泡的非特指外周T细胞淋巴瘤

目的　探讨伴有明显淋巴滤泡增生的非特指外周T细胞淋巴瘤的形态学特点和免疫表型。方法　对 3例特殊形态的外周T细胞淋巴瘤进行形态学观察 ,免疫组化EnVision法检测CD3、CD4 5RO、CD4 3、CD2 0、CD79a、cyclinD1、bcl 2、CD4、CD8、S 10 0等抗体 ,并用PCR方法进行T细胞受体(TCR)基因重排检测。结果　 3例患者均以全身浅表淋巴结肿大为主要表现 ,初发部位为颈部和颌下 ,发病过程中出现低热及全身皮疹 ,并有肝、脾肿大。形态学的显著特点为淋巴滤泡增生 ,套区消失 ,边缘区有中等大小、胞浆透亮的瘤细胞浸润生长。免疫表型标志为T细胞淋巴瘤。基因检测有TCRγ基因阳性条带。结论　部分非特指外周T细胞淋巴瘤可伴有明显淋巴滤泡增生 ,应注意与淋巴结反应性增生、滤泡性淋巴瘤、边缘区淋巴瘤及套区淋巴瘤等鉴别     

The biology of tumor growth in the non-Hodgkin''s lymphomas. A dual parameter flow cytometry study of 220 cases

Dual parameter flow cytometry studies (cell DNA content and electronic cell volume) were performed in 220 cases of non-Hodgkin's lymphoma. All cases were characterized as B or T cell malignancies, based on immunologic surface marker characteristics. Aneuploidy by flow cytometry was more common among the B cell lymphomas than among the T cell lymphomas, and was most common among the large B cell lymphomas and B cell lymphomas of intermediate size. Ploidy index distributions showed a prominent hyperdiploid peak, as well as tumor cell populations with near-tetraploid DNA contents. In serial studies, a decrease in ploidy index was observed in association with clinical and histologic transformation in one case. The highest S fractions were observed among the large and intermediate B cell lymphomas and among the aggressive T cell lymphomas. In clinical samples consisting of mixtures of diploid and aneuploid populations, the data on the aneuploid components could often be separated from other components of the mixture in multiparameter studies on the basis of the larger electronic cell volumes of the aneuploid cells. In each case, the aneuploid large cell component almost invariably had a higher S fraction than the residual component(s) of the mixture. Overall, the data are consistent with a model of clonal selection and clonal evolution in the lymphomas in which early cytogenetic abnormalities that involve little or no change in total cell DNA content are followed by cell tetraploidization that is associated with cytogenetic instability and chromosome loss over the course of time.     

Primary virus-induced lymphomas evade T cell immunity by failure to express viral antigens

T lymphoma induction by the mink cell focus-inducing murine leukemia virus MCF 1233 in C57BL/10 and C57BL/6 mice is influenced by a strongly Th-dependent, H-2I-A-restricted antiviral immune response (25). We compared the MHC class I as well as viral env and gag antigenic cell surface profiles of frequent T lymphomas of H-2I-A nonresponder-type mice to that of rare T lymphomas of H-2I-A responder-type mice. Membrane immunofluorescence studies, with a panel of anti-env mAbs (reactive with the highly conserved gp70f epitope, the p15Ec epitope, and the gp70-p15E complex), a polyclonal anti-p30 serum, and anti-H-2 class I mAbs, showed that all 17 nonresponder tumors tested expressed high levels of both env and gag viral proteins, and 15 of these 17 nonresponder tumors expressed high levels of H-2 class I K and D antigens. In contrast, 10 of 11 responder lymphomas lacked env and/or gag determinants. The only responder lymphoma with both strong env and gag expression failed to express H-2K and -D antigens. Preferential loss of env or gag expression did not correlate with H-2 class I allelic specificities. Both responder and nonresponder T lymphoma DNA contained multiple, predominantly MCF-like, newly acquired proviral integrations. Differences in viral antigen cell surface expression were confirmed at cytoplasmic and RNA levels. The amounts of 8.2- and 3.2-kb viral RNA were greatly reduced in two responder lymphomas when compared with four nonresponder lymphomas. In both responder lymphomas, aberrantly sized viral RNA species were found. Upon in vivo passage of these responder lymphomas in either immunocompetent or T cell-deficient nu/nu mice, it was found that various molecular mechanisms may underlie the lack of viral antigen expression at the cell surface of these lymphomas. One lymphoma re-expressed viral antigens when transplanted with nu/nu mice, whereas the other remained stably gag negative. The combined findings indicate that an H-2I-A-regulated antiviral immune response not only strongly reduces T lymphoma incidence, but also forces T lymphomas that still arise to poorly express viral antigens, thus explaining their escape from immunosurveillance.     

CD45RO阳性的B细胞性淋巴瘤

目的 研究免疫表型异常淋巴瘤的起源。方法 从48例B细胞淋巴瘤中筛选出3例CD45RO、CD20和CD79a( )，CD3(-)的淋巴瘤。采用免疫组化、PCR和基因扫描技术对其免疫表型，免疫球蛋白重链基因重排(IgH)和T细胞受体基因重排(TCR)进行深入研究。结果 3例淋巴瘤均为结外淋巴瘤。1例为滤泡性淋巴瘤，2例为弥漫性大B细胞性淋巴瘤。PCR和基因扫描显示3例均有IgH克隆性扩增；PCR未显示TCR扩增；高敏感性基因扫描显示低峰值TCR克隆性扩增，提示可能为背景T细胞扩增。结论 CD45RO不是T细胞特异性抗原，CD45RO阳性细胞不能完全等同于T细胞，因此，在研究和临床病理诊断中应选用多种抗体配合使用。     

Expression of proliferation-associated nuclear antigen in human malignancies

40 specimens consisting of 27 carcinomas, 9 lymphomas, 2 thymomas, 1 sarcoma and 1 neurinoma were studied by immunoperoxidase technique to demonstrate a cell proliferation-associated antigen defined by proliferating cells (PC) antibody. Though PC antibody expression did not seem to correlate well with histologic grading of carcinomas, positivity to this was consistently more intense in carcinomas than in normal epithelia. Correlation with histologic grading of lymphomas was more significant: the high grade types, e.g. ATL, exhibited greater positivity than intermediate grade types, e.g. diffuse medium lymphoma and certainly much more than low grade types, e.g. follicular lymphomas. These data were compared to another proliferation-associated antigen, the transferrin receptor.     

Imbalanced MHC class II molecule expression at surface of murine B cell lymphomas

To study the role of class II MHC expression in mouse lymphomagenesis, we examined the cell surface expression of I-A/E antigens on 24 spontaneous or murine leukemia virus (MuLV)-induced mouse B10.A (I-Ak, I-Ek) B cell lymphomas. Two primary B10.A B cell lymphomas were observed with strong I-Ek expression but with only minimal cell surface I-Ak expression. Both tumors are readily transplantable in syngeneic mice, with maintenance of their I-A-, I-E+ phenotype. Strikingly, one I-A-, I-E+ B cell lymphoma contains a (11; 17) translocation with a breakpoint on chromosome 17 that is localized within or very close to the H-2 complex. DNA of both tumors contains normal restriction enzyme fragments of the A alpha and A beta genes. Northern blot analyses indicated that one I-A-, I-E+ tumor strongly expressed A alpha, E alpha, and E beta mRNAs but possessed only a weak expression of A beta mRNA. The other B cell lymphoma showed A beta, E alpha, and E beta mRNA expression but only minimal A alpha mRNA expression. In 11 primary B10.A B cell lymphomas with a normal I-A+, I-E+ phenotype, no imbalances in A alpha/A beta mRNA levels were observed. The implications of these findings for the role of class II MHC expression in mouse B cell lymphoma-genesis are discussed.