Malaria hidden in a patient with diffuse large-B-cell lymphoma and sickle-cell trait

We report a case of an African patient with sickle cell trait who was diagnosed in Spain with B-cell lymphoma. Blood smears were negative for malaria, and no plasmodium antigens were detected in the blood. To treat his lymphoma, the patient underwent chemotherapy and autologous stem cell transplantation. Following a splenectomy due to a worsening condition, he developed clinical malaria with detectable parasitemia. This case suggests that the humoral response and parasite removal by the spleen may afford protection from overt disease and may even help maintain subclinical human reservoirs of the disease.     

High throughput tissue microarray analysis of FHIT expression in diffuse large cell B-cell lymphoma from Saudi Arabia.

Recent studies have suggested a potential prognostic role of alterations of the fragile histidine triad (FHIT) gene in diffuse large B-cell lymphoma. To evaluate possible mechanisms of FHIT inactivation and to further clarify its potential prognostic relevance, we analyzed a set of 114 diffuse large B-cell lymphoma with clinical follow-up information. Tissue microarrays were analyzed by immunohistochemistry for protein expression, and corresponding DNA samples were analyzed for FHIT promotor hypermethlyation. Reduced or absent FHIT expression was found in 75 of 114 diffuse large B-cell lymphoma (66%), but was unrelated to clinical tumor stage or patient prognosis. FHIT promotor hypermethylation was observed in 29 of 93 (23%) interpretable diffuse large B-cell lymphoma. Hypermethylation was not significantly correlated to protein expression loss, which could be explained by competing mechanisms for FHIT inactivation in a substantial fraction of non FHIT hypermethylated diffuse large B-cell lymphoma. Hypermethylation was significantly associated with poor prognosis of diffuse large B-cell lymphoma patients and predominantly seen in nongerminal center diffuse large B-cell lymphoma (27%), but less frequent (13%) in germinal center diffuse large B-cell lymphoma. In summary, these data suggest that promotor hypermethylation is responsible for reduced FHIT expression in a substantial subset of diffuse large B-cell lymphoma, which is primarily composed of nongerminal center subtype with poor patient prognosis.     

PKC-beta II expression has prognostic impact in nodal diffuse large B-cell lymphoma.

Recent studies of gene expression and immunohistochemistry have shown that protein kinase C-beta II (PKC-beta II) might have prognostic significance in patients with diffuse large B-cell lymphoma (DLBCL). We sought to determine the prognostic significance of the expression of PKC-beta II in patients with nodal DLBCL. Formalin-fixed, paraffin-embedded tissues were stained with a monoclonal antibody to PKC-beta II protein. A total of 125 patients were studied; 83 patients (66%) were in the low-risk International Prognostic Index (IPI) group. Forty-eight patients (38%) were positive for PKC-beta II. Complete remission was obtained in 70%, and was not influenced by the PKC-beta II status (67 vs 71%). The 5-year event-free survival (EFS) was worse in high-risk patients (14 vs 58%, P<0.001) and in those with PKC-beta II positivity (36 vs 49%, P=0.054). In low-risk IPI patients, PKC-beta II expression was related to a worse 5-year overall survival (OS) (60 vs 76%, P=0.033) and a worse 5-year EFS (48 vs 66%, P=0.014). In a Cox regression analysis for EFS, both PKC-beta II expression (hazard ratio=1.68, P=0.037) and the IPI (HR=3.07, P<0.001) were independent poor prognostic factors. PKC-beta II (HR=1.72, P=0.046) and the IPI (HR=5.16, P<0.001) were also independent poor prognostic factors for the OS. PKC-beta II expression, along with the IPI, were associated with a worse EFS and OS in patients with nodal DLBCL specially in low-risk IPI patients.     

Claudin expression in gastric adenocarcinomas: a tissue microarray study with prognostic correlation

The claudins comprise a multigene family of integral membrane proteins, which play a major role in tight junction formation. Aberrations in the expression of certain claudins have been described in a number of malignancies. Our aims were to determine the expression pattern of claudins 1, 3, and 4 as well as ZO-1 in a large series of US patients with gastric cancer and to correlate expression with clinicopathologic and prognostic variables. Tissue microarrays were created from paraffinized samples from 146 patients with distal gastric adenocarcinomas (61 intestinal and 85 diffuse or mixed subtypes). In addition, cores of normal mucosa and intestinal metaplasia were taken from most cases. The microarrays were stained for claudins 1, 3, and 4 and ZO-1, and the intensity of staining was determined using a 3-point scale. Moderate claudin 1 and ZO-1 membranous staining were present, whereas only focal weak claudin 3 and 4 membranous staining was present in normal gastric epithelium. Moderate to strong staining of claudins 1, 3, 4, and ZO-1 was detected in 74%, 48%, 62%, and 74% of the intestinal but in only 46%, 24%, 45%, and 36% of the diffuse subtype of adenocarcinomas (P < .05). Cox multivariate analysis revealed that tumor stage, diffuse subtype, and moderate to strong claudin 4 staining were associated with decreased survival (P < .02). In conclusion, claudins 1, 3, and 4 and ZO-1 are strongly expressed in most gastric intestinal-type adenocarcinomas but less frequently in diffuse gastric cancers. The up-regulation of claudin expression during gastric carcinogenesis suggests their potential utility as diagnostic biomarkers and possible targets for therapeutic intervention.     

Quantitative gene expression profiling in formalin-fixed, paraffin-embedded tissues using universal bead arrays

We recently developed a sensitive and flexible gene expression profiling system that is not dependent on an intact poly-A tail and showed that it could be used to analyze degraded RNA samples. We hypothesized that the DASL (cDNA-mediated annealing, selection, extension and ligation) assay might be suitable for the analysis of formalin-fixed, paraffin-embedded tissues, an important source of archival tissue material. We now show that, using the DASL assay system, highly reproducible tissue- and cancer-specific gene expression profiles can be obtained with as little as 50 ng of total RNA isolated from formalin-fixed tissues that had been stored from 1 to over 10 years. Further, tissue- and cancer-specific markers derived from previous genome-wide expression profiling studies of fresh-frozen samples were validated in the formalin-fixed samples. The DASL assay system should prove useful for high-throughput expression profiling of archived clinical samples.     

Monoclonal antibodies marking B-cell non-Hodgkin's lymphoma in paraffin-embedded tissue

Traditional methods for the immunophenotypic analysis of the non-Hodgkin's lymphomas require fresh or snap-frozen tissue for flow cytometric or immunohistochemical studies. The monoclonal antibodies LN1, LN2, and L26 have been recently developed to recognize B-cell-specific antigens that survive routine tissue processing and paraffin embedding. In this study, the ability of these three antibodies to mark the neoplastic cells in 160 cases of paraffin-embedded non-Hodgkin's lymphoma relative to frozen section immunophenotype (42 T-cell, 118 B-cell), manner of fixation (B5 versus 10% buffered formalin), and histological subtype was examined. With B5-fixed tissue, the percentages of B-cell lymphoma marking with the antibodies were as follows: L26, 96.6%; LN1, 88.2%; LN2, 93.7%. With formalin-fixed tissue, the percentages of B-cell lymphoma reacting with the antibodies were: L26, 89.1%; LN1, 26.2%; LN2, 57.8%. Each of the antibodies marked a small percentage of paraffin-embedded T-cell lymphomas: L26, 4.7%; LN1, 4.7%; LN2, 7.1%. LN2, and to a lesser extent LN1, stained Reed-Sternberg cells, a feature not seen with L26. Nor did L26 mark nonlymphoid neoplasms, a feature previously reported with LN1 and LN2. Since a high percentage of B-cell lymphomas react with these antibodies and they are relatively specific for B-cells, they should prove highly useful for the evaluation of both diagnostic and experimental pathology specimens. L26 offers the distinct advantage of working well in both B5 and formalin-fixed tissues and seemingly not marking epithelial neoplasms.     

Quantitative RT-PCR assay of HER2 mRNA expression in formalin-fixed and paraffin-embedded breast cancer tissues

Detection of human epidermal growth factor receptor 2 gene (HER2, also known as erbB2) expression is a preparatory process to decide a treatment strategy for breast cancer patients. 20-30% of breast cancer patients have HER2 overexpression, and they usually show poor recovery rate. For detection of HER2 expression, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) methods are conventionally used. Although these methods are accurate and reliable, their time-consuming process and high cost need a concise method with high sensitivity and accuracy. As a complementary method to the current IHC/FISH standard techniques, PCR-based methods have been developed. Here we employed a quantitative PCR method to detect HER2 expression in one hundred ninety nine formalin-fixed and paraffin-embedded (FFPE) breast cancer tissue samples from the patients treated over two years at the Yonsei University Severance Hospital, Republic of Korea. Relative expression of HER2 mRNA in the FFPE samples was analyzed using a quantitative RT-PCR (RT-qPCR) method and the obtained HER2 expression levels were compared with those from IHC/FISH methods. Our results show that the RT-qPCR method was highly concordant with IHC/FISH methods for detecting HER2 expression. Overall sensitivity and specificity of the BrightGen HER2 RT-qDx assay kit (Syantra, Calgary, Canada), which is a kit we used for RT-qPCR analyses, were 93.0% and 89.8% (P < 0.0001), respectively. The diagnostic cut-off value of HER2 RT-qDx for the clinical samples was determined by likelihood ratio, among which the highest likelihood ratio of relative HER2 mRNA levels was over 105.5 (AUC = 0.9466) with the highest sensitivity and specificity. Our study indicates that quantification of HER2 mRNA expression with the RT-qPCR could be an alternative method of conventional IHC/FISH methods.     

Analysis of chromosomal imbalances in de novo CD5-positive diffuse large-B-cell lymphoma detected by comparative genomic hybridization

We recently demonstrated that the prognosis for de novo CD5-positive (CD5+) diffuse large-B-cell lymphoma (DLBCL) is markedly worse than that for CD5-negative (CD5-) DLBCL. Our findings also suggested that on the basis of its clinical features CD5+ DLBCL may constitute a unique disease category. However, the genetic basis for these two categories has not been established. Therefore, we performed comparative genomic hybridization analysis (CGH) of 26 cases of CD5+ DLBCL and 44 cases of CD5- DLBCL. Several identical changes in CD5+ and CD5- DLBCLs were found, such as gains of 3q, 9p, 12q, 13q, and 18q and losses of 1p, 6q, 17p, and 19p. However, distinct differences between the two categories were also detected. These included gains of 11q21-q24 (P=0.032) and 16p (P=0.005) in CD5+ DLBCL, and loss of 16p (P=0.028) in CD5- DLBCL. A comparison with results reported for mantle cell lymphoma, chronic lymphocytic leukemia, and Richter's syndrome demonstrated that the CGH pattern of CD5+ DLBCL was markedly different. This indicates that CD5+ DLBCL constitutes a disease category distinct from that of CD5- DLBCL and other CD5+ malignancies.     

应用组织微阵列技术分析各期鼻咽癌组织p16的表达

目的　构建高通量组织微阵列 /组织芯片 (high throughputtissuemicroarray/tissuechip) ,明确p16基因表达与鼻咽癌的相关性及探讨组织芯片技术的可行性。方法　用组织阵列仪制备按不同临床分期排布的组织芯片 ,然后用免疫组织化学LSAB法检测一张组织芯片上 2 5 9例鼻咽活检组织样本中p16蛋白表达 ,并分析p16蛋白表达在鼻咽癌组织的分布状况及其与临床分期的相关性。结果在正常鼻咽黏膜组织、Ⅰ～Ⅳ期及未分期鼻咽癌组织中p16蛋白的缺失情况分别为 0 / 18、3/ 3、86 3%(38/ 44 )、86 8% (5 9/ 6 8)、82 1% (2 3/ 2 8)、88 8% (87/ 98)。鼻咽癌组织中p16蛋白表达率低于鼻咽非癌组织 (χ2 =82 5 8,P <0 0 0 1) ,p16蛋白表达与鼻咽癌的临床分期无显著相关性 (χ2 =0 0 9,P =0 76 9)。结论　鼻咽癌中p16蛋白的高频缺失提示p16基因在鼻咽癌的发生发展中起重要作用 ,而这种表达缺失在早晚期鼻咽癌中的一致性又提示p16蛋白缺失是鼻咽癌形成过程中的一个早期事件。应用组织芯片大规模高效检测临床组织样本是可行的 ,具有快速、方便、经济、准确的特点。     

Detection of novel gene expression in paraffin-embedded tissues by isotopic in situ hybridization in tissue microarrays

Correlating altered gene expression patterns with particular disease states is a critical step in understanding disease processes and developing treatment strategies. Many thousands of novel gene sequences have recently been annotated in public and private databases and are now available for analysis. Tissue-specific expression patterns of these sequences can be evaluated physically on DNA arrays and other high throughput assays, or virtually by bioinformatics mining of expressed sequence tag (EST) databases. As a secondary screening tool, in situ hybridisation (ISH) not only confirms tissue specificity, but also reveals what is often valuable information about cell-type expression patterns of nov16l sequences. Due to their availability and long-term stability at room temperature, formalin-fixed paraffin-embedded clinical specimens provide an invaluable resource for evaluating expression patterns of novel human genes. We describe a high-throughput approach for identifying and quantifying the expression of novel genes in paraffin-embedded human tissues using isotopic in situ hybridisation and tissue microarrays (TMA).     

弥漫大B细胞淋巴瘤组织bcl-6蛋白表达及基因重排

目的探讨弥漫大B细胞淋巴瘤(DLBCL)中bcl-6和CD10蛋白表达以及基因重排的特点及其临床病理意义。方法应用免疫组织化学EnVision法分析51例DLBCL(22例为淋巴结内,29例为淋巴结外)和10例淋巴结反应性增生石蜡组织中bcl-6蛋白表达,并与CD10的表达作比较分析;应用断裂分离探针及间期核荧光原位杂交(FISH)技术检测32例淋巴结内DLBCL(22例为石蜡组织,10例为新鲜组织)和5例淋巴结反应性增生石蜡组织中bcl-6基因的重排。结果(1)淋巴结内、外DLBCL和淋巴结反应性增生中bcl-6蛋白均呈细胞核表达,阳性率分别为72.7%(16/22)、75.9%(22/29)、100.0%(10/10);CD10的阳性率分别为40.9%(9/22)、41.4%(12/29)、100.0%(10/10),所有CD10阳性肿瘤均共同表达bcl-6蛋白。(2)40.9%(9/22)的淋巴结内DLBCL和41.4%(12/29)的淋巴结外DLBCL为两蛋白共同阳性表达;将之与两蛋白均不表达的DLBCL(27.3%,6/22和24.1%,7/29)相比,前者(Ⅰ或Ⅱ期)的临床分期低于后者(Ⅲ或Ⅳ期,P<0.05)。(3)淋巴结内DLBCL样本中bcl6基因重排阳性率为28.1%(9/32),其中石蜡组织样本阳性率为27.3%(6/22),新鲜组织样本阳性率为30.0%(3/10),P>0.05。5例淋巴结反应性增生样本中均未检测到bcl-6基因重排,与DLBCL相比P<0.05。结论(1)在DLBCL中bcl6有较高的阳性表达率,bcl-6和CD10蛋白的联合检测可以协助DLBCL的诊断和鉴别诊断;bcl6和CD10共同阳性的DLBCL可能具有更好的预后。(2)bcl-6基因重排可能参与了部分DLBCL的发生,并可作为DLBCL诊断的参考指标。     

Cytokeratin expression in hematological neoplasms: a tissue microarray study on 866 lymphoma and leukemia cases

Aberrant expression of cytokeratins (CK) is known to occasionally occur in malignant lymphomas. The monoclonal mouse-anti-human CK cocktail CK22 recognizes keratin polypeptides with a wide range of molecular weights and can be applied in diagnostic panels for tumors of unknown origin. Using tissue microarray technology, we tested 1059 lymphoma and acute leukemia cases, covering the most common disease entities, for aberrant CK expression, using CK22. In total, 866 of the arrayed cases were evaluable (80%), and 13 positive cases (1.5%) were found: 1 out of 230 Hodgkin lymphomas (0.4%), 1 plasma cell myeloma, 2 out of 326 diffuse large B-cell lymphomas (0.6%), 5 out of 18 mantle cell lymphomas (26%), 3 out of 70 small cell lymphomas/chronic lymphocytic leukemias (4%) and 1 out of 27 peripheral T-cell lymphomas, not otherwise specified (4%). Immunostaining was finely granular in most cases, and the total amount of positively staining cells exceeded 10% only in the cases of Hodgkin lymphoma and plasmocytoma. All CK22-positive cases, except for one mantle cell lymphoma, expressed the specific simple epithelial CK8 but not the basal/stratified epithelial CK5/6. Aberrant CK expression can be encountered in a small subset of otherwise characteristic B- and T-cell lymphomas, but not in acute leukemias, which should be considered in difficult differential diagnostic settings.     

Quantitative gene expression analysis in microdissected archival tissue by real-time RT-PCR

Laser-assisted microdissection is a powerful tool for the analysis of morphologically defined cell populations. We report here that the combination of microdissection and real-time RT-PCR technologies together with an optimized RNA microscale extraction procedure allows the reproducible determination of gene expression levels in archival formalin-fixed, paraffin-embedded tissue samples.     

Improved clonality detection in Hodgkin lymphoma using the BIOMED-2-based heavy and kappa chain assay: a paraffin-embedded tissue study

Tapia G, Sanz C, Mate J L, Muñoz‐Mármol A M & Ariza A
(2012) Histopathology  60, 768–773
Improved clonality detection in Hodgkin lymphoma using the BIOMED‐2‐based heavy and kappa chain assay: a paraffin‐embedded tissue study Aims: Although BIOMED‐2 polymerase chain reaction (PCR) standardization protocols allow clonality detection in nearly 100% of non‐Hodgkin B cell lymphomas, they have not been widely validated for Hodgkin lymphoma (HL). Our aim was to assess BIOMED‐2 protocol sensitivity when using non‐microdissected, formalin‐fixed, paraffin‐embedded (FFPE) tissue from HL cases. Methods and results: We studied 69 consecutive HL cases, of which 61 corresponded to classic HL (cHL) and eight to nodular lymphocyte‐predominant HL (NLPHL). CD30‐positive cell numbers (<10, 10–25 or >25 per ×200 field), background CD20‐positive cell density (low or high) and tumour cell immunophenotype were evaluated. IGH and IGK clonality was assessed on FFPE tissue following BIOMED‐2 protocols. Of the 58 assessable cHL cases, 15 (25.9%) exhibited IGH and/or IGK clonality; IGH clonality was shown by nine (15.5%) and IGK clonality by 12 (20.7%). Clonality detection rates in cHL improved as CD30‐positive Reed–Sternberg (RS) cell density increased and CD20‐positive B cell density decreased, although these correlations did not reach statistical significance. Of the eight NLPHL cases studied, none showed clonal rearrangement. Conclusions: Combined study of IGH and IGK rearrangement according to BIOMED‐2 protocols improves clonality detection rate (up to 25% of cases) in HL, even when working on non‐microdissected FFPE tissue.