Intravascular (blood vessel) histiocytosis with haemophagocytosis.

AIMS: Many types of intravascular lymphohistiocytic proliferation have been described recently; this was previously an unnoticed or misinterpreted phenomenon. Intralymphatic lymphohistiocytic aggregates are relatively common, and include benign, malignant and indeterminate conditions. In contrast, all non-endothelial proliferations in the lumina of blood vessels have been interpreted so far as malignant. Herein, we present three cases of histiocytic proliferations in the lumen of blood vessels associated with intracytoplasmic granulocyte debris (haemophagocytosis), a previously undescribed entity. METHODS AND RESULTS: We identified three patients from two institutions with similar cutaneous lesions, both clinically and microscopically. Information regarding clinical history, histological features and immunoprofiles were obtained. The three cases presented intravascular histiocytosis with haemophagocytosis involving blood vessels of the dermis, a process that may be representative of a new entity. The patients were two women and one man who presented a symmetrical reticulated erythema with a tendency to involve the skin of the breasts. The lesions were indolent, did not ulcerate and followed a benign course. CONCLUSION: This seemingly novel condition is characterized by the presence of histiocytic cells inside blood vessels, where they have not been described previously as an entity. The most reasonable explanation for this process is an origin from the non-classical subset of monocytes that 'patrol' the inner face of blood vessels acting as macrophages. The existence of this entity should be kept in mind to avoid overdiagnosis of malignancy.

HLA-I levels correlate with survival outcomes in response to immune checkpoint inhibitors in non-small cell lung cancer.

OBJECTIVES: Immune checkpoint inhibitors (ICIs) have provided a breakthrough in the treatment of non-small cell lung cancer (NSCLC) patients, but only some patients benefit substantively. Identifying definitive predictive biomarkers could overcome this limitation. MATERIALS AND METHODS: We selected 146 metastatic NSCLC patients treated with anti-PD-(L)1. Immunohistochemistry of HLA-I, PD-L1 and CD73 was performed in 122 tumor biopsies at diagnosis. The association with patients, tumor parameters, and the predictive value to ICI treatment were determined. RESULTS: In our cohort, 42 %, 25 %, and 21 % of the tumors exhibited high levels of HLA-I, PD-L1, and CD73, respectively. Lung adenocarcinomas displayed elevated CD73 levels, compared with lung squamous cell carcinomas (P = 0.026). High PD-L1 was significantly correlated with high levels of HLA-I (P = 0.005) and of CD73 (P = 0.025). Patients with high-level HLA-I tumors exhibited more favorable clinical outcomes following ICI, with a median overall survival of 30.7 months (95 % confidence interval [CI]: 18.3 months-not reached), compared with 18.2 months (95 % CI: 12.4-25.2 months) in patients with low-level HLA-I tumors (P = 0.016). The median progression-free survival (PFS) for patients with high-level HLA-I tumors was 18.5 months (95 % CI: 11.1-57.1 months), longer than patients with low-level HLA-I tumors, whose median PFS was 9.2 months (95 % CI: 7.2-11.9 months) (P = 0.006). In a multivariable analysis, high-level HLA-I was independently associated with lower risk of progression to ICI (HR = 0.46, 95 % CI 0.24-0.87; P = 0.018). CONCLUSIONS: High-level HLA-I were associated with better clinical outcomes to ICI in our cohort of NSCLC patients. Therefore, further investigations are warranted to refine this biomarker and validate its efficacy in prospective and larger set of patients.

Gene-expression profiling and not immunophenotypic algorithms predicts prognosis in patients with diffuse large B-cell lymphoma treated with immunochemotherapy.

Diffuse large B-cell lymphomas (DLBCLs) can be divided into germinal-center B cell-like (GCB) and activated-B cell-like (ABC) subtypes by gene-expression profiling (GEP), with the latter showing a poorer outcome. Although this classification can be mimicked by different immunostaining algorithms, their reliability is the object of controversy. We constructed tissue microarrays with samples of 157 DLBCL patients homogeneously treated with immunochemotherapy to apply the following algorithms: Colomo (MUM1/IRF4, CD10, and BCL6 antigens), Hans (CD10, BCL6, and MUM1/IRF4), Muris (CD10 and MUM1/IRF4 plus BCL2), Choi (GCET1, MUM1/IRF4, CD10, FOXP1, and BCL6), and Tally (CD10, GCET1, MUM1/IRF4, FOXP1, and LMO2). GEP information was available in 62 cases. The proportion of misclassified cases by immunohistochemistry compared with GEP was higher when defining the GCB subset: 41%, 48%, 30%, 60%, and 40% for Colomo, Hans, Muris, Choi, and Tally, respectively. Whereas the GEP groups showed significantly different 5-year progression-free survival (76% vs 31% for GCB and activated DLBCL) and overall survival (80% vs 45%), none of the immunostaining algorithms was able to retain the prognostic impact of the groups (GCB vs non-GCB). In conclusion, stratification based on immunostaining algorithms should be used with caution in guiding therapy, even in clinical trials.

MYC status determination in aggressive B-cell lymphoma: the impact of FISH probe selection.

AIMS: To assess how hybridization probe design may affect MYC status determination in Burkitt lymphoma and diffuse large B-cell lymphoma. METHODS AND RESULTS: We compared the results obtained with one dual-fusion and two break-apart commercial probes in a retrospective series of 91 aggressive B-cell lymphomas. All three probes were able to detect the IGH-MYC translocation in every case bearing it (13/13). However, seven of 13 (54%) non-IGH-MYC (light-chain immunoglobulin or non-immunoglobulin-MYC) rearrangements were unambiguously detected by just one of the probes tested. On the other hand, when the IGH-MYC dual-fusion probe was used, nine of 15 (60%) cases with a hybridization pattern suggestive of a non-IGH-MYC translocation were attributable to MYC copy gain rather than MYC rearrangement, as demonstrated by both break-apart probes. CONCLUSIONS: Taking into account the prognostic and therapeutic implications of the MYC translocation, probe design and limitations should be particularly kept in mind when MYC hybridization patterns are interpreted. In our experience, detection of 8q24 abnormalities could be optimized by a two-probe approach involving the application of both IGH-MYC dual-fusion and MYC break-apart selected kits.

MYC protein expression and genetic alterations have prognostic impact in patients with diffuse large B-cell lymphoma treated with immunochemotherapy.

MYC alterations influence the survival of patients with diffuse large B-cell lymphoma. Most studies have focused on MYC translocations but there is little information regarding the impact of numerical alterations and protein expression. We analyzed the genetic alterations and protein expression of MYC, BCL2, BCL6, and MALT1 in 219 cases of diffuse large B-cell lymphoma. MYC rearrangement occurred as the sole abnormality (MYC single-hit) in 3% of cases, MYC and concurrent BCL2 and/or BCL6 rearrangements (MYC double/triple-hit) in 4%, MYC amplifications in 2% and MYC gains in 19%. MYC single-hit, MYC double/triple-hit and MYC amplifications, but not MYC gains or other gene rearrangements, were associated with unfavorable progression-free survival and overall survival. MYC protein expression, evaluated using computerized image analysis, captured the unfavorable prognosis of MYC translocations/amplifications and identified an additional subset of patients without gene alterations but with similar poor prognosis. Patients with tumors expressing both MYC/BCL2 had the worst prognosis, whereas those with double-negative tumors had the best outcome. High MYC expression was associated with shorter overall survival irrespectively of the International Prognostic Index and BCL2 expression. In conclusion, MYC protein expression identifies a subset of diffuse large B-cell lymphoma with very poor prognosis independently of gene alterations and other prognostic parameters.

Impact of Prolonged Ischemia on the Immunohistochemical Expression of Programmed Death Ligand 1 (PD-L1).

Antibodies targeting programmed death receptor 1 or programmed death ligand 1 (PD-L1) have become a standard of care to treat different cancers; for some of these tumors, there is a correlation between tissue expression of PD-L1 and response rates in patients. Although most of the analytical challenges in the evaluation of PD-L1 expression have been standardized, preanalytical issues have been less explored. The objective of this study was to evaluate the impact of time of ischemia on the performance of 2 commonly used antibodies against PD-L1. Sixteen tonsillectomy samples were kept in ischemia for <30 minutes from sample obtention (control) and 1, 3, 6, 12, and 24 hours at room temperature before formalin fixation and paraffin embedding. Selected areas were inserted into TMA paraffin recipient blocks stained with SP142 and SP263 antibodies and evaluated by 2 blind observers. The proportion of suboptimally stained samples was significantly higher for samples with cold ischemia times 6 hours or over ( P <0.0001). False-negative results were 25% in samples exposed to 6 hours of ischemia and raised to 34% for samples remaining in ischemia for 12 or 24 hours. When all observations were pooled, SP142 provided suboptimal results in 24% of observations and SP263 in 12.5%; this is a statistically significant difference ( P =0.042). In conclusion, the quality of staining for PD-L1 in tonsil samples varies with the time of cold ischemia. The SP142 antibody presented a significantly lower tolerance to prolonged cold ischemia than SP263. These results reveal the relevance of controlled preanalytical processing of samples.

MYC activation impairs cell-intrinsic IFNγ signaling and confers resistance to anti-PD1/PD-L1 therapy in lung cancer.

Elucidating the adaptive mechanisms that prevent host immune response in cancer will help predict efficacy of anti-programmed death-1 (PD1)/L1 therapies. Here, we study the cell-intrinsic response of lung cancer (LC) to interferon-γ (IFNγ), a cytokine that promotes immunoresponse and modulates programmed death-ligand 1 (PD-L1) levels. We report complete refractoriness to IFNγ in a subset of LCs as a result of JAK2 or IFNGR1 inactivation. A submaximal response affects another subset that shows constitutive low levels of IFNγ-stimulated genes (IγSGs) coupled with decreased H3K27ac (histone 3 acetylation at lysine 27) deposition and promoter hypermethylation and reduced IFN regulatory factor 1 (IRF1) recruitment to the DNA on IFNγ stimulation. Most of these are neuroendocrine small cell LCs (SCLCs) with oncogenic MYC/MYCL1/MYCN. The oncogenic activation of MYC in SCLC cells downregulates JAK2 and impairs IγSGs stimulation by IFNγ. MYC amplification tends to associate with a worse response to anti-PD1/L1 therapies. Hence alterations affecting the JAK/STAT pathway and MYC activation prevent stimulation by IFNγ and may predict anti-PD1/L1 efficacy in LC.

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.

Immunohistochemical detection of MYC protein correlates with MYC gene status in aggressive B cell lymphomas.

AIMS: MYC gene translocation entails a bad prognosis and a poor response to rituximab-cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) in diffuse large B cell lymphomas (DLBCL), and more intensive chemotherapy regimens could be more effective in those cases. Its evaluation requires cytogenetic or fluorescence in-situ hybridization (FISH) studies, which are expensive and not widely available. The aim of this work was to find an immunohistochemical marker able to be used as a screening tool to identify MYC translocations. METHODS AND RESULTS: Aggressive B cell lymphomas in which MYC status was assessed during their diagnostic work-up between 2007 and 2010 were collected, their immunophenotype was re-evaluated, and were stratified according to the Hans algorithm. Two tissue microarrays were built in order to evaluate MYC protein expression with a commercially available antibody. The study was performed on 56 specimens: nine Burkitt lymphomas (eight translocated), 45 DLBCLs (nine translocated) and two lymphomas with intermediate features (both translocated). Only MYC protein expression detected by immunohistochemistry correlated with MYC translocation. No relationship was seen between MYC gene copies and protein expression. CONCLUSIONS: MYC protein expression detected by immunohistochemistry using a commercially available antibody correlates with MYC gene translocation, and could be used as a screening tool to select those cases in which confirmatory genetic testing is mandatory.

High microvessel density determines a poor outcome in patients with diffuse large B-cell lymphoma treated with rituximab plus chemotherapy.

BACKGROUND: Diffuse large B-cell lymphoma is a clinically and molecularly heterogeneous disease. Gene expression profiling studies have shown that the tumor microenvironment affects survival and that the angiogenesis-related signature is prognostically unfavorable. The contribution of histopathological microvessel density to survival in diffuse large B-cell lymphomas treated with immunochemotherapy remains unknown. The purpose of this study is to assess the prognostic impact of histopathological microvessel density in two independent series of patients with diffuse large B-cell lymphoma treated with immunochemotherapy. DESIGN AND METHODS: One hundred and forty-seven patients from the Leukemia Lymphoma Molecular Profiling Project (training series) and 118 patients from the Catalan Lymphoma-Study group-GELCAB (validation cohort) were included in the study. Microvessels were immunostained with CD31 and quantified with a computerized image analysis system. The stromal scores previously defined in 110 Leukemia Lymphoma Molecular Profiling Project cases were used to analyze correlations with microvessel density data. RESULTS: Microvessel density significantly correlated with the stromal score (r=0.3209; P<0.001). Patients with high microvessel density showed significantly poorer overall survival than those with low microvessel density both in the training series (4-year OS 54% vs. 78%; P=0.004) and in the validation cohort (57% vs. 81%; P=0.006). In multivariate analysis, in both groups high microvessel density was a statistically significant unfavorable prognostic factor independent of international prognostic index [training series: international prognostic index (relative risk 2.7; P=0.003); microvessel density (relative risk 1.96; P=0.002); validation cohort: international prognostic index (relative risk 4.74; P<0.001); microvessel density (relative risk 2.4; P=0.016)]. CONCLUSIONS: These findings highlight the impact of angiogenesis in the outcome of patients with diffuse large B-cell lymphoma and the interest of evaluating antiangiogenic drugs in clinical trials.

The Immunohistochemical Expression of Programmed Death Ligand 1 (PD-L1) Is Affected by Sample Overfixation.

Humanized antibodies targeting programmed death receptor 1 (PD-1) or its ligand (PD-L1) have been approved for the treatment of different cancers. Some of these antibodies show a correlation between the tissue expression of PD-L1 and response. Evaluation of PD-L1 expression presents multiple challenges, but some preanalytical issues such as tissue fixation have been scarcely evaluated. With the hypothesis that immunohistochemical staining of PD-L1 may be impacted by the time of specimen fixation, we evaluated differences in its expression in tonsil samples exposed to predefined fixation times. Random nontumoral tonsillectomy specimens were blindly evaluated in tissue microarray slides after staining with SP142 and SP263 antibodies. With fixation times ranging from 12 to 72 hours, between 2.8% and 6.1% of the samples were considered to be suboptimally stained, with no differences between the 2 antibodies within these fixation times. A significantly higher proportion of samples exposed to a fixation time of 96 hours presented suboptimal immunostaining (15.6%, P<0.0001). In addition, suboptimally stained spots were 20.8% using SP142 and 10.4% using SP263 after 96 hours of fixation (P=0.046). In conclusion, the quality of staining for PD-L1 in tonsil samples decreased with overfixation of the specimen at times >72 hours. Samples exposed to formaldehyde for longer periods presented suboptimal results for both clones, but the SP142 antibody presented a significantly lower tolerance to formalin overexposure than SP263. These results indicate the relevance of a controlled preanalytical processing of samples and particularly the length of fixation of tumor specimens.

Kallikreins Stepwise Scoring Reveals Three Subtypes of Papillary Thyroid Cancer with Prognostic Implications.

BACKGROUND: Papillary thyroid cancer (PTC) is the most common type of thyroid cancer. Unlike most cancers, its incidence has dramatically increased in the last decades mainly due to increased diagnosis of indolent PTCs. Adequate risk stratification is crucial to avoid the over-treatment of low-risk patients, as well as the under-treatment of high-risk patients, but the currently available markers are still insufficient. Kallikreins (KLKs) are emergent biomarkers in cancer, but their involvement in PTC is unknown. METHODS: This study analyzed DNA methylation (HumanMethylation arrays) and gene expression (RNA-Seq) of KLKs, BRAF and RAS mutations, and clinical data from four published thyroid cancer data sets including normal and tumor tissues (n = 73, n = 475, n = 20, and n = 82) as discovery, training, and validation series. The C4.5 classification algorithm was used to generate a decision tree. Disease-free survival was estimated using Kaplan-Meier and Cox approaches. Specific analyses were performed using real-time polymerase chain reaction and immunohistochemistry. RESULTS: The entire KLK family was deregulated in PTC, displaying a specific epigenetic and transcriptional profile strongly associated with BRAFV600E or RAS mutations. Thus, a decision-tree algorithm was developed based on three KLKs with >80% sensitivity and >95% specificity, identifying BRAF- and RAS-mutated tumors. Notably, tumors lacking these mutations were classified as BRAF- or RAS-like. Most importantly, the KLK algorithm uncovered a novel PTC subtype showing favorable prognostic features. CONCLUSIONS: The KLK algorithm could lead to a new clinically applicable strategy with important implications for the risk stratification of PTC and the management of patients.

JC virus early protein detection by immunohistochemistry in progressive multifocal leukoencephalopathy: a comparative study with in situ hybridization and polymerase chain reaction.

In situ hybridization (ISH) for JC virus (JCV) is generally applied for the diagnosis of progressive multifocal leukoencephalopathy (PML). To explore the usefulness of immunohistochemistry (IHC) for JCV early proteins, 14 paraffin-embedded postmortem brain specimens with histologic features compatible with PML were tested for the presence of JCV by means of DNA-DNA ISH with a biotinylated probe corresponding to the entire JCV genome, for JCV early proteins IHC with both PAb 2003 and anti-SV40 large T antigen monoclonal antibodies, and polymerase chain reaction (PCR) amplification of JCV virion protein 3 (VP3) and transcriptional control region (TCR) sequences. ISH was positive in 13 cases and IHC in all 14 cases, the number of IHC-positive cells generally being far in excess of ISH-positive cells. Of the 2 monoclonal antibodies used, PAb 2003 proved to be more sensitive than anti-SV40 large T antigen. Occasional neuronal nuclei were positive for JCV early proteins in 5 cases. As for PCR, VP3 was amplified in all 14 cases and TCR in 9 cases. Consequently, PAb 2003 IHC for JCV early proteins seems to be a powerful tool for viral demonstration in PML and may well become the diagnostic recourse of choice in this setting.

Upregulation of amyloid precursor protein isoforms containing Kunitz protease inhibitor in dementia with Lewy bodies.

Amyloid precursor protein (APP) is involved in the accumulation of alpha-synuclein, the main component of Lewy bodies. It is currently unknown, however, whether any of the APP isoforms is instrumental in alpha-synuclein deposition in dementia with Lewy bodies (DLB). Using real-time RT-PCR, we have studied relative mRNA expression levels of APP isoforms in frozen postmortem frontal cortices of DLB patients, Alzheimer disease (AD) patients, and control subjects. Of the three main APP isoforms, the two with a Kunitz protease inhibitory (KPI) motif (APP770 and APP751) were found to be specifically overexpressed in the frontal cortices of DLB patients when compared with controls and AD patients. These findings suggest a specific role of APP isoforms containing Kunitz protease inhibitor in DLB pathogenesis.

Identification of a protective allele against Alzheimer disease in the APOE gene promoter.

Alzheimer disease (AD) risk is significantly influenced by the APOE2 and APOE4 alleles. In turn, the -491AT and TH1/E47cs polymorphisms alter APOE gene expression levels. To determine whether these two alleles exert any significant effect on AD development we have analysed the genotypes of the APOE promoter -491AT and Th1/E47cs polymorphisms in 163 AD patients and 155 controls divided into three age at onset/age dependent subgroups. Our study has detected a Th1/E47cs-T allele accumulation in healthy individuals over 75 years of age, which suggests it plays a protective role against AD. The Th1/E47cs-T allele may provide greater protection against AD than APOE2, although this awaits proof of Th1/E47cs-T allele overrepresentation in healthy individuals of other populations.

Methionine synthase polymorphism is a risk factor for Alzheimer disease.

Alzheimer disease (AD) patients show increased plasma levels of homocysteine, whose conversion to methionine is catalyzed by methionine synthase (MS). Although altered MS activity may result from the MS A2756G polymorphism, the latter's possible associ-ation with AD remains unexplored. To assess whether the MS A2756G polymorphism holds any influence on AD risk, we have analyzed 172 AD patients and 166 controls. We have also investigated whether the MS-A or MS-G allele interacts with the APOE4 allele. Our results indicate that association with the MS-AA genotype is an APOE4 allele-independent risk factor for AD. These findings provide novel evidence implicating genetic enzymatic alterations of homocysteine metabolic pathways in the pathogenesis of AD.

Cystathionine beta synthase as a risk factor for Alzheimer disease.

One of the known risk factors for developing Alzheimer disease (AD) is hyperhomocysteinemia. The latter may result from mutations of the genes coding for three key enzymes involved in homocysteine metabolism (methylenetetrahydrofolate reductase [MTHFR], methionine synthase [MS], and cystathionine beta-synthase [CBS]). Although MTHFR and MS polymorphisms have been shown to be positively associated with AD in some populations, the relationship of the CBS gene with AD remains undefined. In order to evaluate whether AD is associated with CBS gene changes leading to decreased CBS activity and homocysteine accumulation, we genotyped the CBS 844ins68 mutation and VNTR polymorphisms of the CBS gene in 206 AD patients and 186 age-matched controls. A slight increase in both 844ins68 mutation and VNTR allele 19 frequencies was detected in the whole AD patient group, compared with controls. The division of AD patients and controls into three age-at-onset/age dependent subgroups (<65 years, 65-74 years, > 75 years) revealed that the 844ins68 mutation and VNTR allele 19 are independent risk factors for AD development in subjects aged 75 years or more.

The Th1/E47cs-G apolipoprotein E (APOE) promoter allele is a risk factor for Alzheimer disease of very later onset.

Several polymorphisms in the apolipoprotein E (APOE) promoter region have been recently described. Of interest, APOE gene expression is increased in association with the -491AT polymorphism T-allele and decreased in relation to the Th1/E47cs polymorphism G-allele. In the present study we have investigated both polymorphisms in a series of 183 Alzheimer disease (AD) patients and 169 controls divided into age at onset/age dependent subgroups and the data obtained have been corrected for the presence of both expression-changing alleles in APOE homozygous individuals. Subsequently, the associations among APOE promoter polymorphisms, APOE4, and AD were assessed by chi-square and logistic regression analyses. Significantly, patients whose age at onset of AD was 80 years or more showed an association between the Th1/E47cs-G allele and AD that was independent of the APOE4 allele.

Parkin and synphilin-1 isoform expression changes in Lewy body diseases.

Alternative splicing gives rise to at least seven parkin and eight synphilin-1 isoforms. Since both parkin and synphilin-1 have been involved in Lewy body (LB) formation, we decided to explore whether their isoforms are differentially expressed in LB diseases. With this aim, we studied relative mRNA expression levels of parkin and synphilin-1 isoforms in the frontal cortices of patients with dementia with LBs, the LB variant of Alzheimer's disease and Parkinson's disease and compared the findings with those obtained from Alzheimer's disease patients and control individuals. Duplex real-time PCR reactions, with beta-actin as internal standard, were carried out in a LightCycler. mRNA expression levels of parkin and synphilin-1 isoforms were seen to be specifically altered in each of the LB diseases studied. These findings suggest that parkin and synphilin-1 isoform expression changes play a significant role in the pathogenesis of LB diseases.

Catalyzed signal amplification for cyclin D1 detection in mantle cell lymphoma.

Mantle cell lymphoma is characterized by a t(11;14)(q13;q32) translocation resulting in cyclin D1 protein overexpression. Immunohistochemical detection of the latter, therefore, is a useful marker for the diagnosis of mantle cell lymphoma. Nevertheless, interpretation of results is often hampered by the weak immunoreactivity obtained with routine detection techniques. This problem can be overcome by resorting to highly sensitive catalyzed signal amplification methods based on peroxidase-catalyzed deposition of a biotinylated phenolic compound. The present study compares the results obtained with catalyzed signal amplification, labeled streptavidin biotin, and dextran polymeric conjugate (EnVision+) techniques in cyclin D1 demonstration in mantle cell lymphoma. The study was performed on formalin-fixed, paraffin-embedded archival tissue from 20 mantle cell lymphoma cases. Ten cases of small lymphocytic lymphoma and 10 instances of follicular center cell lymphoma were used as controls. Antigen retrieval was done by autoclaving under controlled pressure (2 bar) and temperature (120 degrees C) conditions. The best results were obtained after 1 minute of exposure with catalyzed signal amplification and after 6 minutes with other detection systems. Regarding cyclin D1 expression in mantle cell lymphoma cases, 17 (85%) were weakly positive and 3 (15%), moderately positive with labeled streptavidin biotin, whereas 15 (75%) were weakly positive and 5 (25%) moderately positive with EnVision+. In contrast, all 20 mantle cell lymphoma cases were strongly cyclin D1 positive with catalyzed signal amplification. No evidence of cyclin D1 immunostaining was obtained in any of the small lymphocytic lymphoma and follicular center cell lymphoma instances with any of the three methods used. In conclusion, catalyzed signal amplification methods provide a very useful tool for cyclin D1 demonstration in cases in which other immunohistochemical techniques yield inconclusive results.