Reactive Lymphoid Infiltrates Research Articles

Sensitivity of an immunoglobulin heavy chain gene polymerase chain reaction primer system for routine diagnosis of lymphomas

Aims Polymerase chain reaction (PCR) and length fragment analyses of the immunoglobulin heavy chain (IgH) gene are useful tools for clonality assessment in malignant Bcell-lymphomas and reactive lymphoid infiltrates. The present investigation analyzes the sensitivity of an IgH gene consensus primer multiplex PCR system for the detection of clonality for routine diagnosis in 109 lymphomas during a period of 3 years (2003–2005) at the Institut of Pathology Klinikum Darmstadt.

Assessment of T-cell Clonality via T-cell Receptor-γ Rearrangements in Cutaneous T-cell–Dominant Infiltrates Using Polymerase Chain Reaction and Single-stranded DNA Conformational Polymorphism Assay

Discerning the pathologic significance of cutaneous T-cell infiltrates can pose a diagnostic challenge for dermatopathologists. Reactive conditions such as drug-associated lymphomatoid hypersensitivity and lymphomatoid lupus erythematosus can demonstrate lymphoid atypia and a phenotype resembling cutaneous T-cell lymphoma (CTCL). Further, lymphoid dyscrasias such as pityriasis lichenoides chronica, large plaque parapsoriasis, and atypical pigmentary purpura confuse the picture because they not only mimic CTCL but also represent prelymphomatous states with inherent malignant potential. Although the emergence of a dominant clone has been considered a clue indicative of a T-cell dyscrasia, there are reports concerning the identification of monoclonality in biopsies of reactive lymphoid infiltrates. We have conducted a modified single-stranded DNA conformational polymorphism (SSCP) assay using paraffin-embedded, formalin-fixed tissue on 92 T-cell-rich biopsies to determine the relative specificity and sensitivity of this methodology. In addition, laser capture microdissection (LCM) was performed on 22 of the 92 samples to isolate the area of interest and to compare its specificity and sensitivity with those SSCP assays performed without LCM. We found that monoclonality or oligoclonality is 86% specific for preneoplastic and neoplastic states, whereas the finding of polyclonality appears to be relatively specific for a reactive process. Some cases of reversible T-cell dyscrasia produced a molecular profile mimicking lymphoma or prelymphomatous states by virtue of monoclonality or oligoclonality. Although LCM appears to improve the sensitivity for detecting preneoplastic conditions, the relative specificity appears to be the same as that encountered with routine SSCP.

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.

Immunohistochemical Detection of Cyclin D1 Using Optimized Conditions Is Highly Specific for Mantle Cell Lymphoma and Hairy Cell Leukemia

Mantle cell lymphoma (MCL) is more aggressive when compared with other lymphomas composed of small, mature B lymphocytes. Cyclin D1 is overexpressed in MCL as a result of the translocation t(11;14)(q13;q32). Cyclin D1 immunohistochemistry in fixed, paraffin-embedded tissue contributes to the precise and reproducible diagnosis of MCL without the requirement of fresh tissue. However, its use in bone marrow biopsies is not well established. In addition, increased levels of cyclin D1 mRNA have been found in hairy cell leukemia but have not consistently been detected by immunohistochemistry. We used a polyclonal antibody and heat-induced antigen retrieval conditions to evaluate 73 fixed, paraffin-embedded bone marrow, spleen, and lymph node specimens with small B-cell infiltrates, obtained from 55 patients. Cyclin D1 was overexpressed in 13/13 specimens of MCL (usually strong, diffuse reactivity in most tumor cells) and in 14/14 specimens of hairy cell leukemia (usually weak, in a subpopulation of tumor cells). No reactivity was detected in five cases of B-chronic lymphocytic leukemia; five cases of splenic marginal zone lymphoma; six cases of nodal marginal zone cell lymphoma; two cases of gastric marginal zone cell lymphoma; or ten benign lymphoid infiltrates in bone marrow, spleen, or lymph nodes. In summary, although the total number of studied cases is small and a larger series of cases may be required to confirm our data, we present optimized immunohistochemical conditions for cyclin D1 in fixed, paraffin-embedded tissue that can be useful in distinguishing MCL and hairy cell leukemia from other small B-cell neoplasms and reactive lymphoid infiltrates.

Clinical relevance of telomerase activity in primary gastric lymphoma.

BACKGROUND: To distinguish between low-grade lymphoma and reactive lymphoid infiltrate can be demanding for pathologists. Demonstration of B-cell monoclonality by polymerase chain reaction (PCR) analysis of immunoglobulin heavy chain is useful as referential data. Telomerase activity, which is frequently detected in malignant tumors while being undetectable in normal tissues, may also have a supportive role in the diagnostic procedure, but has not been investigated in specimens of primary gastric lymphoma. METHODS: Telomerase activity was qualitatively and quantitatively evaluated, using fluorescence-based telomeric amplification assay protocol (TRAP) analysis, in 16 malignant lymphoma and related specimens, including 7 specimens of low-grade mucosa-associated lymphoid tissue (MALT) lymphoma, among which specimens before and after Helicobacter pylori eradication were evaluated from five patients. RESULTS: Telomerase activity was detected more frequently (6 of 7 specimens) at higher levels in high-grade lymphoma compared with low-grade MALT lymphoma. It was detected in 3 of 7 specimens of low-grade MALT lymphoma, but was undetectable either at the stage of Helicobacter pylori-induced gastritis or after H. pylori eradication therapy. CONCLUSION: Telomerase activity evaluated by fluorescence-based TRAP analysis was detectable late in the process of evolution of MALT lymphoma and was undetectable early in the process of regression. It was frequently and overtly elevated in high-grade lymphoma. Telomerase activity may have a role in confirming the histologic diagnosis of primary gastric lymphoma.

Detection of Clonal T-Cell Receptor γ Gene Rearrangements in Paraffin-Embedded Tissue by Polymerase Chain Reaction and Nonradioactive Single-Strand Conformational Polymorphism Analysis

The diagnosis of T-cell lymphoproliferative disorders, which frequently involve the skin and other extranodal sites, is often problematic because of the difficulty in establishing clonality in paraffin-embedded tissue. To this end, we developed a simple, nonradioactive method to detect T-cell receptor gamma (TCR-gamma) gene rearrangements by polymerase chain reaction single-strand conformational polymorphism (PCR-SSCP) in paraffin-embedded tissue. Jurkat and HSB-2 cell lines and peripheral blood samples from normal individuals were used as monoclonal and polyclonal controls, respectively. DNA was extracted from 24 biopsies of T-cell lymphomas, 12 biopsies of reactive lymphoid infiltrates, and 2 biopsies of primary cutaneous large B-cell lymphomas. Vgamma1-8, Vgamma9, Vgamma10, Vgamma11, and Jgamma1/Jgamma2 consensus primers were used for TCR-gamma gene rearrangement amplification and PCR products were analyzed by nonradioactive SSCP. Monoclonal controls yielded a well-defined banded pattern, whereas all polyclonal T-cell controls showed a reproducible pattern of smears. We detected monoclonality in 20/21 (95%) T-cell lymphoma cases, whereas no dominant T-cell clones were found in any of the reactive lymphoid infiltrates or B-cell lymphomas. Sensitivity of 1-5% was demonstrated by serially diluting Jurkat cells in mononuclear blood cells from normal individuals. We conclude that nonradioactive PCR-SSCP for TCR-gamma gene rearrangement analysis is a useful adjunct to routine histological and immunophenotypic methods in the diagnosis of T-cell lymphoproliferative disorders in paraffin-embedded tissue.

B-cell lymphoma of MALT type: a review with special emphasis on diagnostic and management problems of low-grade gastric tumours.

B-cell lymphoma of MALT type: a review with special emphasis on diagnostic and management problems of low-grade gastric tumours.

Detection of clonality of cutaneous T-cell lymphomas using polymerase chain reaction

Differentiation of a cutaneous lymphoma from a reactive lymphoid infiltrate is a demanding challenge for the pathologist. In this retrospective study we examined 24 paraffin-embedded tissue samples from lesions diagnosed as lymphomas and 7 control samples of skin affected by benign changes and with pronounced lymphoid infiltrates for clonal rearrangement of the gamma T-cell-receptor. Using PCR technology we demonstrated clonality in 22 cases of lymphoma (92%). Thus, the primer combination used in this study covering the four main groups (I-IV) of the variable region of the gamma T-cell receptor gene allows high sensitivity. No clonality was demonstrable in any of the 7 control cases. This study demonstrates the growing importance of PCR technology for the diagnosis of lymphoma.

Is there a link between viral hepatitis and lymphoproliferative disorders? From the autopsy room to the PCR thermal cycler

Cases of chronic liver diseases associated with lymphoproliferative disorders (LPD) have been reported since 1872, the first cases being examples of liver cirrhosis and leukaemia. Although well known among German pathologists, this association was not documented in English language literature until the 1950s; with the advent of chemotherapy for malignant lymphoma and leukaemia, a number of publications reported liver fibrosis and cirrhosis in patients with leukaemia. Since most of the patients were children who had been treated with methotrexate, the liver findings were ascribed to treatment. The view that liver disease and lymphoproliferative disorders could occur together in the absence of treatment continued to be expressed occasionally in the literature by a number of workers, including two of the authors. Their suggested pathogenesis was that the same agent, which was responsible for the liver disease, could also induce the transformation of the reactive lymphoid infiltrate into a malignant lymphoma. Lately, and thanks to the use of modern techniques, there have been several reports of a high incidence of hepatitis B virus (HBV) and hepatitis C virus (HCV) in the serum of patients with LPD. Furthermore, HCV has been detected in peripheral mononuclear cells and even in the lymphomatous cells in patients with type II mixed cryoglobulinaemia. These findings support the hypothesis that under certain circumstances, HBV and HCV can induce LPD either directly or indirectly through B-cell immune dysregulation. This paper summarizes the steps which have led us to believe in a link between viral liver diseases and lymphoproliferative disorders.

Update on lymphoid interstitial pneumonitis

Lymphoid interstitial pneumonitis (LIP) involves a clinicopathologic pattern of pulmonary disease characterized by diffuse interstitial reactive lymphoid infiltrates. In adults, it occurs most commonly in autoimmune diseases, such as Sjögren's syndrome (0.9% of these patients) and primary biliary cirrhosis, whereas in children it is usually seen in HIV infection. Dysproteinemias (hyper- and hypogammaglobulinemia) are found in more than 60% of patients. Children can show CD8-lymphocytosis in bronchoalveolar lavage fluid, lung tissue, peripheral blood, and salivary gland, associated with HLA-DR5 haplotype. Radiographically, most patients with LIP have reticulonodular infiltrates, with or without patchy areas of consolidation. CT scans can show both small nodular and ground glass patterns, patterns that are diagnostically nonspecific. Reduced lung volumes and diffusing capacities are consistent and sensitive indicators of disease in LIP. In an experimental model, diffusing capacity was the single most sensitive functional index of disease progression. Microscopically, LIP is part of a spectrum of pulmonary lymphoid proliferations, ranging from follicular bronchitis-bronchiolitis and pulmonary lymphoid hyperplasia (the latter in AIDS patients), proliferations largely limited to airways, to low-grade malignant lymphoma. These patterns may be difficult to differentiate from each other. It appears that LIP sometimes evolves to lymphoma; the frequency of this evolution is probably low but is difficult to assess because low-grade lymphomas may mimic LIP. A relatively high frequency of LIP patients have Epstein-Barr virus DNA in their lungs but not all patients with LIP show this finding, suggesting other possible etiologies.

Lymphoepitheliomalike Carcinoma of the Gallbladder

We report a case of a lymphoepitheliomalike carcinoma arising in the gallbladder of a 60-year-old woman. The gallbladder wall was completely replaced with a tumor that extended into the adjacent liver parenchyma.The tumor was histologically and immunophenotypically indistinguishable from lymphoepithelioma of the nasopharynx, which is an undifferentiated malignant epithelial neoplasm that is histologically distinctive owing to a prominent reactive lymphoid infiltrate. It is believed that the Epstein-Barr virus may play a role in the pathogenesis of nasopharyngeal lymphoepitheliomas; however, our case in the gallbladder was not associated with of Epstein-Barr virus DNA. To the best of our knowledge, this is the first published case of a lymphoepitheliomalike carcinoma of the gallbladder. Int J Surg Pathol 4(3):00-00, 1997

Demonstration of S-100 protein distribution in human lymphoid tissues by the avidin-biotin complex immunostaining method

Immunoreactivity for S-100 protein was investigated immunohistochemically in a series of 49 fixed and paraffin-embedded normal, reactive, and neoplastic human lymphoid tissue specimens. The avidin-biotin complex immunoperoxidase method was used, with overnight (12-hour) incubation with a commercially available antiserum to S-100 protein. In addition, cryostat sections were tested with DRC 1 monoclonal antibody to dendritic reticulum cells (DRCs) in three cases and with OKT6 antibody to interdigitating reticulum cells (IRCs) in nine cases. All tissues, including lymph nodes, tonsils, adenoid, spleens, appendices, thymuses, and tissues containing nodular reactive lymphoid infiltrates, demonstrated a consistent immune staining pattern. A striking network composed of dendritic processes that showed finely granular S-100 protein immunoreactivity was observed in most of the follicular germinal centers; a similar dendritic pattern was observed in the follicular centers when the corresponding frozen sections were immunostained with DRC 1. In the extrafollicular areas, the S-100-positive cells topographically and morphologically resembled the IRCs that were demonstrated by OKT6 antibody in the corresponding frozen sections. The results seem to indicate that cells topographically and morphologically similar to IRCs and DRCs in human lymphoid tissues from different sites share immunoreactivity for S-100 protein. The present study confirms the unexpected presence of S-100 protein in dendritic cells of follicular germinal centers by a simple and currently available method.

Trophoblast-derived immunoreguratory factor

The diagnosis of T-cell lymphoproliferative disorders, which frequently involve the skin and other extranodal sites, is often problematic because of the difficulty in establishing clonality in paraffin-embedded tissue. To this end, we developed a simple, nonradioactive method to detect T-cell receptor γ (TCR-γ) gene rearrangements by polymerase chain reaction single-strand conformational polymorphism (PCR-SSCP) in paraffin-embedded tissue. Jurkat and HSB-2 cell lines and peripheral blood samples from normal individuals were used as monoclonal and polyclonal controls, respectively. DNA was extracted from 24 biopsies of T-cell lymphomas, 12 biopsies of reactive lymphoid infiltrates, and 2 biopsies of primary cutaneous large B-cell lymphomas. Vγ1–8, Vγ9, Vγ10, Vγ11, and Jγ1/Jγ2 consensus primers were used for TCR-γ gene rearrangement amplification and PCR products were analyzed by nonradioactive SSCP. Monoclonal controls yielded a well-defined banded pattern, whereas all polyclonal T-cell controls showed a reproducible pattern of smears. We detected monoclonality in 20/21 (95%) T-cell lymphoma cases, whereas no dominant T-cell clones were found in any of the reactive lymphoid infiltrates or B-cell lymphomas. Sensitivity of 1–5% was demonstrated by serially diluting Jurkat cells in mononuclear blood cells from normal individuals. We conclude that nonradioactive PCR-SSCP for TCR-γ gene rearrangement analysis is a useful adjunct to routine histological and immunophenotypic methods in the diagnosis of T-cell lymphoproliferative disorders in paraffin-embedded tissue.