Evaluation of T cell receptor testing in lymphoid neoplasms: results of a multicenter study of 29 extracted DNA and paraffin-embedded samples.

To evaluate current diagnostic methods used for the evaluation of T cell receptor (TCR) gene rearrangements, 24 different laboratories analyzed 29 lymphoid neoplasm samples of extracted DNA and paraffin-embedded tissue and were asked to complete a technical questionnaire related to the testing. Participating laboratories performed Southern blot and polymerase chain reaction (PCR) testing for rearrangements of the TCRbeta chain gene and PCR for the TCRgamma chain gene rearrangements. Of 14 laboratories performing TCRbeta Southern blot analysis, there was complete agreement in 10 of 14 cases, with some false negative results obtained in 4 cases. No false positive results were obtained by Southern blot analysis. TCRbeta PCR analysis was only performed by two laboratories, and only 47.1% of positive samples were detected. Twenty-one laboratory results were obtained for TCRgamma PCR. This method showed an overall detection rate of 77.9% for T cell gene rearrangements with a 4.1% false positive rate, as compared to both TCRgamma Southern blot analysis results and immunophenotyping. The detection rate for TCRgamma PCR, however, significantly differed when extracted DNA samples from frozen tissue were compared to paraffin-embedded tissue (85.4% versus 65.9%; P = 0.0005). Significant differences in true positive results were obtained when laboratories using primers directed against multiple TCRgamma variable regions (V1-8 plus one to three other primer sets) were compared to laboratories that used only a single set of TCR primers directed against the V1-8 (P < 0.0001). Other technical factors significantly affecting results were also identified. These findings provide useful data on the current state of diagnostic TCR testing, highlight the risk of false negative results for TCR testing directed against only portions of the TCRgamma gene, and identify limitations of testing of paraffin-embedded tissues in some laboratories.

Quantitative real-time reverse transcription-PCR assay for cyclin D1 expression: utility in the diagnosis of mantle cell lymphoma.

BACKGROUND: The t(11;14)(q13;q32) translocation present in the majority of mantle cell lymphomas (MCLs) places the cyclin D1 gene under the control of immunoglobulin transcriptional regulatory elements, causing overexpression of cyclin D1. Quantification of cyclin D1 expression can distinguish MCL from other lymphomas. METHODS: A quantitative real-time reverse transcription (RT)-PCR assay was developed for cyclin D1 mRNA suitable for use with RNA extracted from fresh and formalin-fixed, paraffin-embedded tissues. Specimens were amplified in an Applied Biosystems Model 7700 Sequence Detection System in reactions containing primers and probes for cyclin D1 and a control gene, beta(2)-microglobulin. Relative expression of the two genes was standardized against a control MCL cell line, M02058. RESULTS: The range of cyclin D1 expression among 20 MCLs was substantially higher than that in other lymphomas and reactive lymph nodes. By choosing an optimal cutoff point for assessing overexpression, the sensitivity and specificity of the assay for the diagnosis of MCL in lymph node specimens both approached 100%: Overexpression was detected in 20 of 20 MCLs, but in none of 21 non-mantle-cell lymphomas or 10 reactive lymph nodes. CONCLUSIONS: Quantitative real-time RT-PCR for cyclin D1 overexpression provides a rapid diagnostic test with clinical utility in the diagnosis of MCL.

Malignant lymphoma of the thyroid gland: a clinicopathologic study of 108 cases.

We report a retrospective clinicopathologic study of 108 primary thyroid gland lymphomas (PTLs), classified using the REAL and proposed WHO classification schemes. The patients included 79 women and 29 men, with an average age of 64.3 years. All patients presented with a thyroid mass. The PTLs were classified as marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT) or MZBL (n = 30), diffuse large B-cell lymphoma (DLBCL) with MZBL (n = 36), DLBCL without MZBL (n = 41), and follicle center lymphoma (FCL; n = 1). Excluding the FCL, features of lymphomas of MALT-type were identified in all groups, despite a follicular architecture in 23% of cases. Lymphocytic thyroiditis (LT) was identified in 94%. Ninety-one percent of patients presented with stage IE or IIE disease, whereas 69% had perithyroidal soft tissue infiltration. All patients were treated with surgical excision followed by adjuvant therapy (76%): chemotherapy (15%), radiation (19%), or a combination of radiation and chemotherapy (42%). Disease-specific survival was 82% at last follow up (mean, 82.8 mos) and 79% at 5 years. Statistically, stages greater than IE, presence of DLBCL, rapid clinical growth, abundant apoptosis, presence of vascular invasion, high mitotic rate, and infiltration of the perithyroidal soft tissue were significantly associated with death with disease. No patients with MZBL or stage IE disease died with disease. In summary, PTLs typically occur in middle- to older-aged individuals as a thyroid mass, with a predilection for females. Despite their histologic heterogeneity and frequent simulation of other lymphoma subtypes, virtually all PTLs are lymphomas of MALT-type arising in the setting of LT. Mixed DLBCL and MZBL are common. Overall, PTLs have a favorable outcome with appropriate therapy, but prognosis depends on both clinical stage and histology. MZBL and stage IE tumors have an excellent prognosis, whereas tumors with a large cell component or DLBCL or stage greater than IE have the greatest potential for a poor outcome.

Nodular lymphoid hyperplasia of the lung: a clinicopathologic study of 14 cases.

Nodular lymphoid hyperplasia is a controversial entity in which its existence in the lung has been doubted. The current opinion is that most, if not all, such cases represent extranodal marginal zone B-cell lymphomas masquerading as reactive lesions. We found 14 cases of nodular lymphoid hyperplasia in the files of the Pulmonary Department at the Armed Forces Institute of Pathology from 1974 through 1998. All had clinical histories and hematoxylin-eosin slides. In 12 of 14 with paraffin blocks, we applied immunohistochemical antibodies for CD20, CD3, CD43, CD5, bcl-2, bcl-1, CD45RA, and kappa and lambda immunoglobulin light chains. Molecular genetic analysis was performed on paraffin sections in 10 of 14 by the polymerase chain reaction for rearrangements of the immunoglobulin heavy chain gene and the minor and major break-point regions of the chromosomal translocation t (14;18). There were eight women and six men ranging in age from 19 to 80 years (median, 65 yrs). Most lesions (71%) were incidental findings on routine chest x-rays. Most patients (64%) had a single lesion by chest x-ray whereas the remainder had two to three lesions, except for one patient who had "multiple" lesions. There was associated regional lymphadenopathy in five of 14 cases (36%) which, on biopsy, proved to be reactive follicular hyperplasia. The only treatment was surgical excision. Of the seven patients with follow-up information from 8 months to 6 years (mean, 30 mos), none had clinical recurrence and no patient died of disease. The histology and immunophenotype of the lesions were strikingly similar, including abundant reactive germinal centers, intense interfollicular polyclonal plasmacytosis, and a variable degree of interfollicular fibrosis. No case showed a molecular rearrangement of the immunoglobulin heavy chain gene or the minor or major break-point region of the t (14;18). We conclude that nodular lymphoid hyperplasia of the lung, although rare, does exist and deserves its place in the spectrum of reactive pulmonary lesions that ranges from follicular hyperplasia to diffuse hyperplasia of the bronchus-associated lymphoid tissue (lymphoid interstitial pneumonitis).

Re-evaluation of "reactive lymphoid hyperplasia of the uvea": an immunohistochemical and molecular analysis of 10 cases.

OBJECTIVE: Cases of uveal lymphoid proliferation previously classified as reactive lymphoid hyperplasia (RLH) were studied to correlate pathologic features with clinical outcome. DESIGN: Retrospective case series. PARTICIPANTS: Ten cases of RLH of the uvea on file at the Armed Forces Institute of Pathology with sufficient formalin-fixed, paraffin-embedded tissue for analysis. METHODS: Clinical, histologic, immunohistochemical, and molecular (polymerase chain reaction [PCR]) characteristics of uveal lymphoid proliferations were studied. MAIN OUTCOME MEASURES: Morphologic, immunohistochemical, and PCR characteristics of study cases. RESULTS: Patient age ranged from 40 to 73 years at time of enucleation, with a mean age of 55 years. Retinal detachment was noted clinically in nine patients and glaucoma in eight. All patients were treated with enucleation, and three received radiotherapy. Histologically, two cases were interpreted as RLH and eight were well-differentiated small-cell lymphoma (WDSCL). Systemic lymphoid infiltrate developed in two patients, but there were no deaths with a mean follow-up of 9.9 years. Mature lymphocytes were noted in the iris and angle structures; the atypical cells of uveal lymphoma were found in the choroid and ciliary body. Eight cases were monoclonal by B-cell and T-cell markers and/or immunoglobulin light chain restriction. Amplifiable DNA was present in 6 of 10 cases by PCR. Three cases monoclonal by cell markers were also monoclonal by PCR, but two cases monoclonal by cell markers could not be confirmed by PCR. Lymphoid follicles with germinal centers were found in two cases of RLH and five cases of WDSCL. Nine specimens demonstrated extraocular lymphoid involvement of the episclera and orbit; most appeared more benign morphologically than the choroidal infiltrates. Extraocular infiltrates of WDSCL were monoclonal by immunohistochemistry in five cases, polyclonal in one case, and indeterminate in two cases. CONCLUSIONS: Most cases (8 of 10) previously described as RLH were low-grade B-cell lymphomas histologically and by immunohistochemistry. PCR results agreed with histologic diagnosis in four of six cases. Open-angle glaucoma was common and related mostly to lymphocytic infiltration of the angle structures. Extraocular involvement is common but may not be representative of the choroidal lesion. Prognosis is excellent in low-grade uveal lymphoid neoplasia.

Enhanced sensitivity with a novel TCRgamma PCR assay for clonality studies in 569 formalin-fixed, paraffin-embedded (FFPE) cases.

BACKGROUND: Clonal rearrangement of genes encoding the immunoglobulins (Ig) and T-cell antigen receptors (TCR) are considered to be useful markers for the diagnosis of lymphoma and for determining the clonal origins of B- and T-cell populations in lymphoid neoplasms. METHODS AND RESULTS: Polymerase chain reaction-based clonality assays for TCRgamma, TCRbeta, and immunoglobulin (Ig) heavy chain (IgH) gene rearrangements were evaluated for diagnostic sensitivity and specificity in 569 formalin-fixed, paraffin-embedded (FFPE) tissues. Combined TCRbeta and TCRgamma assays enhanced the routine detection of TCR clonality to 90% of all peripheral T-cell lymphoma (PTCL) cases. IgH clonality was detected in 59% of 241 peripheral B-cell lymphoma (BCL) cases and 6% of 169 PTCL cases. Of 452 lymphomas, 5% could not be classified phenotypically as B or T lineage after immunohistochemical and clonality studies. Of all BCL cases analyzed, 24% had detectable TCRbeta and/or TCRgamma clonality. Of these BCL with biclonal results, 47% were extranodal lymphomas from skin and various tissues. CONCLUSIONS: Clonality assays were useful for distinguishing reactive or benign lymph nodes from neoplastic lymphoid infiltrates in most cases. The inclusion of TCRb and TCRg assays in the assessment of lymphomas results in a significant increase in the sensitivity of clonality detection, but is of limited utility in assessing the T- or B-cell phenotype of the tumor.

Differential expression of cyclin D1 in mantle cell lymphoma and other non-Hodgkin's lymphomas.

Mantle-cell lymphomas are associated with a characteristic chromosomal translocation, t(11;14)(q13;q32). This translocation involves rearrangement of the bcl-1 proto-oncogene from chromosome 11 to the immunoglobulin heavy chain gene on chromosome 14, resulting in an overexpression of cyclin D1 mRNA (also known as bcl-1 and PRAD1). In the current study performed on paraffin-embedded tissue, cyclin D1 mRNA could be detected in 23 of 24 mantle-cell lymphomas by reverse transcription polymerase chain reaction (RT-PCR) whereas only 9 of 24 demonstrated a t(11;14) by PCR. However, we also found that cyclin D1 mRNA could be detected in the majority (11 of 17, 65%) of non-mantle-cell lymphomas and in a minority of atypical lymphoid hyperplasias (3 of 7, 43%). Cyclin D1 mRNA expression was not observed in floridly reactive lymph nodes (0 of 9) or in unstimulated lymph nodes (0 of 20), suggesting that it is a sensitive adjunct marker for malignant lymphoproliferative processes, but not specific for mantle-cell lymphoma. A semiquantitative RT-PCR assay was developed that compared the ratio of cyclin D1 to the constitutively expressed gene beta2-microglobulin. Using this assay on a limited number of our specimens, cyclin D1 overexpression in mantle-cell lymphoma could be reliably distinguished from its expression in other non-Hodgkin's lymphomas. This assay for cyclin D1 expression, designed for formalin-fixed, paraffin-embedded tissue, was a very sensitive and specific marker for mantle-cell lymphoma.

Absence of human herpesvirus 8 DNA sequences in vascular tumors of the liver.

Kaposi's sarcoma-associated herpes virus (KSHV), also designated human herpesvirus 8 (HHV8), has been detected consistently in Kaposi's sarcoma, body cavity lymphoma and multicentric Castleman's disease, both in human immunodeficiency virus (HIV)-positive and -negative patients. Identification of KSHV/HHV8 DNA sequences in various benign and malignant vascular tumors in HIV-negative patients was reported in one study, but was not confirmed in several other studies. The vascular lesions, other than Kaposi's sarcoma, in which sequences could not be detected have included malignant vascular tumors of serous membranes, infantile capillary hemangiomas, and several benign and malignant vascular tumors of the spleen. We studied 30 primary benign and malignant vascular tumors of the liver; KSHV/HHV8 DNA sequences could not be detected in any. We conclude that this virus plays no role in the etiology of vascular tumors of the liver.

Initial genetic characterization of the 1918 "Spanish" influenza virus.

The "Spanish" influenza pandemic killed at least 20 million people in 1918-1919, making it the worst infectious pandemic in history. Understanding the origins of the 1918 virus and the basis for its exceptional virulence may aid in the prediction of future influenza pandemics. RNA from a victim of the 1918 pandemic was isolated from a formalin-fixed, paraffin-embedded, lung tissue sample. Nine fragments of viral RNA were sequenced from the coding regions of hemagglutinin, neuraminidase, nucleoprotein, matrix protein 1, and matrix protein 2. The sequences are consistent with a novel H1N1 influenza A virus that belongs to the subgroup of strains that infect humans and swine, not the avian subgroup.

Separation of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of soluble, aqueous polymers: Ficoll and polyvinylpyrrolidone.

In previous studies we have demonstrated that water soluble polymers of dextran and methylcellulose, when incorporated into sodium dodecyl sulfate (SDS)-polyacrylamide gels, can be used to sharpen the protein bands. We have extended these studies to enhance the separations in the mid-molecular weight range. Enhanced separation of protein molecular weight markers between 30,000-67,000 was best achieved in SDS-gels containing 2.5% polyvinylpyrrolidone (average molecular weight 10,000).

Polyacrylamide gel electrophoresis in vertical, inverse and double-crossing gradients of soluble polymers.

The presence of soluble dextrans, methylcellulose and polyethylene glycol polymers incorporated into vertical sodium dodecyl sulfate (SDS)-polyacrylamide slabs during electrophoresis can have a pronounced effect on protein separations. The effects of various standard and inverse gradients of polymers on the electrophoretic mobility of marker proteins in 10% T, 2.66% C Laemmli-style SDS gels, and the effects of simultaneous pore size and polymer gradients were investigated. These experiments demonstrate that the inclusion of polymers is a new, additional parameter that can be useful in resolving complex mixtures of proteins.

Mesothelial cells produce a chemoattractant for lung fibroblasts: role of fibronectin.

Pleural fibrosis may complicate several types of non-exudative pleural injury. Although the pathogenesis of such lesions is poorly understood, it is conceivable that mesothelial cells may recruit fibroblasts to sites of pleural damage. In order to test this possibility, conditioned medium from cultured rat mesothelial cells was tested for chemoattractant activity towards RL-87 rat lung fibroblasts. For this purpose, rat pleural or pericardial mesothelial cells were maintained in vitro for 6 to 96 h. Conditioned medium from each source was obtained at defined culture times and tested for chemotactic activity in a 48-well microchemotaxis assembly. A progressive, time-dependent increase in fibroblast chemoattractant activity was detected in both pleural and pericardial mesothelial cell conditioned medium samples. This effect was maximal in 96-h cultures. Checkerboard analysis revealed that the conditioned medium was truly chemotactic for lung fibroblasts. Characterization of the chemoattractant demonstrated that it was a nondialyzable (greater than 16 kD), thermolabile (100 degrees C for 15 min), acid-stable (pH 2.5), trypsin-sensitive, and pepsin-sensitive protein. The chemotaxin was shown to be fibronectin, since activity was abolished, in a dose-dependent manner, by treatment with anti-rat fibronectin antiserum as well as by passage through a gelatin agarose affinity column. This product consisted of two bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis of apparent molecular masses 250 and 220 kD. The secretion of a mesothelial cell-derived fibroblast chemoattractant may play a role in the response of the pleura to injury and in the pathogenesis of pleural fibrosis.

Gel electrophoresis in the presence of soluble, aqueous polymers: horizontal sodium dodecyl sulfate-polyacrylamide gels.

Because little is known about the use of aqueous polymers in polyacrylamide gel electrophoresis, we undertook a feasibility study that enables the discontinuous Laemmli-formulated system of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis to be performed in a horizontal format by the addition of large-sized aqueous polymers (i.e., dextrans and methylcelluloses). We studied four parameters: the cross-linking agent (bisacrylamide vs AcrylAide) and the polymer concentration, nature, and size. Three concentrations of each polymer were used. The best differentiation between the standard markers and the sharpest bands were obtained using concentrations of 2.5 and 0.06% (w/v) for Dextran T-500 and methylcellulose 4000, respectively. There was no predictable pattern to the variation in the plots of log Mr vs Rf caused by varying the concentration and length of the dextrans; however, the methylcellulose patterns suggest that gel viscosity is important. The results suggest that the combination of 0.06% methylcellulose 4000 polymers with bisacrylamide is a convenient and inexpensive means of performing flatbed Laemmli SDS-polyacrylamide gel electrophoresis.

Serologic demonstration of the albuminoid nature of the B700 murine melanoma antigen.

Limited available evidence indicates that the B700 murine melanoma antigen is related to serum albumin, but potential relationships to other members of the serum albumin protein family have not yet been established. Using specific antibodies raised against each of the members of the albumin family, we have studied cross-reactivity by solid phase enzyme-linked immunosorbent assay and Western immunoblotting. We demonstrate that B700 is serologically cross-reactive to members of the serum albumin family, which includes alpha-fetoprotein and vitamin D binding protein. Therefore, B700 is part of the serum albumin family of proteins, although the mechanism underlying its specific expression by transformed melanocytes remains unknown.

Homology of the B50 murine melanoma antigen to the Ro/SS-A antigen of human systemic lupus erythematosus and to calcium-binding proteins.

B50 is a murine melanoma-associated antigen found in tight association with B700, a melanoma-specific antigen. B700-like molecules are produced by all melanomas tested to date, including those of murine, human, swine and hamster origin. We have used rabbit antibodies to B50 to determine whether B50 expression is also restricted to melanomas. The results demonstrate that B50 is a commonly occurring protein, or is immunologically cross-reactive to a commonly occurring protein; 29 of 29 cell lines tested bound anti-B50 antibodies. N-terminal amino acid sequence analysis indicates that B50 has significant homology to the Ro/SS-A antigen of human systemic lupus erythematosus and to calcium binding proteins; hence B50 is likely to be an RNA and/or calcium-binding protein.

Absence of murine melanoma 5'-nucleotidase activity is not attributable to a detectable inhibitor.

Previous observations indicated the absence of demonstrable 5'-nucleotidase activity in six of seven cultured murine melanoma cell lines. It could not be determined from those studies whether the enzyme was absent, or whether an inhibitor was present. The current studies indicate that no inhibitor can be demonstrated, therefore the enzyme is absent.