Cystic endocervical tunnel clusters. A clinicopathologic study of 29 cases of so-called adenomatous hyperplasia.

The clinical and histologic features of cystic endocervical tunnel clusters (CETC) are not well known. Unwary pathologists have sometimes mistaken CETC for endocervical adenocarcinoma or interpreted them as "adenomatous hyperplasia." In this study, CETC were identified in 19 (5.9%) of 322 consecutive hysterectomy specimens and three (9.7%) of 31 consecutive cervical conization specimens accessioned during a 1-year period. These 22 cases were analyzed along with seven consultation cases. The 29 patients' ages ranged from 33 to 72 years (mean, 55). All but one (96.6%) were multigravida. Almost 80% of the patients had had at least three previous pregnancies. The mean gravidity and age of the hysterectomy patients with CETC were significantly greater than those without them. CETC typically were discovered incidentally during routine examination of the cervix. The clusters ranged from 0.5 to 18.8 mm (mean, 2.4 mm) in greatest dimension; they were multifocal in 82.8% of cases. CETC consisted of orderly, lobular aggregates of closely packed, dilated tubular endocervical "glands" within the superficial endocervix. The deepest clusters extended to a depth of 9.0 mm. They were commonly associated with multiple Nabothian cysts, which occasionally also penetrated deeply. The lining epithelium was a single layer of flattened or cuboidal endocervical cells. Mitotic figures and significant cytologic atypia were absent. None of the cases had intracytoplasmic CEA immunoreactivity, but in 52% focal positive CEA staining was noted along the luminal border of the endocervical cells. CETC are believed to result from subinvolution of previous episodes of physiologic hyperplasia of the endocervical mucosa, usually due to prior pregnancies. They are unrelated to cervical neoplasms and must be distinguished from adenocarcinoma and other glandular lesions of the endocervix.

Ovarian serous tumors of low malignant potential (serous borderline tumors). The relationship of exophytic surface tumor to peritoneal "implants".

A series of 98 ovarian serous tumors of low malignant potential (LMP) was studied to test the validity of the implantation theory of extraovarian peritoneal spread of tumor by assessing the association between exophytic tumor on the ovarian surface and synchronous peritoneal implants. Patient's ages ranged from 17 to 77 years (mean, 37.8 years). The ovarian tumors were bilateral in 39 cases (40%). Exophytic tumor was present in 47 (48%) cases and involved at least one ovary in 82% of bilateral tumors. Exophytic tumor was found in 29 of 31 patients (94%) with peritoneal implants, but in only 18 of 67 patients (27%) without peritoneal implants. Moreover, 29 of 47 patients (62%) with exophytic tumor had peritoneal implants compared with only 2 of 51 patients (4%) without exophytic tumor. The utility of exophytic tumor as a marker of synchronous peritoneal implants had a diagnostic sensitivity of 94%, a diagnostic specificity of 73%, and an efficiency of 80%. Because of the strongly positive correlation between exophytic tumor and peritoneal implants, the implantation theory remains as a highly likely explanation for extraovarian spread of ovarian serous LMP tumors. The multicentric "field effect" theory, however, cannot be entirely excluded and may be operative in some cases.

Reliable and cost-effective paraffin section immunohistology of lymphoproliferative disorders.

We studied 110 neoplastic and reactive lymphoid proliferations with three monoclonal antibodies--CD20 (L26), CD43 (Leu22), and CD45RO (UCHL1)--on B5-fixed, paraffin-embedded tissue to evaluate the utility of this panel as an immunotypic screen of such lesions. All cases were initially immunotyped by conventional methods. Genotyping by Southern blot hybridization was also done in 54 cases. Seventy-four of 79 malignant lymphomas and both of two hairy cell leukemias were of B-cell origin; and five lymphomas were defined as T-cell lineage. Lineage assignment was identical for paraffin section immunohistology and conventional immunotyping in 73 of 76 B cell and all of five T-cell tumors. CD20 was reactive with 73 of 76 B-cell tumors. CD43 was reactive with 12 of 74 B-cell lymphomas, and CD20/CD43 coexpression was seen in 11 of these cases. CD43 and CD45RO marked all of five and three of five T-cell lymphomas, respectively. Lineage assignment was identical for paraffin immunohistology and genotyping in 48 of 50 cases with identifiable gene rearrangements. Twenty-four nonneoplastic and five Hodgkin's disease cases that were studied also showed similar immunoreactivity patterns by both paraffin and conventional immunotypic methods. This panel of three monoclonal antibodies is an efficient, cost-effective approach for immunotyping most lymphoid proliferations in paraffin sections. Nevertheless, the pathologist should always try to obtain fresh or frozen tissue to aid in resolving occasional discrepant cases, to establish clonality in morphologically ambiguous ones, and to profile prognostically important phenotypic deletions.

Optimal primer selection for clonality assessment by polymerase chain reaction analysis: I. Low grade B-cell lymphoproliferative disorders of nonfollicular center cell type.

Recent polymerase chain reaction (PCR)-based studies focused on the detection of immunoglobulin heavy chain gene (IgH) rearrangements have suggested that clonal populations may be amplified more easily from certain categories of B-cell neoplasia than others and that primer makeup can be a critical factor in successful amplification. However, these particular reports contained relatively few low grade B-cell lymphoproliferative disorders of nonfollicular center cell type (LG-BLPD) and used only a limited panel of available primer sets for PCR amplification of monoclonal B-cell populations. To address this issue more extensively we evaluated 156 samples of LG-BLPD by the PCR to determine optimal primer selection in this setting. All cases were classified according to standard morphological and immunophenotypic criteria, with monoclonality documented by Ig light chain restriction analysis. The LG-BLPD included 33 cases of chronic lymphocytic leukemia (CLL), 57 cases of small lymphocytic lymphoma (SLL), 10 cases of atypical CLL, 32 cases of mantle cell lymphoma (MCL), 17 plasma cell neoplasms (PCNs), and seven cases of hairy cell leukemia (HCL). All primer sets included a 3' IgH joining region consensus primer, whereas the 5' IgH variable region (VH) primer was different in each set. The first-line panel included the following: Set 1, VH-framework III consensus primer, and Set 2, seven separate VH-framework I family-specific primers. A reserve panel of alternate VH consensus primers directed at framework II or III regions was used only when Set 1 showed no evidence of B-cell monoclonality.(ABSTRACT TRUNCATED AT 250 WORDS)

Optimal primer selection for clonality assessment by polymerase chain reaction analysis: II. Follicular lymphomas.

Prior studies have shown variable success in amplification of monoclonal B-cell populations from follicular lymphomas (FLs) of germinal center cell origin using the polymerase chain reaction (PCR). We examined 60 FLs by the PCR to determine optimal primer selection for detection of clonal immunoglobulin heavy chain gene (IgH) rearrangements in this common group of B-cell lymphomas. Each primer set included a 3' IgH joining region consensus primer; the 5' primer was different in each reaction. The first-line panel included the following: Set 1, bcl-2 major breakpoint region (mbr) primer; Set 2, bcl-2 minor cluster region (mcr) primer; Set 3, IgH variable region framework III consensus primer; and Set 4, seven separate IgH variable region framework I family-specific primers. A reserve panel also was used. The efficiency of monoclonal B-cell population amplification differed among primer sets. The bcl-2-targeted primer pairs (Sets 1 and 2) were most efficient and amplified 42 (70%) of 60 cases. Of these, the mbr and mcr primer sets detected monoclonality in 38 and four cases, respectively. Set 3 amplified monoclonal B-cell in 38 and four cases, respectively. Set 3 amplified monoclonal B-cell populations in 31 (52%) cases and Set 4 detected similar populations in only 27 (45%) samples. When results from primer Sets 1, 2, and 3 were combined, 49 of 60 (82%) FLs showed evidence of B-cell monoclonality by the PCR. Three of the 11 negative cases were documented as monoclonal with primer Set 4, and three additional samples were amplified only with our reserve panel.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid polymerase chain reaction-based detection of the causative agent of cat scratch disease (Bartonella henselae) in formalin-fixed, paraffin-embedded samples.

Bartonella (formerly Rochalimaea) henselae (Bh) plays a central role in cat scratch disease. A polymerase chain reaction (PCR)-based assay that can detect Bh DNA in formalin-fixed, paraffin-embedded (FF-PE) samples would have utility in the evaluation of processed lymph nodes suggestive of this disorder. Fresh or FF-PE cultures of Bh and related species were analyzed. Thirteen lymph nodes (12 FF-PE and one fresh cell suspension) with necrotizing suppurative granulomatous inflammation and seven FF-PE negative control lymph nodes were also evaluated. PCR was performed using a novel, hemi-nested protocol. Amplified products were analyzed by gel electrophoresis. The fresh and FF-PE Bh cultures showed a specific PCR product with an analytical sensitivity of 0.5 pg bacterial DNA. Seven (54%) of 13 clinical lymph node samples with morphological features suggestive of cat scratch disease also had detectable Bh DNA, whereas none of the seven negative control lymph nodes yielded positive results. We have designed a rapid and sensitive PCR test that can reliably detect Bh DNA in fresh and FF-PE samples. Our findings indicate that this assay has clinical utility in the diagnosis of cat scratch disease.

Optimal primer selection for clonality assessment by polymerase chain reaction analysis. III. Intermediate and high-grade B-cell neoplasms.

Previous studies have reported that low-grade B-cell lymphoproliferative disorders have variable B-cell monoclonality detection rates by polymerase chain reaction (PCR) analysis. For instance, monoclonal B-cell populations from chronic lymphocytic leukemia/small lymphocytic leukemia and mantle cell lymphoma are most often readily amplified with a single primer pair, whereas follicular lymphomas and plasma cell neoplasms require alternative strategies to approach these higher diagnostic sensitivity standards. Because most published reports have not focused on the variation in PCR B-cell monoclonality detection among subtypes of intermediate and high-grade B-cell neoplasms, additional information is necessary to determine primer selection strategies and identify problematic tumor subtypes within this group. The current investigation, the third part in a series, was aimed at documenting the efficiency of B-cell monoclonality detection by PCR in 71 aggressive B-cell neoplasms of various types using a comprehensive approach. A predetermined panel of primer sets was used in an algorithmic fashion. Specifically, all samples were analyzed with the standard VH-FRIII/JH assay previously shown to have the highest efficiency of monoclonality detection within low-grade B-cell lymphoproliferative disorders. Negative samples were further evaluated with primer sets in the following order until a positive result was observed, or all primer sets were used: (1) bcl-2/JH, (2) VH-FRI family specific/JH, and (3) VH-FRI consensus/JH. Forty-three (61%) of the 71 B-cell neoplasms evaluated with VH-FRIII/JH showed monoclonal B-cell populations. Sequential use of the three reserve primer sets in samples negative with this initial primer pair resulted in an overall improvement in PCR detection from 61% to 82% (58 of 71 specimens) (P < .001). The VH-FRI family specific assay identified B-cell monoclonality in 11 (73%) of these 15 specimens and was the most productive reserve primer set. Individual categories exhibited the following initial (I) and final (F) PCR detection rates: acute lymphoblastic leukemia/lymphoblastic lymphoma, 11 specimens (I = 91% to F = 91%); small noncleaved cell lymphoma, 14 specimens (I = 79% to F = 86% [P > .25]); diffuse large cell lymphoma, 33 specimens (I = 52% to F= 85% [P < .005]) and large cell, immunoblastic lymphoma, 13 specimens (I = 38% to F = 62% [P < .01]). The authors have shown that comprehensive PCR analysis is capable of detecting B-cell monoclonality in a significant proportion of samples from each subtype of intermediate and high-grade B-cell neoplasm. The VH-FRIII/JH assay was an adequate initial primer set, but required augmentation with the reserve PCR assays to attenuate the false negative rate and improve diagnostic sensitivity. The B-cell clonality PCR assay is optimally used as a screening tool and when used in this fashion, the more laborious and time-consuming restriction fragment-Southern blot hybridization (RF-SBH) method for IgH gene rearrangement detection may be limited to a relatively small proportion of PCR-negative aggressive B-cell neoplasms.

Detection of bcl-1 gene rearrangement and B-cell clonality in mantle cell lymphoma using formalin-fixed, paraffin-embedded tissues.

Mantle cell lymphoma (MCL) has recently emerged as a distinct clinicopathologic entity with characteristic molecular genetic features. Specifically, MCL are clonal B-cell neoplasms and often harbor bcl-1 gene rearrangements. Although this genetic profile is well documented, scant or no data are available on the molecular assessment of MCL using formalin-fixed, paraffin-embedded tissue as a sample source. The polymerase chain reaction (PCR) was employed to study bcl-1 and immunoglobulin heavy chain (IgH) gene rearrangements (B-cell clonality) using formalin-fixed tissue from 12 cases of MCL. In addition, 12 cases of low grade B-cell lymphoma and 5 cases of reactive lymphocytic hyperplasia were studied as comparison controls. A hemi-nested PCR assay was developed to identify major translocation cluster (MTC) bcl-1 gene rearrangements, whereas IgH gene rearrangements were evaluated by both a single-step and hemi-nested approach. Bcl-1 gene rearrangements were amplified in 4 of 12 (33%) MCL, but in none of the controls. With the hemi-nested approach, B-cell monoclonality was demonstrated in 11 of 12 (92%) MCL; 6 of 6 (100%) small lymphocytic lymphomas; 1 of 2 marginal zone lymphomas; 1 of 4 follicular lymphomas; and 0 of 5 reactive lymphocytic hyperplasias. When one-step PCR was used for B-cell clonality assessment, the overall detection rate was lower, specifically: 8 of 12 (67%) MCL; 4 of 6 (67%) small lymphocytic lymphomas; 1 of 2 marginal zone lymphomas; 0 of 4 follicular lymphomas; and 0 of 5 reactive lymphocytic hyperplasias were identified as monoclonal. We have demonstrated that MTC bcl-1 gene rearrangements can be amplified from formalin-fixed tissue. In addition, monoclonal B-cell populations from MCL are better amplified with a hemi-nested approach rather than a single-step PCR assay. With specialized nucleic acid isolation techniques and appropriate PCR protocol design, formalin-fixed, paraffin-embedded tissue is an adequate source of DNA for assessing MTC bcl-1 and IgH gene rearrangements.

The "CD43 only" phenotype. An aberrant, nonspecific immunophenotype requiring comprehensive analysis for lineage resolution.

Paraffin section immunohistology of leukocytic proliferations is a routine method of immunophenotyping in many clinical laboratories. Furthermore, a relatively standard antibody screening panel that includes L26 (CD20), Leu22 (CD43), and UCHL1 (CD45RO) appears to be widely used. Although paraffin section immunophenotyping in general, and this panel in particular, have been shown to be very reliable in defining B-cell lineage, characterization of T-cell lineage is less definitive. This is related primarily to the relatively poor specificity of the commercially available T-cell-associated reagents. CD43 (Leu22) in particular has a broad immunoreactivity profile that has not been stressed adequately in some reports. Seventeen cases with a "CD43 only" phenotype were identified during the last several years while using the relatively standard screening panel mentioned above. These cases were quite heterogeneous with respect to cellular differentiation and most were not T-cell proliferations. Specifically, eight cases were extramedullary leukemic infiltrates (five myeloid, two monocytic, one mixed lineage), four cases were T-cell lymphomas, three cases were B-cell lymphomas and two cases were plasmacytomas. Although CD43 has demonstrable utility in a leukocyte screening panel, this report stresses the aberrancy and lack of specificity of the "CD43 only" phenotype. Caution is recommended in assigning a specific lineage to such cellular proliferations without additional immunologic or genotypic analysis. Recommendations for comprehensive diagnostic evaluation of these proliferations are provided.

Frozen sections of cellular lymphoid proliferations provide adequate DNA for routine gene rearrangement analysis.

Optimal use of frozen tissue procured as part of a thorough diagnostic workup of suspected lymphoma is important, and conservation of similar samples is a prerequisite for maintaining a large and varied frozen archive repository. The authors have evaluated a simple tissue-conserving method for the preparation of cellular lymphoid specimens for immunoglobulin and T-cell receptor gene rearrangement analysis. Initially, 16-microns-thick frozen tonsil sections were examined to determine adequacy for DNA extraction. Specimens containing three, six, and nine sections each were evaluated separately. DNA quantitation disclosed yields ranging from 84 to 204 micrograms (mean, 156 micrograms). The authors have used this technique on 24 cellular lymphoid proliferations from their frozen archives. Six to ten 16-microns sections were used, depending on tissue size. DNA quantitation ranged from 0 to 520 micrograms (mean, 135 micrograms). Twenty-one of 24 cases yielded adequate DNA for analysis; each showed appropriate germline or rear-ranged bands with respect to the particular morphologic diagnosis. Attempts to obtain adequate DNA with the use of this technique on skin biopsy specimens with lymphoid infiltrates resulted in overall poor yields; this may be because of dermal collagen or small sample size. This method of sample preparation provides adequate DNA for routine Southern blot hybridization analysis of cellular lymphoid tissues and offers the additional advantage of allowing preservation of frozen tissue for future study.

T-cell lymphocytosis associated with invasive thymomas.

Peripheral T-cell lymphocytosis and bone marrow infiltration is a rarely associated feature of invasive thymomas. Hypotheses for the origin of the circulating lymphocytes include a spillover of tumor lymphocytes into the circulation, a neoplastic lymphoid proliferation, and a reactive proliferation of peripheral lymphocytes resulting from a thymoma-induced immunoregulatory disorder. The authors describe two cases of peripheral T-cell lymphocytosis associated with invasive thymomas with a predominantly lymphocyte-rich, cortical type histologic appearance. In both cases T cells constituted as much as 99% of the circulating lymphoid cell population, according to flow-cytometric analysis before and after treatment, and expressed a mature phenotype consistent with medullary thymic or peripheral T cells (CD2+, CD3+, CD4+ or CD8+, CD5+, CD7+) with predominant expression of the alpha/beta T-cell receptor heterodimer. Bone marrow biopsy specimens showed nodular and interstitial infiltrates of small T cells. No monoclonal rearrangement of the T-cell receptor beta gene was observed. Both cases had a normal female karyotype. Our findings confirm those of previous studies supporting a reactive origin for the peripheral lymphocytosis. Although an immunoregulatory disorder is probably involved in lymphocyte proliferation in situ, we postulate that peripheral lymphocytosis results from augmented release of the more mature thymoma-associated lymphocytes into the circulation through tumor invasiveness. A thymic origin is supported by the presence of a subset of peripheral T cells with low-intensity CD5 expression.

CD30 antigen expression in florid immunoblastic proliferations. A clinicopathologic study of 14 cases.

Reactive immunoblastic proliferations may be confused with certain types of non-Hodgkin's lymphoma and Hodgkin's disease on morphologic grounds. In addition, a characteristic antigen, CD30 (Ki-1; BerH2) on these neoplastic entities may be observed in morphologically atypical yet reactive florid immunoblastic proliferations such as those associated with acute infectious mononucleosis. Although it has been documented, a large series determining the frequency and extent of CD30 antigen expression on a variety of nonneoplastic immunoblastic proliferations is lacking. The authors studied 14 florid immunoblastic proliferations (9 in lymph nodes and 5 in tonsils) for CD30 antigen expression and for B- and T-cell paraffin markers. In situ hybridization to determine the presence of Epstein-Barr virus (EBV) genomes also was performed. Cases were classified into monospot-positive acute infectious mononucleosis (4 cases), EBV-related lymphoproliferative disorder suggestive of acute infectious mononucleosis (5 cases), and other etiologies (5 cases). CD30 Antigen expression was found on the immunoblasts in cases from all three categories and overall in 9 (64%) of 14 specimens. CD30 reactivity in the positive cases varied from occasional to numerous positive cells; 4 samples (3 EBV-related lymphoid proliferations and 1 vaccine-related lymphadenopathy) had numerous CD30-reactive immunoblasts. Expression of CD30 antigen on B or T cells and prominence of B or T cells within a proliferation were variable. Significant "atypia" of immunoblasts was found only in EBV-related disorders and correlated with B-cell prominence of the infiltrate. Appropriate clinical correlation and ancillary laboratory data are necessary to assist in differentiating these CD30(+)-reactive disorders from similar-appearing malignant lymphomas. Most important, a fresh tissue sample should be procured and adequately processed to allow for comprehensive determination of clonality and cellular lineage.

A rapid practical RT-PCR-based approach for the detection of the PML/RAR alpha fusion transcript in acute promyelocytic leukemia.

The t(15;17) and its molecular equivalent, PML/RAR alpha gene fusion, is strongly associated with acute promyelocytic leukemia (APL). Since treatment response to all-trans retinoic acid correlates directly with PML/RAR alpha, expeditious documentation is critical to patient care. We have designed an extremely rapid, practical, polymerase chain reaction (PCR)-based method using a rapid air thermal cycler to detect type A, B, and B-variant fusion patterns of PML/RAR alpha. We examined 15 cases of APL and 13 cases of leukemias other than APL with a nested reverse-transcription PCR assay. Three APL samples were type A, 11 were type B, and 1 was a B variant based on gel band patterns. PCR products exhibited positive probe hybridization signals and had sequences containing type A, B, or B-variant fusion patterns. PCR amplification of PML/RAR alpha was complete in 22 minutes, and the entire test required 4 1/2 hours. This method permits exceptional turnaround time and is an alternative to cytogenetics and slower PCR assays.

Semi-automated ELISA-based detection system for verifying the authenticity of amplified t(14;18)-containing products.

PCR product sizing on ethidium bromide-stained gels, coupled with Southern transfer and hybridization with nonisotopic probes, is an effective way of detecting t(14;18)(q32;q21). We evaluated an alternative ELISA-based test for detecting amplified t(14;18) products. Digoxigenin (DIG)-labeled dUTP is incorporated in a standard PCR method for amplification of bcl-2 major breakpoint region (mbr) rearrangements. The product is hybridized to a specific biotinylated DNA probe internal to the mbr primer, placed in streptavidin-coated wells of a microtiter plate, and detected with a alkaline phosphatase-conjugated anti-DIG antibody and enzyme substrate (pNpp). The colorimetric product is quantitated by an automated optical density (O.D.) reader. We evaluated 13 mbr-positive follicular lymphomas (FL), five mbr-negative B-cell neoplasms (BCN), 16 reactive lymphoid hyperplasias (RLH), 14 cases of Hodgkin's disease (HD), and normal peripheral blood samples from 20 healthy volunteers. All samples were evaluated in duplicate on separate plates. Positive [t(14;18)-containing cell line] and negative [cell line without t(14;18); master mix only] controls, and a standard curve were included with each run. Numerical O.D. readings from the specific hybridization assays revealed differences between FL and the other categories. All FL had an O.D. reading at > 2.0. The vast majority of RLH, HD, BCN, and normal peripheral blood samples showed O.D. readings well below 2.0. Specifically, 13/16 RLH and all HD, BCN, and normal peripheral blood samples had an O.D. of < or = 0.6 in all runs. The three outliers, which were all < 2.0, may represent the low level detection of t(14;18)-containing cells in RLH similar to previous reports. Moreover, all but four RLH had O.D. readings above the background negative controls, suggesting that rare t(14;18)-containing cells may have been present in these samples, as well. Dilution studies estimate that this assay is capable of detecting 1 t(14;18)-containing cell in approximately 10(5) cells, a greater level of sensitivity than can be obtained with gel visualization alone. We conclude that this semi-automated, potentially quantifiable ELISA-based system is a useful, objective and reproducible alternative hybridization procedure for verifying PCR product specificity in this setting.

Primers frequently used for detecting the t(14;18) major breakpoint also amplify Epstein-Barr viral DNA.

We observed a potentially misinterpretable polymerase chain reaction (PCR) amplification product generated with standard primers used to detect the major breakpoint region (mbr) of chromosomal translocation t(14;18). This unexpected phenomenon was initially detected during attempts to transform follicular lymphomas in vitro with Epstein-Barr virus (EBV). Additional studies were performed using the EBV-producing cell line MCUV5, cell lines from EBV-transformed normal B-lymphocytes, and an excised lymph node from a patient with documented EBV-associated infectious mononucleosis. These samples consistently produced a 167-base pair product, which was indistinguishable from a t(14;18) lymphoma product when viewed on ethidium bromide-stained gels. Through DNA sequencing and gene bank analysis, the product was identified as a portion of the EBV genome. A mbr-specific 20-base oligonucleotide probe was able to discriminate between true translocations and the EBV-related amplifications. These results underscore the importance of employing a specific detection system, and comprehensively screening primers when working with PCR.

In situ hybridization analysis of lymphoproliferative disorders. Assessment of clonality by immunoglobulin light-chain messenger RNA expression.

Determination of clonality in B-cell lymphomas is a useful diagnostic adjunct. In situ hybridization (ISH) for the detection of kappa and lambda mRNAs has the potential to overcome some common specimen-related limitations in clonal assessment. Tritium-labeled antisense cRNA probes directed at conserved segments of the constant regions of the kappa and lambda mRNAs were used in an autoradiographic method to detect B-cell clonality. Using these probes, we analyzed 103 formalin-fixed, paraffin-embedded biopsy samples, and the results were subsequently compared to available immunophenotypic (all cases) and genotypic (50 cases) data. Of 103 samples, 82 (80%) had adequate RNA preservation as determined by actin RNA signals, and 73 (89%) of the 82 cases demonstrated concordant clonality assignment by both ISH and immunophenotyping. The remaining nine cases showed a specific form of discordance in that each exhibited no protein (Ig) expression but had evidence of mRNA immunoglobulin light-chain expression. Forty-five (90%) of 50 cases evaluated for immunoglobulin and T-cell receptor beta-gene rearrangements demonstrated concordant results with respect to clonality assignment by ISH. Thus, ISH demonstrates adequate sensitivity with respect to traditional methods of clonality assessment. However, its practical utility awaits the development of nonradioactive detection methods with adequate sensitivity to improve turnaround time.

Cystic medionecrosis of the coronary arteries and fatal coronary vasospasm.

The histopathological basis of coronary vasospasm is not well defined. We report a patient with directly observed coronary artery spasm in whom cystic medionecrosis of the coronary arteries and great vessels and myxomatous degeneration of the mitral valve were evident. We suggest that myxoid connective tissue lesions of the heart may be linked to coronary vasospasm.

Mantle cell lymphoma. Rapid polymerase chain reaction-based genotyping of a morphologically heterogeneous entity.

BACKGROUND: Mantle cell lymphoma is heterogeneous at the morphologic level. Since this B-cell lymphoma may be confused with other entities, ancillary molecular testing may be necessary for definitive diagnosis. A polymerase chain reaction-based method, which is less complicated and more rapid than that generally available for the detection of immunoglobulin heavy chain (IgH) and bcl-1 gene rearrangements, would be helpful in this process. METHODS: Thirty-one mantle cell lymphoma samples (frozen or ethanol-preserved) from 29 patients were studied with two separate polymerase chain reaction assays using an air thermocycler and a low-volume, capillary-tube format for rapid DNA amplification. The reverse primer, JH, was common to both assays. The forward primers were directed to the IgH framework III variable region (VH-FRIII) and the bcl-1 gene major translocation cluster. Agarose gels were used to evaluate amplicon. Additional product verification was also performed. RESULTS: Immunoglobulin heavy chain and major translocation cluster bcl-1 gene rearrangements were detected in all 29 (100%) and in 12 (41%) of 29 mantle cell lymphoma samples, respectively. Each VH-FRIII/JH assay required 26 minutes to complete, whereas the major translocation cluster bcl-1/JH reaction required only 21 minutes. The seemingly low yield of bcl-1 gene rearrangements is not unexpected since this assay only detects major translocation cluster breakpoints. CONCLUSIONS: Presented is an extremely rapid, nonisotopic polymerase chain reaction-based method that detects IgH and major translocation cluster bcl-1 gene rearrangements in mantle cell lymphoma. Each polymerase chain reaction amplification was complete in 26 minutes or less, required only a 10-microL reaction volume, and exhibited adequate and specific product yield. This approach permits superior turnaround time and is thus advantageous in the clinical setting.

Standard polymerase chain reaction analysis does not detect t(14;18) in reactive lymphoid hyperplasia.

There are conflicting data regarding the detection of t(14;18) in reactive lymphoid hyperplasia (RLH) by the polymerase chain reaction (PCR). Although most studies have not detected t(14;18), several groups have definitively shown that a very low number of cells with this translocation (one in 10(5) to 10(6)) are present in a significant proportion of follicular hyperplasias. Review of the methods from these series reveals that modifications of the PCR assay (ie, enhanced sensitivity steps such as seminesting, lengthy autoradiographic exposure times, multiple aliquot reactions of single samples, and/or high concentrations of template DNA) are probably necessary to detect t(14;18) in RLH. We evaluated a diverse set of 111 RLH (85 lymph nodes, 22 tonsils, and four other sites) from patients of different age groups (age range, 9 months to 80 years) to determine if a standard PCR assay would amplify t(14;18). Of these, 61 (55%) specimens had a prominent follicular hyperplastic component. Fifty-seven follicular lymphomas served as a control group. Polymerase chain reaction was performed as a single-run, two-primer-based assay for major breakpoint region bcl-2 translocations (5' major breakpoint region primer and 3' immunoglobulin heavy-chain gene-joining region consensus primer). Two different types of thermocyclers were employed. A metal block thermocycler was used with 35 cycles of amplification on 500 ng to 1 micrograms of genomic DNA, and a separate air thermocycler was used with 45 cycles of amplification on 50 ng of genomic DNA. Product detection was carried out through ethidium bromide staining and UV gel illumination, along with a digoxigenin-alkaline phosphatase-based, internal major breakpoint region oligonucleotide probe system. We found no amplified t(14;18) products in any RLH. In contrast, 36 (63%) of 57 follicular lymphomas showed t(14;18) (published range for detection of major breakpoint region translocations by PCR, 31% to 74%). Moreover, the assay's sensitivity, estimated through dilution studies, was to one in 10(4) to 10(5) cells. Although theoretically possible, our data suggest that there is practically no risk of amplifying a t(14;18) from RLH when utilizing a standard PCR assay.

Angiotropic large-cell lymphoma in a patient with adrenal insufficiency.

Angiotropic large-cell lymphoma is a rare disorder characterized by a proliferation of malignant lymphoid cells within the lumina of small vessels. The skin and central nervous system are typically affected; however, involvement of other organs has been described. We document an unusual case of this disorder in a patient who suffered clinically significant adrenal insufficiency and subsequently died. Autopsy disclosed angiotropic large-cell lymphoma involving both adrenal glands.