Glenn H. Segal

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. Patients 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%) with out 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.

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.

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.

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 Hodgkins 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.

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 K and Λ mRN As 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 β-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.

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.

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 an 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 mbrnegative B-cell neoplasms (BCN), 16 reactive lymphoid hyperplasias (RLH), 14 cases of Hodgkins 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 ≥s0.6 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 ∼105 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.