Prevalence of non-O157:H7 shiga toxin-producing Escherichia coli in diarrheal stool samples from Nebraska.

We determined the prevalence of Shiga toxin-producing Escherichia coli (STEC) in diarrheal stool samples from Nebraska by three methods: cefixime-tellurite sorbitol MacConkey (CT- SMAC) culture, enterohemorrhagic E. coli (EHEC) enzyme immunoassay, and stx1,2 polymerase chain reaction (PCR). Fourteen (4.2%) of 335 specimens were positive by at least one method (CT-SMAC culture [6 of 14], EHEC enzyme immunoassay [13 of 14], stx1,2 PCR [14 of 14]). Six contained serogroup O157, while non-O157 were as prevalent as O157 serogroups.

Using fluorescence-based human androgen receptor gene assay to analyze the clonality of microdissected dendritic cell tumors.

The clonality of dendritic cell proliferations arising in patients with previously diagnosed B-cell non-Hodgkin lymphoma has not been determined. The highly polymorphic human androgen receptor gene (HUMARA) can be used to assess the pattern of X-chromosome inactivation and, hence, the clonality of tumors in female patients. In this study, specimens from 2 female patients with dendritic cell tumor following low-grade B-cell non-Hodgkin lymphoma were analyzed. Microdissection was performed on tissue sections to obtain representative tissues for analysis. The HUMARA polymerase chain reaction was modified to include a fluorochrome (6-carboxyfluorescein)-labeled primer so the product could be assessed with the ABI Genescan Analysis program for the Macintosh (Applied Biosystems, Foster City, Calif). Our results indicate that dendritic cell proliferations associated with low-grade B-cell non-Hodgkin lymphoma are clonal lesions. Previous microdissection is very helpful in obtaining the desired cell populations for study. The use of a fluorescent primer coupled with the Genescan System is a novel, highly sensitive, quantitative system that avoids the use of radioactive materials.

Molecular studies on single cells harvested by micromanipulation from archival tissue sections previously stained by immunohistochemistry or nonisotopic in situ hybridization.

Molecular analysis of isolated single cells is a powerful tool for analyzing heterogeneity within a population of cells and for clarifying issues of cell origin and clonality. Current techniques are limited by the availability of suitable fresh tissue. To broaden the applicability of molecular techniques at single-cell level, we have developed an approach that uses routinely processed archival tissue. Immunoglobulin heavy chain (IgH) gene rearrangement was analyzed in large tumor cells from four cases of diffuse large cell B-non-Hodgkin's lymphoma and in small reactive T and B lymphocytes from three cases of lymphocytic predominance Hodgkin's disease. One case of Epstein-Barr virus (EBV)-encoded RNA (EBER)-positive angiocentric pulmonary T-cell lymphoma was assayed for the presence of the BamHI-W multiple-copy fragment of the EBV genome. T- and B-lymphoid cells were immunostained with anti-CD3 and CD20, respectively. The tissue sections from the EBER-positive T-cell lymphoma were stained by nonisotopic in situ hybridization. Single cells were mobilized after proteolytic treatment under an inverted microscope using a hydraulic micromanipulator at a magnification of 400 x. Isolated cells were aspirated into a micropipette fixed to a second micromanipulator and transferred into a PCR tube. The IgH complementarity determining region (CDR)3 was successfully amplified in 17 of 52 (33%) small B-lymphocytes from lymphocytic predominance Hodgkin's disease using a previously reported semi-nested PCR method, and the products from each case differed in size as expected of a polyclonal population. None of the 49 small T lymphocytes demonstrated any amplifiable IgH CDR3 products, indicating no significant cellular contamination. The IgH CDR3 sequence analysis of the PCR products indicated a clonal relationship among harvested cells. In the T-cell lymphoma case, the harvested EBER-positive cells were amplifiable for the multiple-copy fragment BamHI-W of the EBV genome. Our study indicates that single-cell analysis can be performed on paraffin-embedded archival tissue after being subjected to immunoperoxidase and in situ hybridization procedures.

Clonality analysis of B-lymphoid proliferations using the polymerase chain reaction.

BACKGROUND: Polymerase chain reaction (PCR) based assays are becoming more reliable, simpler, and faster alternatives to traditional Southern blot hybridization (SBH) analysis for the detection of clonal immunoglobulin heavy chain (IgH) gene rearrangements. However, a variety of technical approaches have been reported with markedly different results. METHODS: We analyzed the frozen tissue of 147 neoplastic and hyperplastic lesions on which SBH had previously been performed. Semi-nested and single-step PCR methods were compared. Consensus primers to the joining segments and the framework region (FR) III of the variable segments of the IgH gene were used. All PCR products were analyzed by polyacrylamide gel electrophoresis (PAGE). Thirteen samples were re-analyzed using a denaturing gradient gel electrophoresis (DGGE) system. RESULTS: The overall concordance between SBH and semi-nested PCR assays was 80.2%. In the non-Hodgkin's lymphoma (NHL) group, 75% of the cases with IgH rearrangements by SBH were found to be monoclonal by PCR. Regardless of type of lesion, 71.7% of the cases with IgH rearrangements by SBH were found to be clonal by PCR. The concordance between the semi-nested and single-step procedures was 87.1%. DGGE was helpful in clarifying the results for cases in which the PAGE analysis was difficult to interpret. CONCLUSIONS: PCR analysis of IgH gene rearrangements was found to be an efficient technique for the initial determination of clonality in lymphoid proliferations. The single-step method had an advantage over the semi-nested method because of its simplicity and speed. The DGGE system was useful for the assessment of clonality in cases with equivocal results after PAGE. However, a combination of these techniques in specific cases may achieve higher specificity and sensitivity.

Lymphomatous polyposis. A neoplasm of either follicular mantle or germinal center cell origin.

Lymphomatous polyposis (LP) is generally thought to be an expression of non-Hodgkin's lymphoma (NHL) of follicular mantle cell (MC) origin. We report nine patients with LP from more than 3,500 cases of NHL studied by the Nebraska Lymphoma Study Group. Our patients differed from those reported previously in that LP represented a follicular center cell (FCC) NHL in two of the nine cases, with the remainder consisting of MC NHL. Three patients developed LP during a relapse of previously diagnosed and treated extraintestinal MC NHL (parotid gland, tonsil, and inguinal lymph node, respectively), whereas the other six patients presented with primary LP. In seven of the nine LP cases, a large mass predominated among a myriad of small polyps. The FCC cases were confined to the small intestine, whereas the MC cases were either pan-intestinal or colonic on their localization. Two MC cases studied by Southern blotting exhibited rearrangement of the bcl-1 locus. Bcl-2 rearrangement was not detected in any of the nine cases when studied by either a polymerase chain reaction-based assay (seven cases) or by Southern blotting (two cases). To date, four patients (three MC, one FCC) have experienced recurrent NHL in gastrointestinal sites. With follow-up ranging from 13 to 147 months, the entire group had a median survival of 41 months (primary MC LP:13, 13, 41, and 77 months; primary FCC LP:45 and 147 months; secondary MC LP:17, 41 and 76 months), and only one patient has died. We conclude that LP is a rare manifestation of NHL of either follicular MC or germinal center cell origin.

Clonal relationship between lymphocytic predominance Hodgkin's disease and concurrent or subsequent large-cell lymphoma of B lineage.

The occurrence of a large-cell lymphoma (LCL) concurrent with or subsequent to lymphocytic predominance Hodgkin's disease (LPHD) is well documented. Given the well-characterized B-cell nature of the Reed-Sternberg cell variants in LPHD, there may be a clonal relationship between the LPHD and the associated B-cell LCL. In this study, we adapted a highly sensitive, clonospecific assay to test whether the clone comprising the LCL exists in the corresponding LPHD tumor. Nine cases meeting the histologic criteria of nodular LPHD and B-cell LCL were identified, reviewed, and studied. Initially, clonality of both lesions was assessed using consensus primers to conserved regions in the IgH variable (frame-work III) and joining region genes in a polymerase chain reaction (PCR) assay. The PCR assay detected a clonal B-cell population in five of the LCLs, whereas analysis of eight cases of LPHD did not detect a dominant clone. Clonal products from the LCL were then sequenced, and clonospecific oligonucleotides were designed from the unique nucleotide sequence encoding the complementarity-determining region-III. These were then used as primers and/or probes in sensitive PCR-based assays on the corresponding LPHD tumors. In two cases, the clonospecific assay showed that the LPHD and LCL shared a common clone that was further confirmed by sequence analysis. This finding provides genotypic evidence that, at least in some cases, the LCL represents a clonal progression of LPHD.

Spontaneous complete regression of high grade non-Hodgkin's lymphoma. Morphologic, immunohistochemical, and gene amplification analyses.

BACKGROUND: Spontaneous regression of non-Hodgkin's lymphoma, occasionally reported in low grade groups, is a rare phenomenon in high grade groups. Clonal proliferation has not been confirmed in the majority of reported cases. In this woman, age 58 years, who had been diagnosed as having high grade immunoblastic lymphoma after excision of a single cervical lymph node, the remaining bilateral cervical, inguinal, and axillary adenopathy regressed completely without any cytotoxic treatments 22 days after biopsy. At the time of this writing, the patient has been free of disease for 24 months. METHODS: Clonality of the lymphoproliferation in the case was examined by immunohistochemistry and polymerase chain reaction (PCR) amplification using paraffin embedded biopsy material. Possible implications of Epstein-Barr virus in the pathogenesis of this process was examined also by PCR amplification and in situ hybridization. RESULTS: The proliferating lymphoid cells showed restricted expression of immunoglobulin (Ig) light chain and amplification of clonally rearranged V-D-J regions of Ig heavy chain gene. Epstein-Barr virus did not appear to be involved in the process. CONCLUSION: The present study shows that spontaneous complete regression of clonal lymphoproliferation that is morphologically a high grade lymphoma can occur.

Richter's syndrome with different immunoglobulin light chain types. Molecular and cytogenetic features indicate a common clonal origin.

To investigate the issue of clonality in Richter's syndrome, phenotypic, molecular genetic, and cytogenetic studies were performed on tumor tissue from a patient with concurrent chronic lymphocytic leukemia and diffuse large cell lymphoma in a single lymph node specimen. The tumor was biphenotypic for immunoglobulin (Ig) expression with surface Ig lambda-positive chronic lymphocytic leukemia and surface and cytoplasmic Ig kappa-positive diffuse large cell lymphoma. DNA samples prepared from areas of the lymph node rich in chronic lymphocytic leukemia cells and diffuse large cell lymphoma cells were examined in parallel. Identical Ig heavy chain gene rearrangements were detected in the BamHI and EcoRI digests of the two samples, but the patterns of rearrangement were different in the HindIII and PstI digests. Because it is very unlikely that multiple rearranged Ig heavy chain gene fragments of identical size would be found in more than one enzyme digest from two independently derived B-cell clones, it is probable that the two processes originated from a single clone. Modifications after rearrangement probably accounted for the differing band sizes seen in the HindIII and PstI digests. These conclusions are supported by cytogenetic analysis, which revealed two clones with a common primary abnormality (trisomy 12), one of which also exhibited secondary abnormalities. Therefore, Richter's syndrome may represent a composite tumor of common clonal origin, even when differences in light chain expression are identified.