Immunoglobulin heavy-chain gene rearrangement studies by Southern blot using DNA extracted from formalin-fixed, paraffin-embedded tissue.

BACKGROUND: In most clinical molecular diagnostics laboratories, Southern blots for gene rearrangement studies are not routinely performed on formalin-fixed, paraffin-embedded (FFPE) tissue samples. In this study, immunoglobulin heavy-chain gene rearrangements by Southern blot using DNA extracted from FFPE tissue samples were studied. METHODS AND RESULTS: Eleven paired freshly frozen and FFPE tissue samples were evaluated for immunoglobulin gene rearrangements by PCR and Southern blot analyses. An additional 14 selected samples sent to our laboratory to rule out lymphoma, for which only FFPE tissue (no frozen tissue) was available and for which PCR was interpreted as negative, were evaluated by the same techniques. Southern blots generated from DNA extracted from FFPE tissues were qualitatively identical to those generated from DNA extracted from fresh or freshly frozen tissue and correlated well with the final diagnoses. Ten interpretable Southern blots were generated in the 14 cases in which no frozen tissue was available. Four of these ten blots were interpreted as positive for an immunoglobulin gene rearrangement. Although the number of samples analyzed is small, success with Southern blotting correlated with increased sample size and sample width (1.17 vs 0.49 cm(2); P <.024; 0.71 vs 0.43 cm; P <. 049, respectively). CONCLUSION: DNA extracted from FFPE tissue samples using the simple, efficient, and nontoxic techniques described in this report can be used in many cases for Southern blotting for the detection of clonality by gene rearrangement studies.

Evidence for colorectal cancer micrometastases using reverse transcriptase-polymerase chain reaction analysis of MUC2 in lymph nodes.

Poor survival in patients following resection for early stage colorectal cancer is thought to be due in part to the presence of occult micrometastases at the time of surgery. The MUC2 mucin gene is highly expressed in the colon and associated colorectal tumors and may be a candidate marker for colorectal cancer micrometastases. We have used RT-PCR to detect expression of MUC2 mRNA transcripts in order to identify possible lymph node micrometastases in node negative (Stage I and II, or Dukes A and B) colorectal cancer patients. A total of 396 nodes (histologic stage N0) from 34 colon and nine rectal cancers were studied by RT-PCR analysis with nested primers for MUC2 (an average of 7.6 nodes per case). In the primary tumors, 42/43 (98.1%) were positive for MUC2 by RT-PCR. Evidence of the presence of MUC2 was demonstrated in nodes from 0 of 10 (0%) patients with Tis or T1, one of six (16.7%) from T2, 10 of 25 (40.0%) from T3, and one of two (50%) from T4 tumors. MUC2 RT-PCR was negative in six nodes from three patients with non-malignant colon disease and positive in histologically positive lymph nodes from six of six (100%) stage III colon cancers. In this study, using RT-PCR to detect the presence of MUC2 transcripts, we have found preliminary evidence for possible micrometastatic disease in approximately a third of histologically negative N0 colorectal cancer patients. The increased presence of MUC2 expression also correlated with more advanced T stage. We conclude that MUC2 RT-PCR may be a sensitive and specific marker for occult micrometastases. This technique has the potential to identify a group of colorectal cancer patients at risk for early cancer recurrence.

Peripheral nerve hemangioblastoma.

Capillary hemangioblastoma (HB) is a benign, highly vascular tumor limited almost exclusively to the central nervous system (CNS). It occurs primarily in the posterior fossa and less often in the spinal cord. We report three cases of HB occurring in peripheral nerve, two intradural tumors arising in a C4 and a cauda equina nerve root, respectively, and a third lesion in the sciatic nerve at mid thigh. The patients, 1 woman and 2 men, ranged in age from 25 to 49 years. Two had von Hippel-Lindau disease, an association usually found in one-third of CNS HBs, and one had a family history of pheochromocytoma. In every way, HBs of peripheral nerve were indistinguishable from their CNS counterpart. Ranging in size from 1.5 to 5.5 cm in diameter, the tumors were well circumscribed and contained a myriad of small caliber vessels lined by endothelial cells and surrounded by pericytes. Throughout, the lesions were rich in large, often vacuolated stromal cells. In all of the cases, these stained strongly for vimentin and neuron-specific enolase; only one showed focal S100 protein reactivity. Surgical therapy required excision of the affected nerve roots in the first two cases. In the third case, prominent extension of the tumor within epineurium permitted a microsurgical resection with sparing of sciatic nerve fascicles. No tumor recurred during a follow-up period of 5 to 20 months.

Flow cytometric DNA analysis with cytokeratin gating of formalin-fixed deparaffinized breast cancer nuclei.

The "gold standard" inflow cytometric DNA analysis of breast cancer uses fresh tumor cells simultaneously labeled for cytokeratin (CK) and DNA. We developed a 2-parameter CK-DNA flow assay suitable for archival, paraffin-embedded tissue (PT). Six anti-CK monoclonal antibodies were tested by immunocytochemistry and our assay for staining of nuclei extracted from PT breast cancers by combination pepsin-trypsin digestion. Clone CAM 5.2 was inadequate for PT nuclear suspensions, but a cocktail of 2 anti-CK clones (AE1/AE3 and KL-1) distinguished epithelial from nonepithelial nuclei in 2-parameter flow dot plots. We studied 82 routine PT breast tumors by our assay and used a univariate flow DNA histogram based on fresh biopsy tissue for comparison. Three histogram data quality indicators were improved. A trend toward higher S-phase fractions was found for DNA diploid PT tumors, although when inflammation was evident histologically, the increment in S-phase fraction with gating was often marked. CK gating identified PT tumors containing concurrent CK-positive DNA diploid and nondiploid populations (27 of 56 DNA nondiploid histograms). By excluding nonepithelial nuclei, 2-color CK-DNA flow methods may increase the accuracy of ploidy and S-phase fraction measurements. Our method appears superior to previous techniques using clone CAM 5.2 for labeling of archival breast cancers.

Experimental model of pudendal nerve innervation of a skeletal muscle neosphincter for faecal incontinence.

BACKGROUND: Faecal incontinence is difficult to treat. A variety of reconstructive procedures has been described, but none is entirely satisfactory. This study evaluated the feasibility of cross-innervating a skeletal muscle neosphincter with the pudendal nerve in a canine model. METHODS: Thirty dogs were rendered surgically incontinent (the pudendal nerve was cut and the external sphincter was partially excised). A neosphincter was then created using the semitendinosus muscle. In ten dogs pudendal nerve transposition (PNT) to the nerve to the semitendinosus muscle was performed. Ten dogs were given a dynamic neosphincter by inserting a pulse generator at 6 weeks. The remaining ten dogs served as controls with passive semitendinosus wraps. Anal manometry was performed before operation and monthly for 5 months. Muscle biopsies, performed at the initial operation and at 5 months, were stained for slow- and fast-twitch fibres, and were examined histologically. RESULTS: At 1 month, mean sphincter function was 32 per cent of the preoperative value in the control animals, 34 per cent in the PNT group and 27 per cent in the electrostimulation group; all dogs were incontinent. At 5 months the mean recovery of sphincter function was 42 per cent of the preoperative value in controls, 100 per cent in dogs with PNT (P < 0.001) and 63 per cent in dogs having electrostimulation (stimulator on) (P = 0.02). Six dogs with PNT had squeeze pressures equal to or greater than preoperative levels. At 5 months the ratio of slow to fast fibres was significantly greater in all dogs (control P = 0.01, PNT P < 0.005, electrostimulation P < 0.001). CONCLUSION: Use of the pudendal nerve to innervate a canine skeletal muscle anal wrap produced a functional anal sphincter that was superior to electrically stimulated and passive wraps.

Posttransplant T-cell lymphoproliferative disorders--an aggressive, late complication of solid-organ transplantation.

T-cell non-Hodgkin's lymphomas are an uncommon occurrence after solid-organ transplantation. We describe a morphologically and immunophenotypically distinct group of T-cell lymphoproliferative disorders that occurred late in the course of six patients with solid-organ transplants. The patients ranged in age from 31 to 56 years (median, 43). Three were male; all were splenectomized. The interval from transplant to the diagnosis of lymphoma ranged from 4 to 26 years (median, 15). Symptoms at presentation were related to sites of involvement. Pulmonary, marrow, and CNS involvement were present in five, four, and one case, respectively. No patient had lymphadenopathy. Five patients had an elevated lactate dehydrogenase level (range, 226 to 4,880 IU/L; median, 1,220 IU/L). Five of six patients had a leukoerythroblastic reaction. All cases had large-cell histology and frequently contained cytoplasmic granules. Those cases tested expressed CD2, CD3, and CD8 and were negative for B-cell antigens. T-cell receptor beta- and gamma-chain genes were clonally rearranged in three of three and one of three cases, respectively. All T-cell posttransplant lymphoproliferative disorders (T-PTLDs) studied were negative for Epstein-Barr virus (EBV), human T-cell leukemia/lymphoma virus type 1 (HTLV-1), human T-cell leukemia/lymphoma virus type 2 (HTLV-2), and human herpes virus type 8 (HHV-8) genomes. Treatment with acyclovir (three patients) or chemotherapy (three patients) resulted in two responses. All patients had an aggressive course, with a median survival duration of 5 weeks. In conclusion, a clinically aggressive T-PTLD may be a late complication of solid-organ transplantation and does not appear to be related to EBV, HTLV-1, HTLV-2, or HHV-8 infection.

Follicular large cell lymphoma of the testis in a child.

BACKGROUND: A unique case of primary testicular lymphoma in a child is reported. METHODS: Tumor tissue was studied using immunohistochemical techniques and southern blot hybridization to detect immunoglobulin and bcl-2 gene rearrangement and in situ hybridization for the Epstein-Barr virus (EBV) genome. RESULTS: Light microscopy revealed a lymphocytic infiltrate with a follicular pattern. Immunohistochemical staining revealed lambda light chain restriction and gene rearrangement studies revealed a clonal rearrangement of the immunoglobulin heavy chain, confirming a clonal neoplastic process. Immunostaining failed to detect bcl-2 protein expression, and no evidence of bcl-2 gene rearrangement was noted on southern blot analysis. In situ hybridization for EBV nucleic acid in tumor tissue was negative. CONCLUSIONS: To the authors' knowledge, this is the first report of a case of a primary testicular lymphoma with follicular histology in a child. Despite the follicular histology, no evidence of bcl-2 expression or gene rearrangement was detected.

Inbred guinea pig model of intrauterine infection with cytomegalovirus.

Outbred guinea pigs have previously been utilized in an experimental model for the study of congenital infection with cytomegalovirus (CMV). Development of an inbred model of intrauterine CMV infection would allow analysis of the cells involved in CMV immunity, studies of transplacental CMV transfer, and investigation of the cellular immune factors that participate in intrauterine CMV infections. This study was therefore designed to assess the inbred guinea pig as a model for the study of congenital CMV infection. Intrauterine fetal and placental infection with CMV was demonstrated in inbred Strain 2 guinea pigs, and the maternal factors influencing transplacental transmission of CMV were evaluated. Infectious virus was recovered from placentas and offspring of mothers that experienced primary CMV infection during pregnancy, but not from placentas and offspring of mothers that were inoculated with CMV prior to pregnancy. However, histologic lesions consisting of focal necrosis and inflammation were seen in tissues of offspring from both groups of mothers. Inoculation of seronegative pregnant Strain 2 animals with low doses of virus (2.5 to 3.5 log10 TCID50) resulted in both placental and fetal CMV infection without significant maternal death. Infection of placentas and offspring occurred in utero regardless of the stage of pregnancy. In addition, infectious virus was detectable in fetal tissues at the time of maternal viremia but also later during the course of maternal infection, ie, 4 weeks after inoculation. These findings indicate that the inbred guinea pig model can be used to investigate the pathogenesis of intrauterine CMV infections.

The placenta as a site of cytomegalovirus infection in guinea pigs.

The development of cytomegalovirus (CMV) infection in the placenta was studied in Hartley guinea pigs inoculated at midgestation, and its role in determining the outcome of fetal CMV infection was assessed. A hematogenous spread of CMV from the mother to the placenta occurred early during the course of the infection. However, the virus remained present in placental tissues long after CMV had been cleared from maternal blood (i.e., 3 and 4 weeks postinoculation). At that time, the virus was able to replicate in placental tissues in the presence of specific maternal antibodies. Viral nucleocapsids were seen within nuclei of trophoblastic cells, and virions were present surrounding infected cells. In addition, typical CMV-induced histopathological lesions bearing CMV antigens were consistently localized at the transitional zone between the capillarized labyrinth and the noncapillarized interlobium. Whenever CMV infection of the fetus occurred, virus was isolated from the associated placenta. Among placental-fetal units with CMV-infected placentas, only 27% of the fetuses were found to be infected. In addition, there was a delay in the establishment of the infection in the fetus in relation to the placenta, although frequencies of virus isolation in placental and fetal tissues peaked at 3 weeks after CMV inoculation. These results suggest that during primary CMV infection of pregnant guinea pigs, the placenta not only serves as a reservoir for CMV but also acts to limit transmission of the virus to the fetus.

Alprazolam overdose: clinical findings and serum concentrations in two cases.

Two patients who attempted suicide with alprazolam had markedly elevated serum concentrations but manifested only mild toxicity. Overdose with alprazolam appears much less likely to be life-threatening than overdose with the tricyclic antidepressants.

Nonepithelial neoplasms arising within vesical diverticula.

Two cases of rare vesical tumors, a paraganglioma and a malignant fibrous histiocytoma, are reported. The occurrence of these tumors within the urinary bladder is rare; their occurrence within bladder diverticula appears unique. The clinical, radiologic, and pathologic findings are summarized.

Quantification of alprazolam in serum or plasma by liquid chromatography.

A reversed-phase "high-performance" liquid-chromatographic assay for the quantification of alprazolam in serum or plasma is described. Serum or plasma is extracted with toluene/isoamyl alcohol (99/1 by vol), evaporated, and reconstituted in the mobile phase. The latter is washed with hexane, then subjected to reversed-phase liquid chromatography and ultraviolet detection at 202 nm. Either U-31485, an alprazolam analog, or lorazepam, a 3-hydroxybenzodiazepine, is satisfactory as internal standards. Major alprazolam metabolites and various other commonly used drugs do not interfere. The useful lower limit of sensitivity for quantification is 2.5 micrograms/L. Peak height and alprazolam concentration are linearly related from 2.5 to 100 micrograms/L. For 10 and 20 micrograms/L concentrations, within-run CVs were 1.4% and 0.9% and the between-runs CVs 4.8% and 3.2%. Steady-state serum concentrations ranged from 25 to 55 micrograms/L in patients taking 1.5 to 6.0 mg per day, orally. Preliminary data suggest the method is also suitable for analysis of the structurally similar triazolobenzodiazepine, triazolam.