In vivo detection of monosomy 3 in eyes with medium-sized uveal melanoma using transscleral fine needle aspiration biopsy.

PURPOSE: Cytogenetic prognostication of choroidal melanoma, particularly monosomy 3 detections, is limited to enucleated eyes or resected tumors. The authors developed an in vivo technique to detect monosomy 3 using transscleral fine needle aspiration biopsy (FNAB). METHODS: Eight eyes with medium-sized choroidal melanoma were included in this prospective study. A 25-gauge transscleral FNAB was performed during surgical procedure for brachytherapy, just before applying the radioactive plaque over the tumor base. Sampled material underwent fluorescence in situ hybridization (FISH) with centromeric probes for chromosome 3. Follow-up was >12 months. RESULTS: Transscleral FNAB yielded sufficient material in 7 of 8 eyes (87.5 %). Five of seven eyes had monosomy 3. No early or late complications were detected. CONCLUSIONS: This study demonstrates that medium choroidal melanomas may be safely sampled by intraoperative transscleral FNAB to detect monosomy 3 in vivo.

Tumor outgrowth in peripheral blood mononuclear cell-injected SCID mice is not associated with early Epstein-Barr virus reactivation.

Epstein-Barr virus (EBV)-positive B-cell lymphoproliferative disease develops in severe combined immunodeficient (SCID) mice inoculated with peripheral blood mononuclear cells (PBMC) from EBV(+) individuals (SCID/hu mice). In this study, we investigated the contribution of EBV reactivation and de novo infection of B lymphocytes to tumor outgrowth in SCID/hu mice. Evaluation of BZLF-1, an early EBV activation transcript, in cells recovered from the mouse peritoneal cavity within 16 days following PBMC transfer did not reveal EBV reactivation, while BZLF-1 expression was only detected in tumor masses or in vitro established lymphoblastoid cell lines. To confirm these data by a different strategy, we coinjected PBMC from seropositive donors with purified B cells from seronegative donors of different sex. Fluorescence in situ hydridization analysis of the resulting tumor masses disclosed that the overwhelming majority of lymphoma cells originated from the seropositive donor, implying that no substantial in vivo production and transmission of virus had occurred. Further, treatment of SCID/hu mice with ganciclovir did not prevent lymphoma development. Our results suggest that in the SCID/hu mouse, early EBV replication and secondary infection of bystander B cells does not occur, and that the direct outgrowth of the transformed B lymphocytes present within the PBMC inoculum is the predominant mechanism, which leads to lymphoma generation in this experimental model.

A subgroup of breast carcinomas is cytogenetically characterized by trisomy 12.

Chromosome analysis of short-term cultured breast carcinoma cells revealed trisomy 12 in four cases. In one tumor it was the only abnormality. The second case showed cytogenetic polyclonality, but the mainline had +12 as the sole change. In the remaining two tumors, trisomy 12 was part of more complex karyotypes. These findings, especially when coupled with similar information on previously published cases, show that gain of chromosome 12 is a recurrent and sometimes early event in breast carcinogenesis.

Cytogenetic heterogeneity in a second primary radiation-induced bladder carcinoma: ten karyotypically unrelated clones.

Cytogenetic analysis of a transitional cell carcinoma (TCC) of the bladder, the tumor having developed 32 years after the patient received pelvic irradiation and interstitial radium implantation for an endometrial carcinoma, revealed the presence of 10 cytogenetically abnormal, unrelated clones. Although the tumor was poorly differentiated, all clones were pseudo- or near-diploid with rather simple balanced or unbalanced structural rearrangements or both. The chromosomes involved in structural changes more than once were chromosomes 8, 9, and 11, which were rearranged in three clones, and chromosomes 3 and 17, both rearranged in two clones. No previous TCC of the bladder with cytogenetically unrelated clones has been reported, nor has any such radiation-induced tumor with chromosomal abnormalities been described. The distinct karyotypic and clonal pattern of the case presented here is probably indicative of a carcinogenic field effect due to the previous pelvic irradiation. Postradiation bladder carcinomas thus seem to be distinct cytogenetically in addition to their known unique etiological and clinical features.

The feasibility of statistical parametric mapping for the analysis of positron emission tomography studies using 11C-2-beta-carbomethoxy-3-beta-(4-fluorophenyl)-tropane in patients with movement disorders.

Movement disorders, including Parkinson's disease and parkinsonian syndromes, e.g. progressive supranuclear palsy, multiple system atrophy, and Lewy body dementia, may be difficult to differentiate among each other at an early stage, since they may share similar clinical features and response to dopaminergic drugs. As new tracers for imaging the dopamine transporters become available, the use of positron emission tomography (PET) for the differential diagnosis of movement disorders is gaining clinical relevance. Visual interpretation is generally used for PET image analysis. However, the use of some form of less subjective analysis is desirable in order to detect subtle changes that may be difficult to identify by visual interpretation and to achieve an operator independent analysis. To this end this study was aimed at assessing the feasibility of using statistical parametric mapping (SPM) for the clinical evaluation of single PET scans performed with 2-beta-carbomethoxy-3-beta-(4-fluorophenyl)-tropane ( C-beta-CIT-FE). Eleven healthy volunteers and five patients with movement disorders (Parkinson's disease, essential tremor, PSP and Lewy body dementia) were included in this study. Each subject underwent a PET study after i.v. injection of C-beta-CIT-FE. The PET images of C-beta-CIT-FE distribution acquired between 60 and 90 min were spatially fitted into the Talairach and Tournoux space. A template of normal C-beta-CIT-FE distribution was derived from studies in the 11 normal control subjects. Different patterns of reduction of the uptake of the tracer were detected in the basal ganglia of the five patients, in relation to each pathological condition. The patterns of distribution were all consistent with the severity and type of disease. The results of this study demonstrate the feasibility of differentiating among different states of dopaminergic impairment, due to Parkinson's disease and parkinsonian syndromes, by using PET scans with C-beta-CIT-FE and by using the SPM procedure for analysis of the data.

The lateral reservoir technique of ileal endorectal pull-through for ulcerative colitis and familial polyposis in children.

Total abdominal colectomy and endorectal ileal pull-through have been widely used in children as a definitive continence-preserving procedure for ulcerative colitis (UC) and familial polyposis (FP). Controversy exists in regards to the results achieved with the various techniques utilized to accomplish this procedure, including straight ileal pull-through, pull-through with balloon dilatation of the lower ileal segment, and the construction of a variety of ileal reservoirs: S-shaped, J-shaped, or lateral. We have utilized the two-stage lateral reservoir approach advocated by Fonkalsrud et al, on a total of 12 patients, aged 1 to 17 years, including ten patients with UC and two with FP. Twelve patients have undergone stage 1 and seven of these stage 2. Numerous complications of stage 1 occurred early in the series, including breakdown of the ileoanal anastomosis, 3 patients; cuff abscess, 2 patients; postoperative intestinal obstruction, 2 patients; and intraabdominal abscess, 1 patient. All have fully recovered. The last six stage 1 procedures have been without complications. Five of the seven stage 2 patients are now well, continent, and having four to six controlled bowel movements a day 1 to 2 years postoperatively. Two patients are using small amounts of Immodium and Metamucil. One patient has developed marked dilatation of the pouch at 8 months postoperatively with episodic "pouchitis" requiring operative revision and diversion. One stage 2 patient suffered numerous complications including spontaneous perforation of the reservoir 2 months postoperatively, eventually leading to re-creation of a permanent ileostomy and removal of the pouch. Five patients await further treatment. The lateral reservoir technique is capable of producing a satisfied and continent patient; however, the potential technical problems and early complications are significant. The occurrence of delayed "pouchitis" is worrisome. Although the initial results with the lateral reservoir technique appear encouraging, further longterm evaluation is necessary.

Recent experience with a modified Sawaguchi procedure for biliary atresia.

The treatment of biliary atresia by variations of the original Kasai hepatoportoenterostomy has shown early success with good bile flow and the elimination of jaundice in 50% to 70% of cases in many series. Long-term follow-up in many of these patients shows continued problems with ascending cholangitis and progressive liver disease leading to death. Our recent experience with a modified Sawaguchi hepatoportoenterostomy is encouraging. Twelve patients were operated on before two months of age. All but one became jaundice free within 2 to 4 months and had biliary intestinal continuity reestablished within 3 to 6 months. These 11 patients have remained jaundice free with normal growth and development 1 to 8 years postoperatively. Two patients had one and two episodes of cholangitis, respectively. All have continued mild elevations of hepatocellular enzymes but no patient has obvious signs of liver failure. Serial liver biopsies have shown clearing of bile stasis and continued periportal fibrosis. Size and number of ductules in the excised biliary remnant did not correlate with clinical outcome. One patient remained jaundiced after hepatoportoenterostomy and reoperation, and eventually expired. In contrast, two patients operated at 4 and 9 months of age never drained bile and eventually died of bleeding varices and hepatic failure, respectively. The atypical success and relative lack of cholangitis in this series is not readily explained, but may be related to specific technical modifications of the original Sawaguchi procedure.

Human papillomavirus infections of the genital region in human immunodeficiency virus seropositive women: integration of type 16 correlates with rapid progression.

The efficacy of treatment of HPV infections was evaluated in 14 HIV-1 seropositive women. Disease evolution was evaluated at follow-up examinations, and findings were compared to those in 14 historic HIV-negative women who had received similar treatment. HPV sequences were demonstrated by Southern blot in 13/14 HIV-positive women and by in situ hybridization in 5/13 HIV-negative women. Types 6/11, 16, and 31/33/35 were equally distributed in both groups; type 18 was never detected. In all but one case, the same viral type was present in different lesions, and in recurrences. Among the HIV-positive women, integration of viral sequences had occurred in the dysplastic lesions in 2 of the 3 HPV-16-positive cases; both cases showed progression to carcinoma in situ within 1 to 2 years. Despite comparable treatment, the HIV-positive women showed lesion persistence or progression, while all HIV-negative women experienced regression.

Temozolomide three weeks on and one week off as first line therapy for patients with recurrent or progressive low grade gliomas.

BACKGROUND: Patients with recurrent or progressive low grade gliomas survive for a decade or more following diagnosis, and may be at a higher risk for treatment-related complications, such as cognitive impairment from radiotherapy. PURPOSE: The aim of the present study was to determine in patients with progressive or recurrent low grade gliomas, the response rate and toxicity incurred by a continued schedule of temozolomide chemotherapy administered before radiation therapy, and to explore correlations between response and survival with 1p/19q deletions and MGMT promoter methylation status. METHODS: Progressive radio and chemotherapy naïve low grade glioma patients with O(6)-methyl-guanine-DNA-methyl-tranferase (MGMT) promoter status evaluation were considered eligible. Chemotherapy cycles consisted of temozolomide 75 mg/m(2)/daily for 21 days every 28 days for 12 cycles. RESULTS: A total of 30 patients (median age 45 [range: 24.2-68.6] years) with a median KPS of 90 (range 60-90) were accrued. The overall response rate was 30% (9 partial responses); 17 patients (56.7%) had disease stabilization. CONCLUSION: The prolonged temozolomide schedule considered in the present study is followed by a high response rate; toxicity is acceptable. Further randomized trials should therefore be conducted to confirm the efficacy of this regimen as first-line therapy in patients with progressive low grade glioma.

Atypical meningioma in Werner syndrome: a case report.

INTRODUCTION: Werner Syndrome, or adult progeria, is a rare autosomal recessive disorder caused by a mutation in the Werner Syndrome Gene belonging to the family of RecQ helicase. Malignant mesenchymal tumours and atherosclerosis are typical causes of death. Intracranial meningiomas are frequently described in these patients. CLINICAL PRESENTATION: We present the case of a 46-year-old man with Werner Syndrome and a convexity meningioma. The patient had a 2-year history of paresthesia and paresis in his right leg, which had worsened in recent months. He underwent surgery with Simpson grade II removal, with improvement of the slight paresis and no other neurological defects. The patient then underwent radiotherapy (60 Gy). Histological examination revealed an atypical meningioma. Cytogenetic analysis showed a hypodiploid clone with a complex karyotype characterized by monosomy 22 and deletion 1p. After 3 years' follow-up no relapses had occurred. CONCLUSION: 1p deletion correlates with meningioma progression and in this case correlates with histological examination. The chromosomal instability underlying Werner Syndrome could have fostered the complex karyotype.

Inhibition of wound contraction: comparison of full-thickness skin grafts, Biobrane, and aspartate membranes.

The mechanism by which open wounds contract is unknown. Although myofibroblasts are implicated in this natural process of wound healing, the evidence, however convincing, is only circumstantial. Control of wound contraction has been sought for many years, but only the application of full-thickness skin grafts is able to produce safe and effective clinical results. By comparing the synthetic skin substitute Biobrane and its component parts with full-thickness skin grafts in a rat wound model, we have demonstrated that dermis is not required for inhibition of wound contraction. We postulate that physical properties of skin and its synthetic analogues, including adherence to the wound surface, may be the signals to the open wound that effectively inhibit contraction. Whether myofibroblasts participate in this interaction remains unanswered.

Developmental study of the round window region.

A silicone impression method to study the anatomy of the round window region was used in 102 temporal bones belonging to individuals aged from 4 months of fetal life to 3-year-old children as well as adults. A total of 2,142 measurements of the round window, oval window and round window fossula was made in the molds. The data demonstrate that the round window shows a diameter from 1.21 mm (average for the short axis) to 1.74 mm (average for the long axis) and the circular shape was present in 55% of the adult temporal bones and in 18.18% of the fetal and infantile ones. The adult dimensions of the measured structures are reached during fetal development.

p53 gene alterations and protein accumulation in colorectal cancer.

Aim-To correlate immunohistochemical staining with single strand conformation polymorphism (SSCP) analysis of the p53 gene in colorectal cancer in order to understand how the findings provided by the two techniques complement each other in defining p53 functional status.Methods-Frozen tumour tissue from 94 patients with colorectal cancer was studied for p53 protein accumulation and gene mutations. Accumulation of p53 protein was detected by immunohistochemistry using PAb1801 and BP53-12-1 monoclonal antibodies. The findings were then compared with SSCP analysis of exons 5 to 8 of the p53 gene. All cases with a positive result by SSCP analysis were confirmed by sequencing.Results-Nuclear staining was observed in 51 (54.2%) cases. SSCP analysis of the DNA amplified by PCR revealed that the electrophoretic pattern had shifted in 30 cases; sequence analysis confirmed the occurrence of a mutation in 29 cases and of a polymorphism in one. In 27 cases both assays gave a positive result, and in 40 both were negative; therefore, concordance between PCR-SSCP and immunohistochemistry was seen in 72% of cases.Conclusion-The data indicate that positive immunostaining corresponds with the presence of a mutation in most, but not all, cases studied; other mechanisms could be responsible for stabilisation and accumulation of p53 protein in the nucleus. Nonsense mutations which do not confer stability on the protein will not be detected by immunohistochemistry and false negative results can also occur with SSCP analysis.

Molecular profile of Epstein-Barr virus in human immunodeficiency virus type 1-related lymphadenopathies and lymphomas.

Human immunodeficiency virus type 1 (HIV-1)-infected patients develop a spectrum of lymphoproliferative disorders ranging from nonneoplastic lymphadenopathies to B-cell lymphomas. Although evidence suggests that Epstein-Barr virus (EBV) might be involved, its molecular profile and expression pattern in HIV-1-related lymphoproliferations remain to be defined. Using polymerase chain reaction-based techniques, we studied EBV types and variants in 28 lymphadenopathy lesions and in 20 lymphomas (15 large cell and 5 Burkitt-like). EBV was detected in 89% of lymphadenopathies and in 80% of lymphomas; viral DNA content was significantly higher in the latter. EBNA2 and LMP1 gene analysis indicated that half of the EBV+ lymphadenopathies were coinfected with both EBV type 1 and 2 strains and/or multiple type 1 variants. Conversely, all but one lymphoma carried a single viral variant, consistently type 1 in large cell lymphomas, and type 2 in Burkitt-like tumors. Most lymphomas, but no lymphadenopathies, showed monoclonal Ig heavy-chain rearrangement. Analysis of 5 large cell lymphomas and 9 lymphadenopathies for EBV transcripts identified LMP1 mRNA in most samples, and the EBNA2 transcript in all tumors. These findings provide evidence of a heterogeneous EBV population in lymphadenopathy lesions, strengthen the notion that lymphomas arise from clonal expansion of EBV+ cells, and suggest different roles for EBV types 1 and 2 in HIV-1-related lymphoproliferations.

Co-existence of cutaneous T-cell lymphoma and B hairy cell leukemia.

A primary cutaneous form of peripheral T-cell lymphoma (PTCL) and a low grade B-cell non-Hodgkin's lymphoma that was classified as a variant of hairy cell leukemia (HCL) were simultaneously diagnosed in a 79-year-old woman by both phenotypic and genotypic analyses. The coexistence of a T- and B-cell lymphoma in the same patient is rare, and, to our knowledge, this particular association has not been previously described. The patient was referred to our Department for evaluation of multiple cutaneous itchy, reddish plaques; laboratory analyses disclosed a lymphocytosis, that presented 6 years earlier. A bone marrow aspirate showed a 50% B-cell interstitial infiltrate, while a skin biopsy surprisingly revealed a PTCL. Clonality of both neoplastic processes was assessed by Southern blot analysis. The indolent clinical course of the cutaneous disease, and the low and stable number of circulating neoplastic T cells supported the diagnosis of a mycosis fungoides (MF)-like PTCL. Possible oncogenic events and/or putative underlying viral infections which could have played a role in the occurrence of B- and T-cell non-Hodgkin's lymphomas in the same patient are discussed.

Genital human papillomavirus types in immunocompetent and immunodepressed women in northeast Italy: prevalence and cytomorphological correlations.

OBJECTIVE: We evaluated the prevalence of genital human papillomavirus (HPV) types in correlation with cytomorphological findings in patients at different risk for cervical intraepithelial neoplasia living in northeast Italy. METHODS: Exfoliated cervicovaginal cells from 943 women, who were divided into three groups, were analyzed by polymerase chain reaction. RESULTS: Overall, HPV prevalence rates were 7%, 38%, and 52%, respectively. The single most frequent type was HPV 16 (18%), followed by types 6, 31, 53, 58, 61, and novel/unidentified (5-7%); other types had a frequency <5%. Infection with multiple types was present in 12%. In HIV-infected women, HPV infection was correlated with lower CD4 level and higher viral load; HGSILs were correlated only with a lower CD4 count, and no correlations were found for LGSILs. CONCLUSIONS: HGSILs were associated with high-risk types, mainly HPV 16 (40%). LGSILs, instead, were associated with a broad spectrum of low-risk and high-risk types.

Identification of a human endogenous LTR-like sequence using HIV-1 LTR specific primers.

Using 'consensus' primers derived from the LTR region of 15 HIV-1 isolates, a fragment of 583 bp was amplified from human DNA. Even though specificity was confirmed by Southern blot analysis with a conserved LTR oligonucleotide probe, no significant homologies were detected to either retroviral regions or human or non-human published sequences. Nevertheless, when used as a probe, the 583-bp fragment identified a unique DNA sequence in the human genome on chromosome 1, and cross-reactive sequences in monkey, but not mouse, DNA. This novel, unique and conserved sequence of 583 bp was used to isolate a human HS-1 clone in which the structural property of a viral LTR could be identified.

Epstein-Barr virus-associated post-transplant lympho-proliferative disease of donor origin in liver transplant recipients.

BACKGROUND/AIMS: Post-transplant lymphoproliferative disease, a potential complication of solid organ transplantation, occurs in about 3% of orthotopic liver transplant recipients. We report the genetic and virological characterization of two cases of post-transplant lymphoproliferative disease that occurred early (4 and 6 months) after orthotopic liver transplant as large-cell non-Hodgkin's lymphomas located at the hepatic hilum. METHODS: Lymphomatous tissues were analyzed for clonality and presence of Epstein-Barr virus (EBV) sequences by Southern blot, polymerase chain reaction, and in situ hybridization techniques. RESULTS: The tumors in both cases were sustained by a clonal proliferation of B lymphocytes containing type A EBV DNA. Moreover, in situ hybridization with a digoxigenin-labeled EBV-specific probe evidenced a strong nuclear signal in most of the neoplastic cells. DNA microsatellite analysis at three different loci detected alleles of donor origin in both tumor samples, suggesting that the neoplastic B cells were of donor origin. CONCLUSIONS: EBV-infected donor B lymphocytes might be responsible for intragraft post-transplant lymphoproliferative disease in orthotopic liver transplant recipients. As 20 to 30% of post-transplant lymphomas involve the graft itself, donor-derived post-transplant lymphoproliferative disease might be more frequent than presently appreciated. Prospective studies are needed to assess its real incidence and identify possible risk factors.