Search

Splenic hematopoiesis is a major feature in the course of myelofibrosis (MF). In fact, the spleen of patients with MF contains malignant hematopoietic stem cells retaining a complete differentiation program, suggesting both a pivotal role of the spleen in maintaining the disease and a tight regulation of hematopoiesis by the splenic microenvironment, in particular by mesenchymal stromal cells (MSCs). Little is known about splenic MSCs (Sp-MSCs), both in normal and in pathological context. In this work, we have in vitro expanded and characterized Sp-MSCs from 25 patients with MF and 13 healthy subjects (HS). They shared similar phenotype, growth kinetics, and differentiation capacity. However, MF Sp-MSCs expressed significant lower levels of nestin, and favored megakaryocyte (Mk) differentiation in vitro at a larger extent than their normal counterpart. Moreover, they showed a significant upregulation of matrix metalloprotease 2 (MMP2) and fibronectin 1 (FN1) genes both at mRNA expression and at protein level, and, finally, developed genetic abnormalities which were never detected in HS-derived Sp-MSCs. Our data point toward the existence of a defective splenic niche in patients with MF that could be responsible of some pathological features of the disease, including the increased trafficking of CD34+ cells and the expansion of the megakaryocytic lineage., (© 2018 Wiley Periodicals, Inc.)

We generated human induced pluripotent stem cells (hiPSCs) from dermal fibroblasts of a 51years old female patient homozygous for the mutation c.535 G>A p.G179S on the KCNQ1 gene, causing a severe form of autosomal recessive Long QT Syndrome type 1 (AR-LQT1), not associated with deafness. The hiPSCs, generated using four retroviruses each encoding for a reprogramming factor OCT4, SOX2, KLF4, cMYC, are pluripotent and can differentiate into spontaneously beating cardiomyocytes (hiPSC-CMs)., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

We report the generation of human induced pluripotent stem cells (hiPSCs) from dermal fibroblasts of a female patient carrier of the two compound heterozygous mutations c.568 C>T p.R190W (maternal allele), and c.1781 G>A p.R594Q (paternal allele) on the KCNQ1 gene, causing Jervell and Lange-Nielsen Syndrome (JLNS). To obtain hiPSCs, we used the classical approach of the four retroviruses each encoding for a reprogramming factor OCT4, SOX2, KLF4, cMYC. The obtained hiPSC clones display pluripotent stem cell characteristics, and differentiate into spontaneously beating cardiomyocytes (hiPSC-CMs)., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

hTERT component is the key regulator of telomerase. Alternatively spliced variants of hTERT generate different telomerase activity. The goal of the study was to determine the role of different hTERT isoforms in the regulation of telomerase expression in AML patients. Among the 97 studied patients, 45 had a complex karyotype and 52 a normal karyotype. hTERT isoforms expression was determined in bone marrow samples by q-RT-PCR, using SYBR Green I. hTERT expression was lower in AML patients than controls (median 2.5 vs. 10.1, p = .003), though no difference was observed between the complex and normal karyotype (median 3.2 vs. 2.3, p = .37). High trans-dominant negative isoform expression increased the response rate by two. High expression of inactive product (-α - β) was shown to increase the risk of relapse by about three times. In conclusion, our data suggest an intriguing link between the control of hTERT isoforms expression and AML outcome.

Background: Long QT Syndrome type 2 (LQT2) is caused by mutations in the KCNH2 gene that encodes for the α-subunit (hERG) of the ion channel conducting the rapid delayed rectifier potassium current (I Kr ). We have previously identified a disease causing mutation (IVS9-28A/G) in the branch point of the splicing of KCNH2 intron 9. However, the mechanism through which this mutation causes the disease is unknown., Methods and Results: We generated human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from fibroblasts of two IVS9-28A/G mutation carriers. IVS9-28A/G iPSC-CMs showed prolonged repolarization time, mimicking what observed at the ECG level in the same patients. The expression of the full-length ERG1a isoform resulted reduced, whereas the C-terminally truncated ERG 1aUSO isoform was upregulated in mutant iPSC-CMs, with consequent alteration of the physiological ERG 1aUSO /ERG1a ratio. Importantly, we observed an impairment of hERG trafficking to the cell membrane. The severity of the alterations in hERG expression and trafficking correlated with the clinical severity of the disease in the two patients under study. Finally, we were able to revert the trafficking defect and reduce the repolarization duration in LQT2 iPSC-CMs using the proteasome inhibitor ALLN., Conclusion: Our results highlight the key role of the KCNH2 intron 9 branch point in the regulation of KCNH2 isoform expression and hERG channel function, and allow to categorize the IVS9-28A/G mutation as LQT2 class 2 mutation. These findings may result in a more personalized clinical management of IVS9-28A/G mutation carriers., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In myelodysplatic syndromes and acute myeloid leukemia (MDS/AML) deletion of the 11q14 region is a rare chromosomal defect (incidence: 0.6-1.0%), included within the intermediate risk criteria by the International Prognostic Scoring System. No fluorescence in situ hybridization (FISH) study has yet been performed to identify a common breakpoint region (CBR). In our study through FISH with bacterial artificial chromosomes and commercial probes, we analyzed seven patients with MDS/AML harboring 11q14 deletion on conventional cytogenetic analysis. FISH revealed deletions in five patients and amplifications in two. Three patients with deletion carried a CBR, two had a deletion involving a more centromeric breakpoint. These five patients exhibited multilineage dysplasia, blast cells with large round nuclei, loose chromatin, small and abundant nucleoli, and vacuolated cytoplasm with very thin Auer bodies. In conclusion, the morphological features which occur independently of the extent of the deletion are of multilineage dysplasia in MDS and leukemic blasts strongly reactive to peroxidase in AML; despite the variable size of the deleted area, some patients harbor a CBR., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Objectives: Imatinib is a cornerstone of treatment of chronic myeloid leukemia. It remains unclear whether transient treatment discontinuation or dose changes affect outcome and this approach has not yet been approved for use outside clinical trials., Methods: We conducted a retrospective single-institution observational study to evaluate factors affecting response in 'real-life' clinical practice in 138 chronic myeloid leukemia patients in chronic phase treated with imatinib. We used a novel longitudinal data analytical model, with a generalized estimating equation model, to study BCR-ABL variation according to continuous standard dose, change in dose or discontinuation; BCR-ABL transcript levels were recorded. Treatment history was subdivided into time periods for which treatment was given at constant dosage (total 483 time periods). Molecular and cytogenetic complete response was observed after 154 (32%) and 358 (74%) time periods, respectively., Results: After adjusting for length of time period, no association between dose and cytogenetic complete response rate was observed. There was a significantly lower molecular complete response rate after time periods at a high imatinib dosage., Discussion: This statistical approach can identify individual patient variation in longitudinal data collected over time and suggests that changes in dose or discontinuation of therapy could be considered in patients with appropriate biological characteristics.

Fanconi anaemia (FA) is an inherited disorder characterized by pancytopenia, congenital malformations and a predisposition to develop malignancies. Alterations in the haematopoietic microenvironment of FA patients have been reported, but little is known regarding the components of their bone marrow (BM) stroma. We characterized mesenchymal stromal cells (MSCs) isolated from BM of 18 FA patients both before and after allogeneic haematopoietic stem cell transplantation (HSCT). Morphology, fibroblast colony-forming unit (CFU-F) ability, proliferative capacity, immunophenotype, differentiation potential, ability to support long-term haematopoiesis and immunomodulatory properties of FA-MSCs were analysed and compared with those of MSCs expanded from 15 age-matched healthy donors (HD-MSCs). FA-MSCs were genetically characterized through conventional karyotyping, diepoxybutane-test and array-comparative genomic hybridization. FA-MSCs generated before and after HSCT were compared. Morphology, immunophenotype, differentiation potential, ability in vitro to inhibit mitogen-induced T-cell proliferation and to support long-term haematopoiesis did not differ between FA-MSCs and HD-MSCs. CFU-F ability and proliferative capacity of FA-MSCs isolated after HSCT were significantly lower than those of HD-MSCs. FA-MSCs reached senescence significantly earlier than HD-MSCs and showed spontaneous chromosome fragility. Our findings indicate that FA-MSCs are defective in their ability to survive in vitro and display spontaneous chromosome breakages; whether these defects are involved in pathophysiology of BM failure syndromes deserves further investigation., (© 2015 John Wiley & Sons Ltd.)

This study evaluated whether the NCCSS truly improves the prognostic stratification of 630 consecutive de novo MDS patients and established which cytogenetic grouping [NCCSS or International Prognostic Scoring System (IPSS)], when combined with the WHO classification, best predicted the clinical outcome of myelodysplastic syndromes (MDS). The frequency of chromosomal defects was 53.8%. Clinical parameters, including number of cytopenias, WHO classification, IPSS cytogenetic categories and scores, NCCSS were all relevant for overall survival (OS) and leukemia-free survival (LFS) and were included in six distinct multivariate models compared by the Akaike Information Criterion (AIC). The most effective model to predict OS included the number of cytopenias, the WHO classification and the NCCSS, whereas the model including the number of cytopenias, blast cell percentage and the NCCSS and the model including the number of cytopenias the WHO classification and the NCCSS were almost equally effective to predict LFS. In conclusion, the NCCS (i) improves the prognostic stratification of the good and poor IPSS cytogenetic categories by introducing the very good and the very poor categories; (ii) is still incomplete in establishing the prognostic relevance of rare/double defects, (ii) applied to patients who receive supportive treatment only identifies five different prognostic subgroups, but applied to patients treated with specific therapies reveals only a trend toward a significantly different OS and LFS when patients of the poor and intermediate cytogenetic categories are compared, (iii) combined with the WHO classification is much more effective than the IPSS in predicting MDS clinical outcome., (Copyright © 2012 Wiley Periodicals, Inc.)

TET2 haplo-insufficiency occurs through different molecular mechanisms and is promptly revealed by array comparative genomic hybridization, single nucleotide polymorphism (SNP) array, and next-generation sequencing (NGS). Fluorescence in situ hybridization (FISH) can effectively demonstrate TET2 deletions and is often used to validate molecular results. In the present study 41 MDS patients with and without 4q abnormalities were analyzed with a series of bacterial artificial chromosome (BAC) probes spanning the 4q22.3-q25 region. On conventional cytogenetic (CC) studies, a structural defect of the long arm of chromosome 4 (4q) was observed in seven patients. In three, one each with a t(1;4)(p21;q24), an ins(5;4)(q23;q24qter), and a t(4;17)(q31;p13) as the sole chromosomal abnormality, FISH with the RP11-356L5 and RP11-16G16 probes, which cover the TET2 locus, produced one signal only. Unexpectedly, this same result was achieved in 3 of the remaining 34 patients. Thus, a TET2 deletion was observed in a total of six patients (14.6%). TET2 deletion was not correlated with any particular clinical findings or outcome. These findings demonstrate that 1) FISH is an effective and economical method to reveal cryptic abnormalities of band 4q22-q24 resulting in TET2 deletions; 2) in these patients, TET2 deletion is the unifying genetic event; and 3) the different breakpoints within the 4q22-q25 region suggest that deletions are not mediated by repetitive sequences., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The present study was designed to establish the incidence of cytogenetic evolution (CE), defined as the acquisition of chromosomal defects during the course of MDS, in order to correlate it with the WHO classification and IPSS score, and to assess its impact on overall survival (OS) and risk of MDS/AML evolution (progression-free interval, PFI) by means of Cox models for time-dependent covariates. Adjustments for known risk factors were achieved by performing a bivariable analysis. The study was carried out in 153 MDS patients who were followed for a median period of 45.2 months. Disease progression occurred in 42.4% of patients after a 65.2-month median PFI, while CE occurred in 30.7% of patients. Our study shows that (1) CE was more common in advanced than in early MDS, and advanced MDS presented secondary chromosomal defects distinct from those of early MDS; (2) CE significantly affected OS and PFI independently of other prognostic variables; (3) del(7)(q31q34) was the only secondary chromosomal defect which significantly affected PFI; trisomy 8 had only a moderate influence.

This study correlated chromosomal defects with French-American-British (FAB)/World Health Organization (WHO) classification subtypes, proposed a revised International Prognostic Scoring System (IPSS) cytogenetic grouping; and established which classification, when used with the IPSS cytogenetic categories, best predicted clinical outcome in the myelodysplastic syndromes (MDS). A higher prevalence of chromosomal defects and distinct defects were observed in patients with multi-lineage dysplasia and a blast cell percentage >10%. Abnormalities of the long arm of chromosome 3, del(7)(q31q35), trisomy 8, del(11)(q14q23), del(12p) and 20q- could be segregated from their respective IPSS cytogenetic categories and used to develop new cytogenetic subgroups. Clinical parameters, FAB/WHO classification, IPSS score and standard or revised cytogenetic categories were statistically relevant for overall survival (OS) and progression-free intervals (PFI) and were included within five distinct multivariate models compared by the Akaike Information Criterion. To predict OS, the best models included age, WHO classification and standard or revised IPSS cytogenetic categories; to predict PFI, the best model included the same variables and revised cytogenetic categories. In conclusion, (i) the WHO classification was associated with a more homogeneous cytogenetic pattern than the FAB classification, (ii) WHO classification and standard/revised IPSS cytogenetic categories were much more effective than IPSS for predicting MDS clinical outcome, (iii) revised cytogenetic subgroups predicted PFI more effectively than standard categories.

Myelodysplastic syndromes (MDS) are a group of heterogeneous stem cell disorders with different clinical behaviors and outcomes. Conventional cytogenetics (CC) studies have demonstrated that the majority of MDS patients harbor clonal chromosome defects. The probability of discovering a chromosomal abnormality has been increased by fluorescence in situ hybridization (FISH), which has revealed that about 15% of patients with a normal chromosome pattern on CC may instead present cryptic defects. Cytogenetic abnormalities, except for the interstitial long-arm deletion of chromosome 5 (5q-), are not specific for any French-American-British (FAB)/World Health Organization (WHO) MDS subtypes, demonstrate the clonality of the disease, and identify peculiar morphological entities, thus confirming clinical diagnosis. In addition, chromosome abnormalities are independent prognostic factors predicting overall survival and the likelihood of progression in acute myeloid leukemia.

ABL1 amplification, due to a cryptic episomal translocation NUP214/ABL1, is a novel finding in T-cell acute lymphoblastic leukemia (ALL). Here we report on the incidence and clinical features of this genetic defect in a series of 30 consecutive adult T-cell ALL patients. Multiple copies of the ABL1 gene were detected in two patients (6.6%), one with the karyotype 46,XY,t(1;3)(p36;p21),del(6)(q23)/46,XY and the other without analyzable metaphases. Metaphase/interphase fluorescence in situ hybridization (FISH) detected multiple uncountable signals corresponding to ABL1 in mitotic cells and nuclei from both patients. In one patient, no signals corresponded with the 9p21 chromosomal region, which contains the p16INK4a gene, and in the other one signal was observed. Quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) demonstrated that in these patients ABL1 gene expression was 14- and 18-fold greater than in normal controls, and returned to normal levels only when complete remission was achieved. We reached the following conclusions: (1) FISH is the only technique that promptly identifies T-cell ALL patients with ABL1 amplification, (2) quick identification with FISH is fundamental in the clinic because this T-cell ALL subset is imatinib sensitive but may become resistant due to development of additional mutations, and (3) ABL1 quantitative RT-PCR may be easily applied to monitor minimal residual disease.

The impact of clinical parameters, International Prognostic Scoring System (IPSS) scores/cytogenetic categories, and some single cytogenetic defects on overall survival (OS) and time to myelodysplastic syndromes (MDS)/AML progression (progression-free interval (PFI)) was evaluated in 331 MDS patients. Statistical analysis demonstrated that OS and PFI were significantly affected by all these parameters. Since single 7q- showed a better survival than the poor IPSS cytogenetic category (P=0.009), it was considered as a new prognostic entity ('modified IPSS categories'). In multivariate analysis OS was significantly influenced by age, marrow blast cell percentage, number of cytopenias and either modified or standard IPSS cytogenetic categories; hazard ratios for MDS/AML progression were influenced by all the former, except for age and cytopenias. Multivariate analysis of del(7)(q31q35) confirmed the results of univariate analysis, but the Akaike Information Criterion showed no difference in evaluating OS and PFI between the modified and standard IPSS cytogenetic grouping. In conclusion, (i) chromosome defects as grouped by IPSS and blast cell percentage are the most relevant parameters for predicting OS and PFI; (ii) the prognostic power of the IPSS cytogenetic grouping is not ameliorated by the introduction of del(7)(q31q35) as a new entity; (iii) complex karyotypes have a prognostic value independent of blast cell percentage., (Leukemia (2005) 19, 1424-1431.)

Objective: To evaluate the expression of bcl-2, p-53, Ki-67, and apoptosis as evidenced by nuclear DNA fragmentation in cervical biopsies obtained from human immunodeficiency virus (HIV)-seropositive and HIV-seronegative women with a history of intravenous drug abuse., Methods: We investigated 109 consecutive cervical biopsies (73 from HIV seropositive and 36 from HIV seronegative patients), including 86 cervical intraepithelial neoplasia (CIN) lesions and 23 normal cervical epithelium. The markers of apoptosis and proliferation were detected using immunostaining methods on paraffin sections. The associations between HIV status and the intensities of immunostaining were tested by univariate and multivariable methods., Results: The severity of CIN correlated directly with the intensities of p-53 (Spearman Rho = 0.19; P = 0.05) and Ki-67 immunostaining (Spearman Rho = 0.46, P < 0.001) and with apoptosis and Bcl-2 expression (chi-square for trend = 10.6 and 3.91 , P < 0.001 and P = 0.048, respectively).After adjustment, by logistic regression analysis, for the potential confounding effect of severity of CIN and Human Papillomavirus (HPV) infection, apoptosis was five times more common in cervical biopsies of HIV seropositive compared to HIV seronegative patients (27 out of 73 as compared to 4 out of 36, adjusted Odds Ratio = 5.0; 95% confidence intervals = 1.56-16.33, P = 0.007). The adjusted odds ratio of increasing p53 immunodetection was significantly higher in biopsies obtained from HIV-seropositive patients compared to HIV seronegative controls (OR = 8.7; 95% confidence intervals = 2.97-25.3, P < 0.0001)., Conclusion: Markers of apoptosis such as nuclear DNA fragmentation and p-53 immunoreactivity are more intensely expressed in cervical biopsies of normal and dysplastic epithelium of HIV-seropositive compared to HIV-seronegative women.

Meaningful progress has been made toward clarifying the molecular steps in the pathogenesis of acute myeloid leukemia (AML). Chromosome studies have established that translocations/inversions are the most common cytogenetic defects in AML. Cloning of chromosome breakpoints has shown that genes involved in the chromosome abnormalities are transcription factors, functional loss of which alters chromatin configuration and results in the disruption of myeloid differentiation. However, transgenic animal models have demonstrated that AML-specific translocations/inversions alone are insufficient to cause overt leukemia, which occurs only when point mutations affecting receptor tyrosine kinases (RTKs) develop. Therefore, development of AML is now considered a two-step process in which RTK mutations provide a proliferative and a survival advantage to a clonal cell population already marked by impaired differentiation. In addition, more accurate definition of such genetic lesions has led to a more precise insight as to how such lesions interact with cellular signaling pathways that are aberrantly regulated in AML. All these new data have profound clinical and therapeutic implications and will surely translate into the development of molecules that target specific mutations or signal transduction pathways.

A case-control study was performed to investigate the risk of cervical cancer associated with p53 polymorphism at codon 72, encoding either arginine or proline. It has been recently suggested that the arginine isoform increases the susceptibility to invasive cervical cancer; however, data remain controversial. The polymorphism was examined by both allele-specific PCR and RFLP analysis in 101 patients with primary cervical cancer and in 140 healthy women of the same age and from the same geographical area. The distribution of p53 genotypes in cervical cancer patients and in controls was not significantly different (P = 0.445), and homozygosity for arginine at residue 72 was not associated with an increased risk for cervical cancer (odds ratio, 0.81; 95% confidence interval, 0.47-1.42; P = 0.52). Similarly, different genotype distribution and increased risk were not observed when patients versus controls were analyzed according to human papillomavirus status and cancer histotype. Therefore, no evidence of association between homozygosity for p53 arginine and cervical cancer was found in our population sample.

Objective: To evaluate the risk of perinatal human papillomavirus (HPV) transmission from mothers with latent infections to the oropharyngeal mucosae of their infants., Methods: Seven hundred eleven mother-newborn pairs were tested. Polymerase chain reaction was done with MY09/MY11 consensus primers to identify HPV DNA in maternal cervicovaginal lavages and newborn nasopharyngeal aspirates. Positive cases were further amplified with type-specific primers for HPVs 6, 11, 16, 18, and 33. All infants born to HPV-positive mothers were observed to 18 months for appearance of HPV in oropharyngeal mucosae., Results: Human papillomavirus DNA was detected in 11 neonates born vaginally to HPV-positive women, a vertical transmission rate was 30% (95% confidence interval [CI] 15.9, 47). Nasopharyngeal aspirates were HPV-negative in all 11 cases in which rupture of membranes occurred less than 2 hours before delivery. When rupture preceeded delivery by 2-4 hours, and when it occurred after more than 4 hours, the respective rates for HPV positivity were seven of 21 and four of five (chi2 for trend = 10.7, P = .001). At follow-up, virus was cleared from the oropharyngeal samples as early as the 5th week., Conclusion: Pregnant women with latent HPV infections have low potential of transmitting the virus to the oropharyngeal mucosae of their infants. The time between rupture of the amnion and delivery seems to be a critical factor in predicting transmission. Human papillomavirus-positive infants should be considered contaminated rather than infected since virus is cleared over several months after birth.

Mutant p53 is frequently detected in endometrial and ovarian carcinoma, but it is rare in cervical cancers. Previous reports focused on cervical squamous cell carcinoma, whereas cervical adenocarcinoma was given little attention. We searched for p53 gene mutations in 74 primary cervical adenocarcinomas with known human papillomavirus (HPV) status. Our aim was to evaluate the prevalence of p53 mutations and to investigate their possible role as an independent prognostic factor. We found mutations in 13.5% with a high rate of G:C --> A:T transitions as observed in endometrial adenocarcinoma. As p53 mutations are more frequently detected in malignancies of high grade, high stage, and large size, this molecular event seems to play a role in the progression rather than in the induction of cervical adenocarcinoma. In our series, patients with HPV-negative tumors and patients with mutated neoplasms, irrespective of HPV infection, had a shorter survival. Yet the absence of HPV infection and presence of p53 mutations are not independent risk factors for tumor-related death after adjustment for clinicopathological confounders. The only significant and independent predictors of survival are age of patient, stage of disease, tumor grade, and presence of lymph node metastases.

Cervicovaginal lavages from 752 pregnant women at term were investigated by polymerase chain reaction to evaluate human papillomavirus (HPV) infection prevalences and were compared with cervicovaginal samples from two series of nonpregnant subjects (504 healthy women attending a family planning service and 560 symptomatic patients attending a vaginitis outpatient service). The odds ratios (ORs) of HPV infection were computed by conditional logistic regression analysis on age-matched sets. In pregnant women, the overall risk of HPV infection was about the same as in nonpregnant healthy subjects (adjusted OR, 0.90; 95% confidence interval [CI], 0.51-1.58) and was 50% less than in patients with symptomatic vaginitis (adjusted OR, 0.48; 95% CI, 0.30-0.76). Moreover, the prevalence of oncogenic HPV types 16 or 18 (or both) was lower in pregnant women (P = .015 and P = .0018 respectively).

The authors investigated by PCR 138 infiltrating cervical adenocarcinoma (27 grade 1, 76 grade 2, and 35 grade 3) for the presence of human papillomavirus (HPV) 16 and 18 infection. They included 95 (68.8%) mucinous and 43(31.2%) non-mucinous tumors. The overall prevalence of HPV infection was 84.8%; 28.3% of the cases were positive for HPV 16, 29.7% for HPV 18, and 26.8% for both HPVs. Amplification of HPV 16 and 18 negative cases with consensus primers MY09/MY11 failed to yield any additional tumors with HPV DNA sequences. Patients with HPV infection were younger than the patients who were HPV-negative (P = .001). The type of HPV was unrelated to age. Human papillomavirus infection was found in 95.8% mucinous and in 60.5% non-mucinous tumors (P < .001), with even distribution among grade 1, 2 and 3 adenocarcinoma. Our findings confirm the key role of HPV 16 and 18 in the development of cervical adenocarcinoma, particularly in mucinous histotypes. The absence of HPV infection, the old age of patients and the non-mucinous differentiation may identify a subset of cervical adenocarcinoma with different etiopathogenesis.

Seventy-nine transitional cell carcinomas (TCCs) of the urinary bladder (25 grade 1, 22 grade 2, and 32 grade 3 tumours) were examined for p53 overexpression by immunohistochemistry with a monoclonal antibody and for human papillomavirus (HPV) infection by the polymerase chain reaction (PCR). Positive immunostaining for p53 was detected in 40.5 per cent of the cases; the percentage of positive cases was significantly lower in low-grade (G1 and G2) TCCs than in high-grade (G3) tumours (10.6 per cent vs. 84.4 per cent; P < 0.0001). The overall rate of HPV infection was 32.9 per cent; 20.3 per cent of the cases were positive for HPV 16, 3.8 per cent for HPV 18, and 8.9 per cent for both. Consensus primers as well as type-specific primers for HPV types 6, 11, and 33 failed to detect any additional case with HPV infection. The prevalence of HPV 16 and/or HPV 18 infection was significantly higher in low-grade than in high-grade tumours (44.7 per cent vs. 15.6 per cent; P = 0.0061). p53-positive cases were more common among papillary, deeply infiltrating tumours, and HPV-positive cases among papillary, non-infiltrating lesions. According to these data, p53 overexpression and HPV 16/18 infection are common findings in bladder TCC and there appears to be an inverse correlation of p53 overexpression and of HPV infection with tumour aggressiveness. The possibility of different molecular pathways in superficial low-grade and in invasive high-grade tumours is suggested.

Experimental models indicate that activated ras genes and HPV oncogenic sequences may cooperate in inducing a completely transformed phenotype in epithelial cells. We searched for K-ras gene mutations and HPV type-16 and -18 sequences in 67 primary adenocarcinomas of the uterine cervix by analyzing DNAs from formalin-fixed, paraffin-embedded tissue samples. Target sequences were amplified by PCR and analyzed by denaturing gradient gel electrophoresis (DGGE) and sequencing for the detection of K-ras gene mutations and by Southern blotting for the detection of HPV infection. We found 16 mutations in 15 cases; 14 were at codon 12 and 2 at codon 13; 11 were base transitions and 5 were transversions. Mutations were more frequent in mucin-secreting than in non-mucinous tumors. HPV oncogenic sequences were detected in 58 cases with no significant difference between K-ras-mutated and wild-type tumors. HPV oncogenic sequences were also more frequent in mucin-secreting than in non-mucinous tumors. Both molecular events were present simultaneously in 13 out of 58 cases, all of which had histologically grade-2 and grade-3 tumors. Clinico-pathological parameters of the disease and the overall survival, however, were independent of K-ras mutations and of HPV-16 and -18 infection, as shown by univariate and multivariate analysis. In contrast, stage of disease, lymph-node metastases, deep infiltration, clear-cell histology and low grade of differentiation were risk factors for tumor-related death.

Objective: Our purpose was to evaluate the proliferative activity of cervical intraepithelial neoplasia lesions in human immunodeficiency virus-seropositive women., Study Design: The proliferative activity of cervical intraepithelial neoplasia lesions was measured by nucleolar organizer regions-associated proteins count. Twenty-two biopsy specimens of cervical intraepithelial neoplasia lesions from patients positive for human immunodeficiency virus were compared with 22 matched biopsy specimens obtained from controls negative for the virus., Results: The mean count of nucleolar organizer regions-associated proteins per cell was 9.5 +/- 3.7 (SD) in human immunodeficiency virus-positive patients and 7.6 +/- 2.8 in human immunodeficiency virus-negative controls (p < 0.0001 by Poisson test of heterogeneity of counts). The difference in counts between the two groups, which was confirmed by log-linear analysis, persisted within each severity stratum of cervical intraepithelial neoplasia and was independent of associated human papillomavirus infection. In human immunodeficiency virus-positive patients log-linear analysis showed that high-grade cervical intraepithelial neoplasia, the presence of human papillomavirus infection, and the severity of human immunodeficiency virus disease were independently correlated with increased nucleolar organizer regions-associated protein counts per cell., Conclusion: The results of this study indicate that the proliferative activity of cervical intraepithelial neoplasia lesions of human immunodeficiency virus-positive patients was increased compared with matched lesions from human immunodeficiency virus-negative women. This finding suggests the possibility of an increased oncogenic progression of cervical intraepithelial neoplasia lesions in human immunodeficiency virus-positive patients.

Frequency and pattern of expression of eight markers of gastric, intestinal, and pancreatobiliary duct epithelial cells have been investigated by histochemical and immunohistochemical techniques in 85 cases of cervical adenocarcinomas. M1, a mucin antigen, and Cathepsin E (CaE), an aspartic proteinase, markers of normal gastric superficial/foveolar epithelial cells, are expressed in 40 and 55 tumors, respectively. Periodic acid-concanavalin A-reactive mucin or Pepsinogen (PG) II, markers of gastric mucus neck and pyloric gland cells, are found in 24 tumors, 13 of which also express M1 and CaE. CAR-5 and M3SI, markers of intestinal mucin, are expressed in 68 and 12 tumors and DU-PAN-2, marker of normal pancreatobiliary duct cells, is found in 46 tumors. All but two tumors express at least one of the eight markers studied, none express PG I, marker of gastric chief cells. The different histologic subtypes of cervical adenocarcinomas expressed to a variable degree both gastrointestinal and pancreatobiliary markers. Only endocervical type tumors, however, showed the full spectrum of mucosal pyloric type differentiation, including the expression of PGII which is not present in any other histotype. A correlation between expression of gastroenteropancreatic markers and tumor grade is not apparent in our series.

Special immunohistochemical stains for the identification of gastroenteropancreatic antigens in two cases of primary retroperitoneal mucinous cystoadenocarcinomas (PRMC) show that these tumours have patterns similar to ovarian mucinous tumours. Markers of pyloric type gastric mucosa differentiation (M1, cathepsin E, concavavalin A, pepsinogen II) are mostly positive in benign and borderline areas with endocervical type differentiation, while immunoreactivity for intestinal cell markers (M3SI and CAR-5) and for DU-PAN-2 is present mainly in frankly malignant areas, regardless of differentiation type. DNA analysis shows a point mutation of K-ras oncogene at codon 12 (GGT to CGT) in one case. The immunohistochemical and genotypic similarity of PRMC and ovarian mucinous tumours may indicate similar mechanisms in their histogenesis.

The mean counts of Langerhans' cells were evaluated in cervical biopsies obtained from 30 patients with human immunodeficiency virus (HIV) infection and cervical intraepithelial neoplasia (CIN) and from 30 HIV-seronegative control patients. Each HIV-seronegative control was matched to a seropositive case with respect to grade of CIN, age, and smoking habits. Langerhans' cells were identified by immunohistochemical staining for S-100 protein. In situ hybridization with biotinylated probes was performed to detect human papillomavirus (HPV) DNA 6/11, 16/18, and 31/35/51. The mean counts of S-100 positive cells per 100 basal cells were lower in HIV-seropositive patients than in controls (0.99 +/- 0.08 vs 1.9 +/- 0.2 P = 0.024). These differences occurred independent of any coexisting HPV infection. Positive correlations between S-100 positive cell counts and CD4+ and CD8+ cell counts were found in HIV-infected women. AIDS patients had lower Langerhans' cell counts compared both to patients with AIDS-related complex or asymptomatic HIV infection. Our results suggest that local cervical immunity, as evaluated by Langerhans' cell counts, is impaired in HIV-seropositive women. The severity of impairment seems to correlate with the stage of the HIV disease.

In 100 mucinous tumors of the ovary (37 benign, 24 borderline, and 39 malignant), the authors determined by histochemical and immunohistochemical techniques the frequencies and patterns of expression of a total of nine markers of gastric, intestinal, and pancreatobiliary duct epithelial cells. M1, a mucin antigen, and cathepsin E (CaE), an aspartic proteinase, two markers of normal gastric superficial/foveolar epithelial cells, were expressed in 95 and 92 tumors, respectively. Periodic acid-concanavalin A-reactive mucin or pepsinogen (PG) II, markers of gastric mucus neck and pyloric gland cells, were found in 79 tumors. All of these tumors also expressed M1 or CaE. DU-PAN-2 and the N-terminal epitope of gastrin-releasing peptide, markers of normal pancreatobiliary duct cells, were found in 70 and 49 tumors, respectively, and CAR-5 and M3SI, markers of intestinal mucin, were expressed in 51 and 30 tumors, respectively. All tumors expressed at least two of the nine markers studied; none expressed PG I, a marker of gastric chief cells. The mucopeptic cell marker, PG II, was significantly more common in benign and borderline than in malignant tumors (P less than 0.005), whereas CAR-5 and M3SI, markers of intestinal mucin, were expressed significantly more often in malignant than in benign and borderline tumors (P less than 0.001). By electron microscopic examination, many tumor cells had fine structural features characteristic of gastric superficial/foveolar and pyloric gland cells, intestinal columnar or goblet cells, and endocervical cells. The results indicate that gastroenteropancreatic cell differentiation--and, in particular, gastric type differentiation--is a prominent feature of ovarian mucinous tumors.

The prevalence of lower genital neoplasia and Human Papilloma-virus-related genital lesions were evaluated in a cohort of 75 women with Human Immunodeficiency Virus type 1 (HIV-1) infection at different stages of HIV disease. The overall rate of cervical intraepithelial neoplasia (CIN) in the group studied was 29.3% (22/75). Eight out of 10 high-grade CIN lesions contained 'high-risk' HPV-DNA 16/18 and/or 31/35/51 as demonstrated by 'in situ' hybridization with biotinylated probes. Vulvar and/or perianal condylomata were histologically diagnosed in 14 patients (18.7%); nine of these biopsies contained detectable HPV-DNA which was always related to HPV 6/11. The rate of high-grade CIN in symptomatic HIV-infected patients was 28% (7/25) as compared to 6% (3/50) of the other cases (P = 0.022). CD4 lymphocyte counts, white blood cell counts, CD4+/CD8+ cell ratio and percentage of CD4+ lymphocytes were lower in patients with high-grade CIN in comparison to the patients with negative colposcopical and/or cytological examination. After adequate standard treatment (cryotherapy, electrocauterization, cold-knife conization) only one case of CIN 2 recurred during the 2 years of follow-up period. The prevalence of lower genital neoplasia and HPV-related lesions among HIV-infected women is high and seems to correlate with the severity of HIV disease.