Genetic alterations in primary mediastinal B-cell lymphoma: an update.

Primary mediastinal B-cell lymphoma (PMBL) is a distinct clinical entity among non-Hodgkin's lymphoma. The malignancy has received little attention from a standpoint of basic research due in part to its rarity. However, based on recent studies consistent trends are beginning to emerge regarding the molecular and chromosomal alterations commonly observed in this disease. By both CGH and AP-PCR, genetic gains involving chromosomes 2, 5, 7, 9p, 12, and Xq are among the most frequently observed events. From a molecular standpoint, alterations in the c-myc, p16(INK4) and p53 genes have been observed in up to 30% of cases. This information along with the well-established histological, immunological, and clinical features should convince the few remaining disbelievers that PMBL is a distinct pathological entity among non-Hodgkin's lymphomas.

Alteration of chromosome arm 6p is characteristic of primary mediastinal B-cell lymphoma, as identified by genome-wide allelotyping.

Five cases of primary mediastinal B-cell lymphoma (PMBL) each have been studied with 375 microsatellite markers from all 22 autosomes. Of the 151 genomic alterations among the 1,875 assays, only five were allelic losses. The remainder of the microsatellite alterations consisted of 114 allelic imbalances and 32 instabilities. Microsatellite alterations were found in all cases on chromosomal arms 6p and 9p. These allelic imbalances most likely are indicative of genetic amplification, a finding agreeing well with those of studies using either comparative genomic hybridization or arbitrarily primed polymerase chain reaction, in which amplification of chromosome arm 9p in PMBL has been found. The allelic imbalances on chromosome arm 6p always included marker D6S276 located at 6p21.3-p22.3, where the MHC class I genes reside. These allelic imbalances may be reflective of alterations in the expression of the MHC gene products, characteristic of PMBL. Allelic anomalies close to the MYB gene locus on 6q were detected in two cases and prompted the analysis of MYB rearrangements in a series of 30 lymphomas. One rearrangement was detected in one of 18 cases of PMBL and in none of 10 diffuse, large B-cell lymphomas and two T-cell lymphomas. Our genome-wide microsatellite analysis provides independent confirmation that PMBL is characterized by infrequent chromosomal losses and by frequent genetic alterations involving chromosomal arm 9p. For the first time, chromosomal arm 6p has been identified as a highly frequent target of genetic alterations in this tumor type. Finally, MYB may also be involved occasionally in PMBL pathogenesis.

High resolution allelotype of nonfunctional pancreatic endocrine tumors: identification of two molecular subgroups with clinical implications.

A high resolution allelotype for nonfunctional pancreatic endocrine tumors (NF-PETs) has been generated by microsatellite analysis of DNA from 16 frozen cases, each probed with 394 markers. Two subgroups of NF-PETs were found. Seven cases showed frequent, large allelic deletions [loss of heterozygosity (LOH)] with an average fractional allelic loss (FAL) of 0.55, whereas nine cases showed a small number of random losses with a FAL of 0.15. Designated high or low FAL, respectively, these genetic phenotypes showed correlation with the ploidy status: high-FAL tumors were aneuploid, low-FAL were diploid. Chromosomes 6q and 11q showed LOH in >60% of cases. About 50% of cases had losses on 11p, 20q, and 21. Selected LOH analysis on an additional 16 paraffin-embedded NF-PETs confirmed the high frequency of 6q and 11q LOH. The allelotype of NF-PET is markedly different from that of either ductal or acinar tumors of the pancreas as well as from that of functional-PETs. Moreover, whereas deletions involving chromosome 11 also are a feature of functional-PETs, the involvement of chromosome 6q is characteristic of NF-PETs. Survival analysis showed that none of the single chromosomal alterations was associated with outcome, whereas ploidy status is an independent factor adding prognostic information to that furnished by the proliferative index measured by Ki-67 immunohistochemistry.

Pancreatic tumours: molecular pathways implicated in ductal cancer are involved in ampullary but not in exocrine nonductal or endocrine tumorigenesis.

Alterations of K- ras, p53, p16 and DPC4/Smad4 characterize pancreatic ductal cancer (PDC). Reports of inactivation of these latter two genes in pancreatic endocrine tumours (PET) suggest that common molecular pathways are involved in the tumorigenesis of pancreatic exocrine and endocrine epithelia. We characterized 112 primary pancreatic tumours for alterations in p16 and DPC4 and immunohistochemical expression of DPC4. The cases included 34 PDC, 10 intraductal papillary-mucinous tumours (IPMT), 6 acinar carcinomas (PAC), 5 solid-pseudopapillary tumours (SPT), 16 ampulla of Vater cancers (AVC) and 41 PET. All tumours were also presently or previously analysed for K- ras and p53 mutations and allelic loss at 9p, 17p and 18q. Alterations in K- ras, p53, p16 and DPC4 were found in 82%, 53%, 38% and 9% of PDC, respectively and in 47%, 60%, 25% and 6% of AVC. Alterations in these genes were virtually absent in PET, PAC or SPT, while in IPMT only K- ras mutations were present (30%). Positive immunostaining confirmed the absence of DPC4 alterations in all IPMT, SPT, PAC and PET, while 47% of PDC and 38% of AVC were immunonegative. These data suggest that pancreatic exocrine and endocrine tumourigenesis involves different genetic targets and that among exocrine pancreatic neoplasms, only ductal and ampullary cancers share common molecular events.

Molecular characterization of pancreatic serous microcystic adenomas: evidence for a tumor suppressor gene on chromosome 10q.

Pancreatic serous microcystic adenomas (SCAs) are rare, benign tumors with a striking female preference. Virtually no information is available about chromosomal or genetic anomalies in this disease. We performed extensive molecular characterization of 21 cases of formalin-fixed, paraffin-embedded sporadic SCAs consisting in genome-wide allelic loss analysis with 79 microsatellite markers covering all 22 autosomes, assessment of microsatellite instability, and mutational analysis of the VHL, K-ras, and p53 genes in nine cases for which frozen tissue was available. Although no case showed microsatellite instability of the type seen in mismatch repair-deficient tumors, a relatively low fractional allelic loss of 0.08 was found. Losses on chromosome 10q were the most frequent event in SCAs (50% of cases), followed by allelic losses on chromosome 3p (40% of cases). Moderately frequent losses (>25% of cases) were found on chromosomes 1q, 2q, and 7q. The VHL gene, located on chromosome 3p, had somatic inactivating mutations in two of nine cases (22%), whereas no mutations were found in either K-ras or p53, in agreement with the finding that all 21 cases stained negative for p53 by immunohistochemistry. Our study indicates that the involvement of chromosomal arms 10q and 3p is characteristic of SCAs and that the VHL gene is involved in a subset of sporadic cases.

Allelotype of pancreatic acinar cell carcinoma.

Pancreatic acinar cell carcinoma (PAC) is a rare pancreatic tumor for which no information about chromosomal and gene anomalies is available. We performed genome-wide allelotyping of 9 PACs using DNA from 5 frozen and 4 paraffin-embedded samples and 76 PCR-amplified, chromosome-specific microsatellite markers. High degrees of allelic loss were found, with a mean fractional allelic loss of 0.33. Chromosomes 1p, 4q and 17p showed loss of heterozygosity in >70% of cases and chromosomes 11q, 13q, 15q and 16q, in 60% to 70% of cases. Chromosomes 3q, 6q, 8q, 18q and 21q showed loss in 50% to 60% of cases. All of the remaining chromosomes showed no or few allelic losses. The resulting allelotype of PAC is markedly different from that of either ductal or endocrine tumors of the pancreas, and the involvement of chromosomes 4q and 16q appears to be characteristic of this tumor type. High-resolution mapping of the 12 frequently altered chromosomes in 5 cases with 222 markers permitted subchromosomal localization of regions of consensus loss on 5 chromosomes, including 1p36.31, 3p25.2, 4q26-31.1, 15q15-22.1 and 16q21-q22.1. Our findings suggest that PAC tumorigenesis involves molecular pathways different from those occurring in more common pancreatic tumor types.

A mortality study among workers in a French aluminium reduction plant.

OBJECTIVE: A mortality study on the association between lung cancer and occupational exposure to polycyclic aromatic hydrocarbons (PAHs) was carried out in a French aluminium reduction plant. This study updated a previous mortality study. METHOD: The historical cohort included every male worker who had been employed in the plant for at least 1 year between 1950 and 1994. Workers were followed-up for mortality from 1968 to 1994. Causes of death were obtained from death certificates. Standardised mortality ratios (SMRs) and 95% confidence intervals (CI) were computed using regional mortality rates as external reference to compare observed and expected numbers of deaths, adjusted for gender, age and calendar time. RESULTS: The cohort comprised 2,133 men, of whom 335 died during the follow-up period. The observed mortality was lower than expected for all causes of death (SMR = 0.81, CI 0.72-0.90) and for lung cancer (observed = 19, SMR = 0.63, CI 0.38 0.98). No lung cancer excess was observed in workshops where PAH exposure was likely to have occurred, and no trend was observed according to duration of exposure and time since first exposure. This low lung cancer mortality could be partly explained by a marked healthy worker effect and a possible negative confounding by smoking. An excess was observed for bladder cancer (observed = 7, SMR = 1.77, CI 0.713.64) in the whole cohort, that was higher among workers employed in workshops where PAH exposure was likely to have occurred (observed = 6, SMR = 2.15, CI 0.79-4.68). In addition, an SMR higher than unity was observed for "psychoses and neuro-degenerative diseases" (observed = 6, SMR = 2.39, CI 0.88-5.21), that could not be related to occupational aluminium exposure. CONCLUSION: No lung cancer risk was detected. Non-significant excesses were observed for bladder cancer and for psychoses and neuro-degenerative diseases.

Renal angiomyolipoma with epithelioid sarcomatous transformation and metastases: demonstration of the same genetic defects in the primary and metastatic lesions.

Angiomyolipoma (AML) is a benign neoplasm that occurs either sporadically or in patients with tuberous sclerosis complex (TSC) and shows frequent allelic losses at chromosome arm 16p. It has been suggested recently that the melanogenesis marker-positive perivascular epithelioid cell (PEC) has been found consistently in AML. The authors report a 50-year-old woman without evidence of TSC affected by classic renal AML containing an area composed of atypical epithelioid cells with the same morphoimmunophenotypic characters of PEC. After 7 years from surgical removal of the lesion, the patient developed a local recurrence and successive lung and abdominal metastases that showed morphologic and immunohistochemical features overlapping those of the epithelioid area of the previously removed AML. Genetic analysis showed that the classic AML and its epithelioid area as well as the pulmonary and abdominal metastases shared the same allelic loss on chromosome arm 16p. Based on these findings, the authors view this case as evidence of a malignant transformation of a classic AML with morphologic, immunophenotypic, and genetic demonstration of its clonal origin.

Pancreatic acinar carcinoma shows a distinct pattern of chromosomal imbalances by comparative genomic hybridization.

Pancreatic acinar cell carcinoma (PAC) is a rare pancreatic tumor for which no information about chromosomal anomalies is available. We examined six primary PACs by comparative genomic hybridization (CGH). All cases showed chromosomal changes. A total of 106 gains and 48 losses was detected. Consensus regions of gain were identified on chromosomes 1, 12, and X: 1q21 in four cases, 1q42 in three cases, 12p11.2 in four cases, and Xq12-21 in three cases. Recurrent losses were found at 16p13.2-p13.1 in three cases and at 16q23 in three cases. To verify these chromosomal imbalances, microsatellite analysis of matched normal and tumor DNA was performed using PCR-amplified markers for chromosomes 1, 12, and 16 in the regions showing nonrandom gains or losses. This analysis showed allelic imbalances in tumor DNA consistent with the CGH profiles. Our CGH study suggests that PAC shows a characteristic pattern of chromosomal alterations, involving gain at 1q, 12p, and Xq and loss of sequences at 16p and 16q. This pattern appears unique among solid tumors and is markedly different from that detected in pancreatic ductal carcinomas by the same technique. This suggests that PAC tumorigenesis involves different molecular pathways than those involved in the more common pancreatic ductal tumors. Genes Chromosomes Cancer 28:294-299, 2000.

Molecular features of primary mediastinal B-cell lymphoma: involvement of p16INK4A, p53 and c-myc.

Primary mediastinal B-cell lymphoma (PMBL) shows chromosome 9p anomalies in 50% of cases. Based on reports that p16INK4A gene, located on this chromosomal arm, is frequently altered in aggressive lymphomas, we analysed for alterations of this gene in 27 cases of PMBL, which were part of a series of 32 PMBL cases that have been characterized for alterations in c-myc, p53, N-ras, bcl-1, bcl-2, bcl-6 and for Epstein-Barr virus (EBV) infection. Four cases showed p16INK4A gene anomalies, including three with promoter methylation and one homozygous deletion. Eight PMBLs showed c-myc rearrangements. Three additional cases showed sequence variations in the c-myc P2 promoter, two of which consisted of the same germline variation involving a novel polymorphic XhoI site. Four tumours contained p53 gene mutations and three had clonal EBV infection. One case had a bcl-6 rearrangement. In conclusion, our study shows that p16INK4, c-myc and p53 alterations occur in 15%, 25% and 13% of PMBLs, respectively. EBV monoclonality was found in 9% of cases, whereas no abnormality was detected in bcl-1, bcl-2 and N-ras. Thus, none of the common genetic aberrations seen in other types of non-Hodgkin's lymphomas appears to be stringently involved in the pathogenesis of this unique lymphoma type.

Nonrandom chromosomal imbalances in primary mediastinal B-cell lymphoma detected by arbitrarily primed PCR fingerprinting.

We used arbitrarily primed polymerase chain reaction (AP-PCR) fingerprinting to identify chromosomal imbalances in six primary mediastinal B-cell lymphomas (PMBLs). Seventy-four chromosomal imbalances were detected, consisting of 49 sequence gains and 25 losses. Amplifications on chromosome X were seen in five cases, four of which involved the same chromosomal locus. Nonrandom gains at the same locus were also identified on chromosomes 2 and 7 in four cases and on chromosomes 5, 9, and 12 in three cases. Five PMBLs were also analyzed by comparative genomic hybridization (CGH), which found chromosome arm 9p amplification as the only nonrandom imbalance. Our data demonstrate that chromosomal amplifications outnumber losses in PMBL. These mainly involve chromosomes 9 and X and may reflect more complex phenomena, such as translocations or other chromosomal rearrangements, as AP-PCR found coexistent gains and losses on these chromosomes. Comparison between AP-PCR and CGH suggests that anomalies affecting the same chromosomal regions may occur at much higher frequencies than expected by CGH, suggesting that genomic amplifications are usually confined to DNA segments smaller than the megabase long segments required for detection in CGH. Modest increases in genetic material may be as effective as higher-level amplifications when affecting sites where a proto-oncogene resides.

In vivo protein-DNA interactions on a glucocorticoid response unit of a liver-specific gene: hormone-induced transcription factor binding to constitutively open chromatin.

Transcription from the liver promoter of a 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) gene depends on the presence of glucocorticoids that act via a glucocorticoid response unit (GRU) located in the first intron. The promoter and the GRU are in a constitutively open chromatin configuration. To determine how glucocorticoids would affect factor binding to the GRU in absence of chromatin remodeling, we have used a combination of in vitro DNA-binding assays and in vivo genomic footprinting in rat hepatocytes and hepatoma cells. We found that, in the absence of glucocorticoids, the GRU binds nuclear factor-I (NF-I). Glucocorticoid treatment modified factor binding to the NF-I site and induced the binding of hepatocyte nuclear factor-3 (HNF-3). Transfection assays showed that HNF-3 cooperates with the glucocorticoid receptor in stimulating transcription. In contrast with the lack of effect of glucocorticoids on factor binding to constitutively open GRUs of other genes, HNF-3 binding to the open PFK-2 GRU was hormone-dependent. Therefore, the PFK-2 GRU behaves as a novel type of GRU.

In vivo footprinting of the interaction of proteins with DNA and RNA.

Analysis of the interaction of proteins with either DNA or RNA sequences by in vivo footprinting involves two steps: (i) the in situ modification of nucleic acids by the footprinting reagent and (ii) the visualization of the footprints. Ligation-mediated PCR (LM-PCR) procedures provide a level of sensitivity and specificity that is suitable for visualization of footprints of single-copy genes or low-abundance mRNAs in higher eukaryotes. In this article, we discuss several of the technical aspects of these multistep procedures that contribute to the quality of the results, particularly the parameters that affect the specificity and fidelity of the reactions: (i) the design of the primers, which is important to achieve optimal specificity; (ii) the choice of polymerases so that the amplified material represents faithfully the initial nucleic acid population; and (iii) the impact of the plateau effect within the PCR on the interpretation of the data. We then discuss aspects of in vivo nucleic acid manipulation that may affect the quality of the footprinting image, in particular the choice of the footprinting reagent and its condition of use (e.g., on intact or permeabilized cells or prepared nuclei) and the extent of nucleic acid modification. Finally, we provide detailed experimental procedures corresponding to the techniques we have developed or modified: LM-PCR, reverse ligation-mediated PCR, and nuclease treatment of RNAs in vivo.

Isolation of a B-cell-specific promoter for the human class II transactivator.

The class II transactivator (CIITA) is essential for the expression of major histocompatibility complex (MHC) class II antigens. The tissular patterns of CIITA and MHC class II gene expression are tightly correlated: CIITA mRNA is highly expressed in B cells, and is induced by interferon gamma (IFN-gamma) in macrophage and epithelial cell lines. We first isolated two overlapping cosmids encoding human CIITA which, when co-transfected, are able to restore MHC class II expression in a B-lymphoblastoid cell line (B-LCL) defective for CIITA. Subsequently, a 1.8 kilobase (kb) fragment of the CIITA promoter was isolated and sequenced. A motif presenting a strong similarity to an initiator was detected, as well as putative binding sites for Sp1, GATA-2, LyF-1, ets-1, AP1, and MZF1 transcription factors, and two GAS motifs. When introduced in front of a luciferase reporter gene, this promoter is able to direct a high luciferase activity in a human B-LCL. In contrast, luciferase expression was not stimulated after IFN-gamma treatment when the construct was transfected in macrophage or in epithelial cell lines. However, an induction of the human CIITA gene was observed in mouse macrophage and fibrosarcoma cell lines, when the cells were transfected with a cosmid containing the human CIITA gene, but lacking the 1.8 kb promoter described above. Taken together, these data suggest the existence of an intragenic promoter driving an IFN-gamma-inducible expression of CIITA.

The glucocorticoid receptor regulates the binding of C/EPBbeta on the alpha-1-acid glycoprotein promoter in vivo.

A complex interaction between the Glucocorticoid Receptor (GR), C/EBPbeta, and other transcription factors activate the Alpha-1 Acid Glycoprotein (AGP) promoter in HTC(JZ-1) rat hepatoma culture cells. This effect is mediated by the so-called Steroid Responsive Unit (SRU) of the AGP promoter that contains several binding sites for C/EBP transcription factors, some of which overlap with the Glucocorticoid Responsive Element (GRE). Our in vivo footprinting experiments revealed that the GRE- and the C/EBP-binding sites were already occupied glucocorticoid dependently in HTC(JZ-1) cells 10 min after dexamethasone administration (10(-6) M). Furthermore, local changes in the chromatine structure shown by the appearance of DNAse I hypersensitive sites (HS sites) also took place. These changes were probably dependent on a tissue-specific organization of the chromatine at the SRU because they were not detectable in a different glucocorticoid-responsive cell line (PC12) that did not express AGP. Here, we have also shown that withdrawal of dexamethasone or addition of the anti-glucocorticoid RU486 were able to revert the pattern induced by dexamethasone in vivo. The disappearance of the protected region and the hypersensitive sites, typical of the hormone activated promoter, confirmed the necessity of the GR to be bound by the agonist and the inability of the GR-antagonist complex to bind the DNA. By functional assays, we showed that the occupancy of the SRU by these transcriptional proteins in vivo correlated with the activation of the AGP gene transcription. With these results, we have shown that one of the functions of the GR to activate transcription of the AGP gene is to recruit C/EBPbeta and to maintain it bound at its target DNA sequences (SRU). This process was not accomplished by RU486.

Induction of CIITA and modification of in vivo HLA-DR promoter occupancy in normal thymic epithelial cells treated with IFN-gamma: similarities and distinctions with respect to HLA-DR-constitutive B cells.

In this study, the IFN-gamma induction of MHC class II gene expression in primary cultures of thymic epithelial cells (TEC) was analyzed. This cellular system offers the advantage that MHC class II induction is studied in a "physiologic" cell lineage that, as a result of this expression within the thymus, is thought to participate to the selection and maturation of the T cells. It was found that the MHC class II gene expression was associated with the de novo transcription of the gene encoding the CIITA trans-activator, a crucial MHC class II gene regulatory factor. Furthermore, the anatomy of interaction between the MHC class II DRA promoter and corresponding binding factors was analyzed by in vivo DNAse I footprint. It was found that treatment with IFN-gamma induces changes in the occupancy of the DRA gene regulatory sequences by nuclear factors. The resulting occupancy displays strong similarities with the one observed in the MHC class II-constitutive B cells, represented by both the Burkitt lymphoma line Raji and normal tonsil- derived B cells. However, some peculiar differences were observed between the TEC, either IFN-gamma-induced or not, and the constitutive B cells. These results suggest that both common mechanisms, such as the one mediated by the CIITA trans-activator, and distinct tissue-specific constraints contribute to the transcriptional control of constitutive and IFN-gamma-induced MHC class II gene expression.

The role of hyaluronidase on lidocaine and bupivacaine pharmacokinetics after peribulbar blockade.

Orbital regional anesthesia is the only circumstance where hyaluronidase is routinely added to local anesthetics to accelerate the onset of the block. The aim of this study was to compare the pharmacokinetics of lidocaine and bupivacaine with or without hyaluronidase for peribulbar blockade. Twenty-one patients scheduled for cataract surgery with lens implantation were included in this prospective randomized study. Peribulbar blocks were achieved with plain bupivacaine 0.5% (5.5 mL), lidocaine 2% (5.5 mL), and hyaluronidase (100 IU = 2 mL) (n = 10) ir sterile water (2 mL) (n = 11). Plasma bupivacaine and lidocaine concentrations were measured by high-performance liquid chromatography at regular intervals from the end of the local anesthetic injection until the 360th minute. Maximum plasma concentration (Cmax) and time to reach Cmax (Tmax) were obtained for all the patients except one who needed a supplementary injection and was excluded from the study. The time to onset and duration of the analgesia and akinesia were monitored at the times of sampling. Motor blockade was incomplete in two patients in each group without affecting surgery. The Tmax and absorption half-life (t1/2a) of lidocaine and bupivacaine were not different within each group (P > 0.05). The Tmax of lidocaine was shorter in the presence of hyaluronidase (17.1 +/- 2.6 min vs 32.7 +/- 6.0 min) as well as the Tmax of bupivacaine (16.8 +/- 3.0 min vs 26.5 +/- 4.4 min). The Cmax of lidocaine and bupivacaine were not modified by the addition of hyaluronidase. The clearance, terminal half-life, and volume of distribution were not different between groups. The absorption of lidocaine and bupivacaine from the peribulbar space are hastened by the addition of hyaluronidase. The Tmax of lidocaine is not different from that of bupivacaine within each group suggesting that the absorption of local anesthetics is minimally influenced by the liposolubility of the drugs. Moreover, hyaluronidase influences the absorption kinetics of both lidocaine and bupivacaine in the same manner.

Divergent evolution in the mechanisms controlling major histocompatibility complex class II gene transcription in mouse and human.

The expression of the major histocompatibility complex (MHC) class II gene family is developmentally regulated and, in general, in a coordinate manner. In this study, we show that the expression of the entire repertoire of human class II genes, otherwise transcriptionally silent in the bare lymphocyte syndrome-derived BLS1 cell line, can be rescued by somatic cell hybridization with normal mouse spleen cells. The analysis of the interspecies cell hybrids revealed a particularly important and unprecedented aspect. A return to the BLS1-like, human MHC class II-negative phenotype due to segregation of mouse chromosomes was accompanied in certain hybrids by loss of IE, but not IA cell surface antigen expression. At the molecular level, this was the result of lack of E alpha-specific mRNA in the presence of E beta-, A alpha- and A beta-specific mRNA. Thus, the mouse trans-acting function operating across species barriers and able to complement the defect of human BLS1 cells diverged in mice to control Ea, but not Eb, Aa and Ab gene expression. These findings suggest that evolutionary pressure has maintained the expression of the MHC class II multigene family under the control of quite distinct species-specific transcriptional mechanisms.