Promoter hypermethylation of mismatch repair genes, hMLH1 and hMSH2 in oral squamous cell carcinoma.

OBJECTIVES: Major risk factors of oral squamous cell carcinoma (OSCC) are environmental and can lead to DNA mutagenesis. Mismatch repair (MMR) system functions to repair small DNA lesions, which can be targeted for promoter hypermethylation. We therefore wanted to test whether hypermethylation of MMR genes (hMLH1, hMSH2) could contribute to oral carcinogenesis by correlating the information to patient clinical data. METHODS: Genomic DNA was extracted from 28 OSCC and six normal oral epithelium samples. The methylation status of the two MMR genes was assessed using Methylation Specific PCR after DNA modification with sodium bisulfite. Serial sections of the same tissues were immunostained with antibodies against hMLH1 and hMSH2 protein. RESULTS: Promoter hypermethylation was observed in 14/28 OSCC cases. Remarkably, 100% of patients with multiple oral malignancies showed hypermethylation in hMLH1 or hMSH2 compared with 31.5% of single tumor patients. In 10 cancer cases, expression of the hMLH1 and hMSH2 genes by immunostaining showed reduced or absence of expression of one of the genes, although some did not reflect the methylation status. CONCLUSIONS: Hypermethylation of hMLH1 and hMSH2 might play a role in oral carcinogenesis and may be correlated with a tendency to develop multiple oral malignancies.

Early hydrocephalus in Listeria meningitis: Case report and review of the literature.

Listeria monocytogenes is a gram-positive bacterium with a predilection to infect the central nervous system, often affecting immunocompromised or elderly patients. The most common manifestations are meningitis and rhomboencephalitis. We report two cases of Listeria meningitis complicated by acute hydrocephalus several days after presentation and we further review the literature of similar cases. We conclude that acute hydrocephalus is a significant, not often recognized, complication of Listeria meningitis, usually occurring several days from onset when coverage did not include anti-Listeria antimicrobials. In high risk patients, meningitis combined with acute hydrocephalus is suggestive of LM infection.

bic, a novel gene activated by proviral insertions in avian leukosis virus-induced lymphomas, is likely to function through its noncoding RNA.

The bic locus is a common retroviral integration site in avian leukosis virus (ALV)-induced B-cell lymphomas originally identified by infection of chickens with ALVs of two different subgroups (Clurman and Hayward, Mol. Cell. Biol. 9:2657-2664, 1989). Based on its frequent association with c-myc activation and its preferential activation in metastatic tumors, the bic locus is thought to harbor a gene that can collaborate with c-myc in lymphomagenesis and presumably plays a role in late stages of tumor progression. In the present study, we have cloned and characterized two novel genes, bdw and bic, at the bic locus. bdw encoded a putative novel protein of 345 amino acids. However, its expression did not appear to be altered in tumor tissues, suggesting that it is not involved in oncogenesis. The bic gene consisted of two exons and was expressed as two spliced and alternatively polyadenylated transcripts at low levels in lymphoid/hematopoietic tissues. In tumors harboring bic integrations, proviruses drove bic gene expression by promoter insertion, resulting in high levels of expression of a chimeric RNA containing bic exon 2. Interestingly, bic lacked an extensive open reading frame, implying that it may function through its RNA. Computer analysis of RNA from small exon 2 of bic predicted extensive double-stranded structures, including a highly ordered RNA duplex between nucleotides 316 and 461. The possible role of bic in cell growth and differentiation is discussed in view of the emerging evidence that untranslated RNAs play a role in growth control.

Therapy-related leukemia: clinical characteristics and analysis of new molecular risk factors in 96 adult patients.

Therapy-related leukemia or myelodysplasia (t-leuk/MDS) is a serious problem that is increasing in frequency. We studied the clinical characteristics of 96 patients (pts) with a mean age of 48 years, and analyzed the molecular parameters that could predispose to t-leuk/MDS. Hematological malignancies were the most common primary (53%), followed by breast and ovarian cancer (30% combined). The mean latency until the development of t-AML was 45.5 months. Median survival was 10 months. Cytogenetics was abnormal in 89% of pts. FLT3 internal tandem duplications were found in six of 41 (14.6%) pts, of whom four had an abnormal karyotype. Analysis of drug metabolism and disposition genes showed a protective effect of the CYP3A4 1*B genotype against the development of t-leuk/MDS, whereas the CC genotype of MDR1 C3435T and the NAD(P)H:quinone oxidoreductase1 codon 187 polymorphism were both noncontributory. Microsatellite instability (MSI) analysis using fluoresceinated PCR with ABI sequence analyzer demonstrated that 41% of pts had high levels of MSI in four or more of 10 microsatellite loci. Immunohistochemistry demonstrated reduced expression of MSH2 and MLH1 in 6/10 pts with MSI as compared to 0/5 of pts without MSI. In conclusion, genetic predisposition as well as epigenetic events contribute to the etiology of t-AML/MDS.

The tryphostin AG17 induces apoptosis and inhibition of cdk2 activity in a lymphoma cell line that overexpresses bcl-2.

Tyrphostins are low molecular weight compounds that specifically inhibit protein tyrosine kinases. We studied the effects of tyrphostins on OCI-Ly8, a cell line derived from a patient with immunoblastic lymphoma that carries the t(14;18) translocation and overexpresses the B-cell lymphoma/leukemia-2 gene (bcl-2). To test the possibility that tyrphostins induce apoptosis in these cells, overcoming the protection rendered by bcl-2, we screened 16 tyrphostins representing different families at a concentration of 0.5-50 microM. We found that AG17 was the most potent in this regard. Cell cycle analysis demonstrated that AG17 induces arrest at the G1 phase followed by apoptosis with general reduction of the intracellular level of tyrosine-phosphorylated proteins. To further elucidate the mechanism of action of AG17, we investigated its effect on some of the key proteins that regulate the cell cycle. Bcl-2 and cdk2 protein levels were not altered with AG17, whereas cdk2 kinase activity, as well as p21 and p16 protein levels, were reduced markedly. These results suggest that the target of AG17 is inactivation of cdk2. Because lymphoma cells with the t(14;18) translocation and bcl-2 overexpression are resistant to chemotherapy, novel drugs selectively able to induce apoptosis in these cells could offer a new approach to the treatment of lymphoma patients.

Image-guided core-needle biopsy in malignant lymphoma: experience with 100 patients that suggests the technique is reliable.

PURPOSE: In an initial evaluation of 1,500 computed tomography (CT)-guided core-needle biopsies performed at our institute during the period from 1989 to 1994, we encountered 100 patients with the diagnosis of lymphoma. Here, we review the clinical impact of 109 image-guided needle biopsies in these 100 patients with non-Hodgkin's lymphoma (NHL) and Hodgkin's disease (HD). PATIENTS AND METHODS: NHL was diagnosed in 71 patients, and 29 had HD. Among the NHL patients, 17 (24%) had proven lymphoma diagnosed before the biopsy was performed; in 54 (76%) core-needle biopsy was performed as the first diagnostic procedure. Of 29 HD patients, nine (31%) were already established cases of HD, and in 20 (69%) core-needle biopsy was the first diagnostic procedure attempted. Most of the biopsies were performed under CT control using a 20- or 18-gauge Turner biopsy needle. RESULTS: Eighty-six patients received therapy based on the results of the needle biopsy alone. Fourteen patients received therapy after undergoing surgical biopsy for a suspected diagnosis of lymphoma, which could not be established with certainty on the basis of an earlier core-needle biopsy alone. In 78% of the patients, the needle biopsy saved a further surgical procedure that may have been difficult to perform because of the primary location of the tumor. CONCLUSION: From our experience in this study, image-guided core-needle biopsies provide sufficient information for the diagnosis of and subsequent therapeutic decision to treat most cases of lymphoma.

Absence of microsatellite instability during the progression of chronic myelocytic leukemia.

Microsatellites are highly polymorphic, short-tandem repeat sequences dispersed throughout the genome. To test the occurrence of genetic instability in the progression of chronic myelocytic leukemia (CML), we studied microsatellite instability (MSI) in 17 patients with CML. The DNAs from both chronic phase and blast crisis were analyzed at 10 loci. No MSI was observed in any of the 17 cases of blast crisis. These results indicate that MSI is rare and is not associated with progression to blast crisis in most cases of CML.

ABL1 methylation in Ph-positive ALL is exclusively associated with the P210 form of BCR-ABL.

In human Ph-positive leukemia there is a clear association of different forms of the BCR-ABL oncogene with distinct types of leukemia. The P190 form of BCR-ABL is rarely observed in chronic myeloid leukemia (CML) but is present in 50% of Ph-positive acute lymphoblastic leukemia (ALL). In contrast, the P210 form is observed both in CML and 50% of Ph-positive ALL. Methylation of the proximal promoter of the ABL1 gene has been shown to be a nearly universal event associated with clinical progression of CML. This raises the question of whether methylation of the ABL1 promoter is an epigenetic modification also associated with Ph-positive ALL. To study this issue, we used methylation-specific PCR and bisulfite sequencing to determine the methylation status of the ABL1 promoter in 18 Ph-positive ALL samples. We report here that gene-specific ABL1 promoter methylation is associated mainly with the P210 form of BCR-ABL and not the P190 form. While six out of the seven P210-positive ALL samples had ABL1 promoter methylation, none of the 11 P190-positive ALL samples demonstrated ABL1 promoter methylation. In addition, we estimated the extent and relative abundance of ABL1 promoter methylation in several Ph-positive ALL samples and compared it to the methylation pattern in chronic, accelerated and blastic crisis phases of CML. We put forth a model that correlates the different types of leukemias with the different levels of ABL1 promoter methylation.

Two splicing variants of a new inhibitor of apoptosis gene with different biological properties and tissue distribution pattern.

Using homology searches, we identified a novel human inhibitor of apoptosis (IAP) gene. This gene has two splicing variants that contain open reading frames of 298 and 280 amino acids and both contained a single copy of baculovirus IAP repeat (BIR) and RING domain. We refer here to the longer and shorter variants as Livin alpha and beta, respectively. Semiquantitative reverse transcriptase-polymerase chain reaction demonstrated a tissue-specific and non-correlated expression pattern in both adult and fetal tissues. Both mRNA variants were detected in various transformed cell lines. Despite their very close similarity, the two isoforms have different antiapoptotic properties. Both isoforms have a significant antiapoptotic activity in the Jurkat T cell line after triggering apoptosis via tumor necrosis factor and CD95 receptors. The Livin alpha but not beta protects cells from apoptosis induced by staurosporine, but in contrast, apoptosis initiated by etoposide was blocked only by the beta isoform. This difference in biological activities may indicate the presence of critical amino acids outside the BIR and RING domains. These functional and tissue distribution differences of Livin alpha and beta suggest that Livin may play a complex role in the regulation of apoptosis.

Ageing and the mismatch repair system.

Age-related accumulation of mutations has been extensively documented, and it has been proposed as one of the prominent causes of malignancies in old age. The present review is focused on the particular case of DNA mismatch repair system (MMR), that has drawn increased attention for its possible relevance to malignancy. We also report on our own observations on an age-associated genomic instability that develops with age in the MMR system. Our study was performed on DNA samples that were prepared from peripheral blood cells, obtained at a 10-year interval from young and old human subjects. The two DNA samples from each individual were examined comparatively. The older individuals showed a significantly higher rate of microsatellite instability (MSI) in several loci examined, whereas no difference was found between the paired samples of any of the young subjects. We suggest that this increase in MSI with age may indicate an overall genomic instability in the elderly.

Salvage therapy of refractory and relapsed acute leukemia with high dose mitoxantrone and high dose cytarabine.

We have assessed the outcome of 66 refractory and relapsed acute leukemia patients treated with high dose mitoxantrone and cytarabine. Therapy consisted of a total dose of 40-60 mg/m2 mitoxantrone and 3 g/m2 of cytarabine daily on 5 consecutive days. A total of 28 patients were treated for primary resistant and 38 patients for early or late relapsed leukemia. A total of 35 patients achieved CR. Four patients died during the induction course. Toxicity was acceptable and comparable to other salvage regimens. The median disease-free and overall survivals were 4 and 6 months, respectively. Although this regimen is effective in achieving remission in refractory leukemia, its duration is short.

dup(10q) lacking alpha-satellite DNA in bone marrow cells of a patient with acute myeloid leukemia.

A marker chromosome was identified in leukemic cells on an AML patient. The G-banding pattern resembled on i(10q), but its centromeric position was not clear; in some cells it had a telocentric shape, in others a metacentric or acentric shape. The origin of the marker chromosome was confirmed by FISH, using chromosome-10-specific painting. To determine the centromeric position, C-banding and alpha-satellite probes were applied in FISH, and none of them gave a positive signal. Despite the absence of the centromeric alpha-satellite sequences and the constricted feature of the centromere, the essential centromeric activity was retained in this chromosome, namely, the separation of sister chromatids in anaphase.

Reversed BCR/ABL rearrangement detected by FISH in Philadelphia negative chronic myelocytic leukemia.

We investigated leukemic cells from a patient with chronic myelocytic leukemia (CML) and a normal 46,XX karyotype. Molecular studies revealed rearrangement of the M-bcr region and formation of BCR/ABL fusion mRNA with b3a2 configuration. Fluorescence in situ hybridization (FISH) using the abl probe showed signal on both chromosomes 9 band q34, while the bcr probe hybridized to one chromosome 22 and to one chromosome 9. In this case, as in three other cases recently described (Hagemeijer et al. and Nachava et al.), the bcr/abl rearrangement is shown to be on 9q34, instead of the usual location on 22q11.

A novel translocation (1;2)(p34;p21-22) in acute myelomonoblastic leukemia.

A novel and as yet unrecorded translocation, (1;2)(p34;p21-22), detected in a patient with acute myeloid leukemia (AML) is reported. The leukemia--in this case, AML-M4--showed a rapidly progressive fatal course despite an early transient response to aggressive chemotherapy. In this patient, the leukemic cells showed a novel balanced translocation, (1;2)(p34;p21-22), in most of the metaphases at the time of diagnosis and during subsequent relapse. Interferon-inducible double-stranded RNA-dependent protein kinase (ds RNA-PK) is located in the chromosome region, 2p21-22, that was involved in the translocation in this case. The possible role of ds RNA-PK in leukemogenesis is briefly mentioned.

Clinical implications of fluorescence in situ hybridization analysis in 13 chronic myeloid leukemia cases: Ph-negative and variant Ph-positive.

Thirteen chronic myeloid leukemia (CML) patients, 10 with variant Philadelphia (Ph) translocations and 3 Ph negative cases, were analyzed by fluorescence in situ hybridization (FISH) with the use of BCR and ABL cosmid probes and a chromosome 22 painting probe. In the variant Ph translocations, the BCR-ABL fusion gene was located on the Ph chromosome; in 1 CML Ph-negative patient, the BCR-ABL fusion gene was located on the Ph chromosome; and, in 2 patients, it was located on chromosome 9. The chromosome 22 painting probe was detected on the third-party chromosome of the variant translocation, and in none of the variant translocations was there any detectable signal on chromosome 9. In CML patients with clonal evolution of a simple Ph, a signal of the chromosome 22 painting probe was detected on the der(9) of the Ph translocation. It was concluded that the variant Ph translocations evolved simultaneously in a three-way rearrangement. The clinical parameters of the 13 patients were similar to those of a large group of CML patients with a simple Ph translocation. It is suggested that, to determine the prognosis of CML patients with a complex karyotype, FISH analysis with a chromosome 22 painting probe be performed.

Epidural Spinal Cord Infiltration: A Rare Complication of Acute Myeloblastic Leukaemia.

Epidural infiltration by leukemic cells in Acute Non Lymphoblastic Leukemia (ANLL), and extradural granulocytic sarcomas as a cause of spinal cord compression are extremely rare phenomena. Very few cases have been recorded in the literature. In this brief case report we describe a young patient with acute myeloblastic leukemia (M4, FAB) who presented with sudden spinal cord compression during relapse after bone marrow transplantation. The literature on this subject is also reviewed.

The association of inflammatory bowel disease and leukemia--coincidence or not?

Inflammatory bowel diseases, (IBD) including Crohn's disease and ulcerative colitis, are chronic systemic disorders associated with intestinal and other systemic features. Common hematological manifestations of IBD include: anemia, hypercoagulable state, leukocytosis and thrombocytosis. Recently it has been recognized that lymphoma and leukemia can also be associated with both Crohn's disease and ulcerative colitis. Careful review of the literature reveals more than 30 cases of leukemia reported in patients with IBD. Epidemiological data show that this association is statistically significant (relative risk of 5.3; p < 0.01; 95% confidence interval). This review attempts to characterize the clinical features of this association. The etiology of leukemia in these cases seems to be multifactorial and may involve: genetic susceptibility, environmental factors, immune abnormalities, prior exposure to diagnostic radiation and a variety of therapeutic modalities. No definitive time interval, specific pattern or correlation between the extent of bowel involvement and type of leukemia was found. Treatment of leukemia might affect the activity and severity of the inflammatory bowel disease, but there is very little data relating to this issue. There is some evidence suggesting that bone marrow transplantation performed for leukemia may induce remission of both diseases. Further investigation is still required in the future in order to establish a definite relationship between these two disorders.

Expression of EBV encoded nuclear small non-polyadenylated RNA (EBER) molecules in 32 cases of childhood Burkitt's lymphoma from Israel.

We have analyzed paraffin sections from 32 children with histologically confirmed Burkitt's Lymphoma (BL) for the presence of EBV using in situ hybridization to detect expression of the EBV-encoded early RNAs (EBERs). EBV was present in the tumors of 11 patients (34%). Sixty nine percent of the children presented with abdominal disease, 19% had bone marrow infiltration and only one child had jaw involvement. There was no statistically significant difference between EBV positive and EBV negative children with regard to age, gender, origin, primary site at presentation, or clinical stage of disease. However, there was a trend for younger age in the children with EBV positive BL with a median age of 4, compared to 7 years in children with EBV negative BL. None of the 7 children of Ashkenazi Jewish origin had EBER positive disease. There was no difference in the treatment outcome between the EBV positive patients (estimated survival at 24 months of 82%) and EBV negative children (estimated survival rate of 71% (p=0.58)). In conclusion, although this is only a small series it seems that childhood BL in Israel has the clinical characteristics of sporadic, non-African type with 34% EBV association and a low incidence of jaw tumors. Our data suggest that Ashkenazi Jewish children with BL are less likely to have EBV positive tumors than other ethnic groups. However, more patients will need to be studied in order to assess the validity of this observation.

Disseminated visceral fusariosis treated with amphotericin B-phospholipid complex.

Fusariosis, a rare infectious disease of the immunocompromised host, is relatively resistant to amphotericin B (AmB) or other antifungal agents. We describe a 5-year follow-up of a 40 year old woman with T-type acute lymphoblastic leukemia who following chemotherapy developed prolonged high fever, chills, night sweats, and severe weakness. Liver function tests were impaired and abdominal computerized tomography (CT) showed multiple lesions in the liver and abnormal structure of the spleen. A laparotomy revealed multiple granulomas containing Fusarium sp. in the liver, and the spleen was heavily infiltrated by the same fungus. The patient failed to respond to the conventional AmB dosage form (Fungizone) even after a total dose of 3.0 g was given, and developed significant renal impairment. AmB was complexed (in a mole ratio of 1:16) with a mixture of the phospholipids dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol (mixed in 7:3 mole ratio). The resulting drug complex, AmB-PLC, was then administered (1-4 mg/kg/day, total dose 4.2 g) and subsequently the patient was cured of all symptoms of fusariosis. There were only mild side effects and no nephrotoxicity was evident. On the contrary, marked improvement of the renal function tests occurred during AmB-PLC treatment. Eight months later, she developed a spinal lesion with dense consistency in L5 and S1, and after receiving another course of AmB-PLC (3.1 g) she recovered completely. In a 2 year follow-up period the patient had no further relapse of the fungal disease. Subsequent chemotherapy given for relapse of the leukemia was followed by a new fungal infection, which was treated with AmB-cholesteryl sulfate complex (Amphocil).(ABSTRACT TRUNCATED AT 250 WORDS)

Candida abscess of the thyroid in a patient with acute lymphocytic leukemia.

A case of Candida abscess of the thyroid in a patient with acute lymphoblastic leukemia is described. The patient developed this rare complication after treatment with steroids and combination chemotherapy, during therapy with broad spectrum antibiotics for febrile neutropenia. Prior to the thyroiditis the patient had pulmonary aspergillosis. The abscess developed during treatment with high dose Amphotericin B. Unlike previous cases, the Candida was isolated to the thyroid, with no evidence of Candidemia or Candida infection in other sites.