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Competing Interests: Conflict-of-interest disclosure: C.S.C. holds stock in Adaptive Biotechnologies, Inc. The remaining authors declare no competing financial interests.

Burkitt lymphoma (BL), the most common pediatric cancer in sub-Saharan Africa, is a malignancy of antigen-experienced B lymphocytes. High-throughput sequencing (HTS) of the immunoglobulin heavy ( IGH ) and light chain ( IGK / IGL ) loci was performed on genomic DNA from 51 primary BL tumors: 19 from Uganda and 32 from Ghana. Reverse transcription polymerase chain reaction analysis and tumor RNA sequencing (RNAseq) was performed on the Ugandan tumors to confirm and extend the findings from the HTS of tumor DNA. Clonal IGH and IGK / IGL rearrangements were identified in 41 and 46 tumors, respectively. Evidence for rearrangement of the second IGH allele was observed in only 6 of 41 tumor samples with a clonal IGH rearrangement, suggesting that the normal process of biallelic IGHD to IGHJ diversity-joining (DJ) rearrangement is often disrupted in BL progenitor cells. Most tumors, including those with a sole dominant, nonexpressed DJ rearrangement, contained many IGH and IGK / IGL sequences that differed from the dominant rearrangement by < 10 nucleotides, suggesting that the target of ongoing mutagenesis of these loci in BL tumor cells is not limited to expressed alleles. IGHV usage in both BL tumor cohorts revealed enrichment for IGHV genes that are infrequently used in memory B cells from healthy subjects. Analysis of publicly available DNA sequencing and RNAseq data revealed that these same IGHV genes were overrepresented in dominant tumor-associated IGH rearrangements in several independent BL tumor cohorts. These data suggest that BL derives from an abnormal B-cell progenitor and that aberrant mutational processes are active on the immunoglobulin loci in BL cells., Competing Interests: Conflict-of-interest disclosure: C.S.C. holds stock in Adaptive Biotechnologies, Inc. The remaining authors declare no competing financial interests.

Background and Purpose: ALL is the most common pediatric cancer. The causes of the majority of pediatric acute leukemia are unknown and are likely to involve an interaction between genetic and environmental factors. Therefore, unfavourable gene-environmental interactions might be involved in the genesis of ALL. The aim of this work was to evaluate, in a case-control study, whether the common polymorphisms in 5, 10-methylenetetrahydrofolate reductase (MTHFR) namely (C677T and A1298C) and methionine synthase (MS) (A2756G) genes may play a role in altering susceptibility to pediatric ALL as individual genes and in combination., Patients and Methods: DNA of 88 ALL patients (age < or = 18 years) and 311 healthy control subjects was analyzed for the polymorphisms of MTHFR and MS genes using PCR-RFLP method., Results: The frequencies of the wild types of MTHFR 677CC, MTHFR 1298AA and MS 2756AA, the homozygous genotypes of MTHFR 677TT, MTHFR 1298CC and MS 2756GG and heterozygous genotypes of MTHFR 677CT and MS 2756AG showed no statistically significant differences between patients and controls. The frequency of the MTHFR 1298AC heterozygous genotype was 25% among patients compared to 45.0% among controls; the difference was found to be statistically significant (p value =0.001, O.R=0.382 & 95% C.I=0.222-0.658). The frequency of the MTHFR1298AC heterozygous genotype plus 1298CC homozygous genotype was 34% among patients compared to 54.3% among controls and the difference was statistically significant (p value =0.001). A synergistic effect of 677CT and1298AC (CTAC) was observed, (p value=0.002) with 3.65 fold protection (OR 0.273 & 95% C.I=0.155-0.9) compared to 2.6 folds for MTHFR 1298AC alone. This protective effect of CTAC polymorphism was abolished when combined with MS 2756AA or AG., Conclusion: The present study provided further evidence for the protective role of MTHFR 1298AC mutant alleles in acute lymphoblastic leukemia in children (2.6 fold protection). This suggests that folate and methionine metabolism play an important role in the pathogenesis of pediatric ALL. In contrast to the main bulk of literature, we did not find any protective role of either MTHFR C677T or MS A2756G polymorphisms. This may reflect the ethnic variation in both the polymorphism frequencies, variation in plasma level of folate, in addition to the possible role of gene-environment interaction mainly dietary availability of folate. The synergistic effect of MTHFR 1298AC and 677CT and its abolishment by MS 2756AA or AG further emphasizes that the interaction of genes, rather than the polymorphism in any single one, determines risk susceptibility to disease.

Microarray analysis is a powerful technology, but its impact on routine diagnosis for the near future maybe in revealing individual genes, which are useful diagnostic markers. Recently microarray analysis has identified a novel subgroup of childhood precursor-B acute lymphoblastic leukaemia (ALL) from a unique gene expression profile of over 30 genes. We have evaluated the four most highly expressed genes from this profile, by quantitative real time RT-PCR, to determine whether any of these genes by itself could be useful as a diagnostic indicator. The levels of expression of N-acetylglucosamine-6-O-sulfotransferase (GN6ST), protein tyrosine phosphatase receptor M (PTPRmu), G protein-coupled receptor 49 (HG38) and KIAA1099 protein were determined in childhood precursor-B ALL samples from a cohort of 116 Indian patients. In nine cases, three or four of these genes exhibited very high expression levels, but only GN6ST was consistently over-expressed. We suggest that very high level expression of GN6ST is a useful diagnostic marker for a subgroup of previously unclassified ALL.

The mechanisms that regulate induction of the antiapoptotic state and mitogenic signals in primary effusion lymphoma (PEL) are not well known. In efforts to identify novel approaches to block the proliferation of PEL cells, we found that curcumin (diferuloylmethane), a natural compound isolated from the plant Curcuma Ionga, inhibits cell proliferation and induces apoptosis in a dose dependent manner in several PEL cell lines. Such effects of curcumin appear to result from suppression of the constitutively active STAT3 through inhibition of Janus kinase 1 (JAK1). Our data also demonstrate that curcumin induces loss of mitochondrial membrane potential with subsequent release of cytochrome c and activation of caspase-3, followed by polyadenosin-5'-diphosphate-ribose polymerase (PARP) cleavage. Altogether, our findings suggest a novel function for curcumin, acting as a suppressor of JAK-1 and STAT3 activation in PEL cells, leading to inhibition of proliferation and induction of caspase-dependent apoptosis. Therefore, curcumin may have a future therapeutic role in PEL and possibly other malignancies with constitutive activation of STAT3.

Purpose: Phosphatidylinositol 3'-kinase (PI3'-kinase) can be activated by the K1 protein of Kaposi sarcoma-associated herpes virus (KSHV). However, the role of PI3'-kinase in KSHV-associated primary effusion lymphoma (PEL) is not known. To assess this, we studied survival and apoptosis in PEL cell lines following inhibition of PI3'-kinase., Experimental Design: Constitutive activation of several targets of PI3-kinase and apoptotic proteins were determined by Western blot analysis using specific antibodies. We used LY294002 to block PI3'-kinase/AKT activation and assess apoptosis by flow cytometric analysis., Results: Blocking PI3'-kinase induced apoptosis in PEL cells, including BC1, BC3, BCBL1, and HBL6, whereas BCP1 was refractory to LY294002-induced apoptosis. LY294002-induced apoptosis did not seem to involve Fas/Fas-L but had an additive effect to CH11-mediated apoptosis. We also show that AKT/PKB is constitutively activated in all PELs and treatment with LY294002 causes complete dephosphorylation in all cell lines except BCP1 where a residual AKT phosphorylation remained after 24 hours of treatment. FKHR and GSK3 were also constitutively phosphorylated in PELs and treatment with LY294002 caused their dephosphorylation. Although inhibition of PI3'-kinase induced cleavage of BID in all cell lines, cytochrome c was released from the mitochondria and caspase-9 and caspase-3 were activated in LY294002-induced apoptotic BC1 but not in resistant BCP1. Similarly, XIAP, a target of AKT, was down-regulated after LY294002 treatment only in sensitive PEL cells., Conclusions: Our data show that the PI3'-kinase pathway plays a major role in survival of PEL cells and suggest that this cascade may be a promising target for therapeutic intervention in primary effusion lymphomas.

Colorectal cancer CRC is one of the most common malignancies worldwide. Advances in molecular techniques have provided deep insight into the molecular pathogenesis, biologic and genetic changes occurring in colon cancer patients. Current theories of malignant transformation postulate that development of colon cancer is related to 2 main pathways; the loss of heterozygosity pathway, which is usually due to a defect in the adenomatous polyposis coli APC gene and microsatellite instability, which is usually due to a defect in mismatch repair MMR genes. This review summarizes the role of the wingless signaling pathway genes including APC and MMR genes in the development of CRC.

We examined the functional role of the phosphatidylinositol 3'-kinase pathway in the growth and survival of cell lines of T-cell origin. Pharmacological inhibition of PI3'-kinase using LY294002 resulted in apoptosis of acute lymphoblastic T-cell leukemia (T-ALL) cell lines including CEM, Jurkat, and MOLT-4. On the other hand, the cutaneous T-cell lymphoma cell line HUT-78 was found to be refractory to LY294002- inducible apoptosis. Sensitivity or resistance to pharmacological inhibitors of PI3'-kinase correlated with tumor suppressor PTEN gene expression, as sensitive T-ALL cells do not express PTEN and have high level of activated AKT, in contrast to HUT-78 cells. Our data demonstrate that inhibition of PI3'-kinase results in dephosphorylation of AKT and partial inhibition of Bcl-xL expression in T-ALL cells, but not in HUT-78 cells. Interestingly, HUT-78 cells were also found to express higher levels of Bcl-xL protein as compared to T-ALL cells. Inhibition of PI3'-kinase also induces release of cytochrome c from mitochondria and activation of caspase-3 and PARP in all T-ALL cell lines tested, but not in HUT-78 cells. Taken altogether, our data demonstrate that the PI3'-kinase/AKT pathway plays a major role in the growth and survival of PTEN-null T-ALL cells, and identify this cascade as promising target for therapeutic intervention in acute T-cell leukemias.

In patients (pts) with non-Hodgkin's lymphoma (NHL) under 25 years, treatment with MCP-842 protocol, a short duration intense protocol, yields worse survival in pts with lymphoblastic lymphoma (LL) compared to other high grade lymphomas. In order to identify both favourable and unfavourable subgroups in pts with T-cell LL (T-LL) with respect to relapse free survival following treatment with MCP-842 protocol, we analysed the expression of p53 and bcl-2 proteins in 22 pts with T-LL treated at the Tata Memorial Hospital, Mumbai by immunohistochemistry. p53 protein overexpression was noted in 59% cases and bcl-2 overexpression was noted in 29.4% cases. p53 expression correlated with a higher rate of relapse (p = 0.03; RR 7.9). The 5-year relapse free survival (RFS) was better in p53 negative patients compared to positive patients (70 vs 38%) (log-rank sigma = 0.04). In conclusion, in this study, overexpression of p53 protein was common in patients with T-LL. T-LL pts negative for p53 are likely to benefit from the short intense protocol--MCL-842. Bcl-2 protein overexpression was not a prognostic factor in these patients.

Patients suffer with various abnormalities of body tissues. "Leiomyoma Uteri" is a solid tumor of uterus, one of such abnormalities. To treat such tumors, basic physical and biologic investigations are required to be carried out. In the present work, ultrasonic characterization of soft tissues, in this case, uterine tumor in vitro, are studied. A double probe through-transmission technique is used for the measurement of these propagation parameters, viz., velocity and attenuation. Other parameters like acoustic impedance, dynamic modulus of elasticity and compressibility are also determined. The average velocity and attenuation are found to be 1550 ms(-1) and 433 dBm(-1), respectively, at 3.5 MHz frequency and room temperature 28 degrees C. The present investigation is useful in tissue differentiation and tissue identification to enable the doctors to give proper treatment.

Purpose: Langerhans cell histiocytosis (LCH) is a disorder of unknown etiology involving the proliferation and accumulation of cells with the phenotype of a bone marrow-derived antigen-presenting cell of the skin, the Langerhans cell. We have studied p53 expression, an element in the control of cell proliferation, to determine whether it plays a role in the pathogenesis of LCH., Patients and Methods: LCH lesions from 10 patients with either localized (n = 5) or multisystem disease (n = 5) were studied. p53 protein expression was assessed by immunohistochemistry, and p53 gene mutation by the single strand conformation polymorphism (SSCP) technique., Results: p53 protein expression was detected in all 10 LCH biopsy specimens examined. It was restricted to Langerhans cells (LCH cells), absent from adjacent cells, and localized to the cell nuclei. No mutations of the p53 gene were detected, nor was there abnormal expression of the p53 binding protein, mdm2., Conclusions: p53 is readily detectable in LCH cells but not in normal cells. This is either caused by an unusual mechanism (given the absence of mutations in the p53 gene and of mdm2 expression in LCH cells) or by overexpression or posttranslational changes of normal p53 in response to an as yet unidentified cellular stress. Stabilization and inactivation of p53 could lead to the uncontrolled proliferation of LCH cells, or the abnormality could lead to the induction of programmed cell death.

Burkitt's lymphoma (BL) cell lines carry a translocated c-myc gene and, in 60-80% of cases, exhibit mutations in the p53 tumor suppressor gene. We examined the potential role of the p53 gene in BL tumorigenicity using an in vitro assay that measures p53-dependent cell cycle arrest in the G1 phase of the cell cycle and an in vivo athymic murine model that detects differences in the tumorigenicity of BL cell lines. A highly significant inverse correlation was found between the ability of BL cells to arrest in G1 after irradiation and their tumorigenicity in athymic mice, consistent with the notion that loss of p53 function is associated with increased tumorigenicity. Inactivation of wild-type (wt) p53 function by expression of the human papillomavirus E6 protein in the AG876V BL cell line, which carries both wt and mutant p53 proteins, rendered the cell line significantly more tumorigenic in athymic mice. Transfection of the wt p53 gene into the p53 mutant and highly tumorigenic BL-41 cell line caused it to acquire wt p53 function and rendered it less tumorigenic in mice. In addition to confirming a role for the loss of p53 function in tumor progression, the data demonstrate that wt p53 protein can reduce BL tumorigenicity in vivo.

About one fourth of patients with Hodgkin's disease relapse after therapy. The mechanisms that lead to resistance to treatment in these patients are poorly understood. The authors describe the differential protein expression of p53, proliferating cell nuclear antigen (PCNA), and p21 at initial presentation and relapse, and discuss their role in disease progression and resistance to therapy. Thirty-four patients with Hodgkin's disease who had relapsed after standard chemotherapy and radiotherapy regimens were assessed for the expression of p53 protein, PCNA, and p21 protein (waf/cip 1). In 14 of these cases, sequential biopsies performed both at presentation and at relapse were available for the study. Seventy-five percent of the cases were positive for the p53 protein. Tumors at relapse had higher p53 and PCNA scores than those at initial presentation. In the paired samples, a significant increase was noted in the number of p53 and PCNA-positive cells and in the intensity of staining with p53 antibody. Six of seven paired samples tested for p21 showed an increased p21 expression at relapse. These results suggest that, at relapse, Reed-Sternberg (RS) cells and their variants positive for p53, PCNA, and p21 are increased in number and individually have an increased expression of p53, PCNA, and p21 proteins. These findings suggest that therapy failure and relapse may, at least in part, be associated with altered p53, p21, and PCNA pathways. HUM PATHOL 28:549-555. This work was carried out during an exchange fellowship program at the National Cancer Institute, Bethesda. There are no restrictions on its use

Purpose. Rhabdomyosarcoma (RMS) is an embryonal tumor thought to arise from skeletal muscle cells that fail to differentiate terminally. The majority of RMSs express MyoD, a protein essential to the differentiation of skeletal muscle. It was recently shown that during myogenesis, MyoD activates the expression of the cyclin-dependent kinase inhibitor (CDKi), p21, which itself plays a critical role in normal muscle development. To investigate the integrity of the MyoD/p21 pathway in RMS, we analyzed p21 and its relationship to MyoD expression in RMS.Methods. A panel of RMS samples was assembled from primary biopsies and from cell lines. Integrity of p21 was analyzed by single-strand conformation polymorphism (SSCP) and sequencing. Expression of p21 and MyoD was determined by Northern blot analysis, and the ability of exogenous p21 to arrest the cell cycle of RMS cell line was determined by transfection studies.Results. Our analysis indicates that although p21 is wild type in RMS, there is an inverse correlation between the levels of p21 and MyoD in these tumors. Tumors that express significant amounts of MyoD fail to express p21. This does not appear to be the result of mutations within the potential CACGTG sites present in the p21 promoter region or in the coding region of p21. An additional group of RMSs express very high levels of p21 but express little, if any, MyoD. Furthermore, RD, a RMS cell line which expresses high levels of endogenous p21, undergoes withdrawal from the cell cycle following forced expression of p21, suggesting that the pathway which would lead to G(1) arrest from endogenous p21 activity is defective.Discussion. These data suggest that the interaction between p21 and MyoD is defective in RMS although the precise nature of the defect remains to be elucidated.

We describe an EBV-driven lytic system (LySED) that can be used to specifically target therapy to EBV- containing tumors. This system takes advantage of the transactivating properties of EBNA-1, a latency protein expressed in all EBV-containing cells, to drive the expression of Zta, a gene sufficient for inducing the EBV lytic cycle. Thus, EBV provides both the target and the executor for mediating tumor-specific cell death, markedly increasing the specificity of the system. Transfection of EBV-positive cell lines with the LySED construct resulted in a switch to lytic cycle and subsequent cell death, even in the presence of an inhibitor of EBV thymidine kinase (acyclovir) without an increase in virion production. In contrast, growth of EBV-negative B-cell lines was not affected.

p53, a tumor suppressor gene, is frequently mutated in sporadic human cancer, and inherited mutations in p53 predispose to the early onset of cancer. p53 mutations occur frequently in sporadic lymphoma, and, in mice deficient for p53, lymphoma is the most common type of malignancy. Families with an increased incidence of lymphoma have been described, suggesting an inherited predisposition to lymphoma in these circumstances. To determine whether the predisposition to lymphoma in these families results from germline mutations in p53, we analysed exons 4-11 of the p53 gene in 35 individuals from 19 lymphoma-prone kindreds. We found no germline p53 mutations in any of the individuals tested. However, p53 expression assessed by immunohistochemistry, which suggests mutation, was observed in 35% of the tumor samples from the familial Hodgkin's disease cases and in 13% of the familial non-Hodgkin's lymphoma cases. These results suggest that p53 mutations do not play a critical role in heritable susceptibility to lymphoma. p53 may act by different, non-mutation related mechanisms in this setting, or be involved in late events in the pathogenesis of these tumors.

The poly(ADP-ribose) polymerase (PADPRP) gene (13q33-qter) depicts a two-allele (A/B) polymorphism. In the noncancer population, the frequency of the B allele is higher among blacks than among whites. Since the incidence of multiple myeloma and prostate and lung cancer is higher in the U.S. black population, we have analyzed the B-allele frequency in germ-line DNA to determine whether the PADPRP gene correlates with a polymorphic susceptibility to these diseases. For multiple myeloma and prostate cancer, an increased frequency of the B allele appeared to be striking only in black patients. In contrast, the distribution of the B allele in germ-line DNA did not differ among white patients with these diseases, when compared with the control group. An elevated B-allele frequency was also found in germ-line DNA in blacks with colon cancer. These observations suggest that the PADPRP polymorphism may provide a valid marker for a predisposition to these cancers in black individuals. To determine the genomic structure of the polymorphic PADPRP sequences, a 2.68-kb HindIII clone was isolated and sequenced from a chromosome 13-enriched library. Sequence analysis of this clone (A allele) revealed a close sequence similarity (91.8%) to PADPRP cDNA (1q42) and an absence of introns, suggesting that the gene on 13q exists as a processed pseudogene. A 193-bp conserved duplicated region within the A allele was identified as the source of the polymorphism. The nucleotide differences between the PADPRP gene on chromosome 13 and related PADPRP genes were exploited to develop oligonucleotides that can detect the difference between the A/B genotypes in a PCR. This PCR assay offers the opportunity for analyzing additional black cancer patients, to determine how the PADPRP processed pseudogene or an unidentified gene that cosegregates with the PADPRP gene might be involved with the development of malignancy.

Available evidence suggests that, among hematological malignancies, p53 is most often mutated in Burkitt's lymphoma (BL). However, much of the published data is based on cell lines. We have, therefore, analyzed BL biopsies to determine more accurately the frequency and pattern of p53 mutations in primary tumors and to determine whether there are differences among the various subtypes of BL. Among 27 BL biopsies from South Africa, we have observed mutations in the p53 gene (exons 5 through 8) in 37% of tumors. The higher frequency of mutations in cell lines (70%) suggests that mutation of p53 may be associated with tumor progression. Summarizing available data we conclude that the presence of mutated p53 in BL is independent of the geographic origin of the tumor, the 8;14 chromosomal breakpoint locations and Epstein-Barr virus association. We also find that the mutational spectrum of p53 in BL differs from that observed in nonlymphoid tumors. More than 50% of mutations in BL are clustered in a small stretch of 33 amino acids (codons 213 to 248). Interestingly, codon 213 appears to be as frequently mutated as codon 248. Conversely, codon 273, often mutated in solid tumors, is rarely involved in BL.

The nuclear enzyme poly(ADP-ribose) polymerase (PADPRP) is thought to play a role in DNA recombination, replication, and repair. In view of the implication of these processes in tumorigenesis, and based on preliminary evidence which indicated the presence of an extraneous polymorphic restriction fragment for murine PADPRP loci in strains of mice susceptible to plasmacytomas, we investigated correlations between the restriction fragment length polymorphism of the PADPRP gene(s) and human Burkitt lymphoma. No increase in the frequency of polymorphisms on chromosome 1 (containing the active gene) or on chromosome 14 (a pseudogene) was observed. However, restriction fragment length polymorphism analysis of PADPRP sequences on chromosome 13 (either a processed pseudogene or a gene with extensive identity to PADPRP) revealed that of 19 DNA samples derived from endemic Burkitt lymphoma all contained at least one copy of a rare allele (B). Simple two-allele (A/B) polymorphisms in this PADPRP-like locus were identified by digestion with a number of restriction enzymes including HindIII, PstI, KpnI, and MspI. These restriction fragment length polymorphisms always segregated together, suggesting that they identify a deletion within or close to the PADPRP sequences on chromosome 13, which we mapped precisely to 13q33-qter. Based upon family studies the A and B alleles were shown to be transferred in a Mendelian codominant fashion. Subsequently, this probe was used as a linkage marker to study the frequency of this deletion in various tumors including B-cell follicular lymphomas, small cell lung carcinomas, breast carcinomas, and colorectal carcinomas. In noncancer control populations, the frequency of this deletion was 3-fold higher among Blacks as compared to Caucasians. When DNA from various tumors was compared to normal DNA from racially appropriate noncancer controls, the frequency of this deletion was still 2- to 3-fold higher in the tumor DNA. Matched samples provided instances of tumor-specific loss of heterozygosity but also revealed that the predominant source of this deletion is the germ line, suggesting that the chromosome 13 region neighboring the PADPRP locus may harbor a gene whose loss may predispose individuals to malignancy.

Ewing's sarcoma (ES) is a highly malignant childhood bone tumor and is considered curable by moderate doses of radiotherapy. The addition of chemical inhibitors of the activity of the nuclear enzyme poly(adenosine diphosphate ribose) [poly(ADPR)] polymerase to ES cells in culture results in increased cell killing, a phenomenon called "inhibitor sensitization." Since poly(ADPR) polymerase is thought to be associated with DNA repair, it has been suggested that ES cells and other inhibitor-sensitized cells may have a reduced capacity for polymer synthesis resulting in deficient postirradiation recovery. We present here the unexpected observation that in comparison to other cell lines tested, ES cells exhibit a high enzyme activity, higher constitutive levels of the protein, and elevated levels of its mRNA transcript for poly(ADPR) polymerase. No gross amplifications or rearrangements of the gene were observed; however, regulation of poly(ADPR) polymerase in these tumor cells takes place at the level of the gene transcript.

The cytotoxic effects of acetylated oil of Semecarpus anacardium nuts on the cells of P388 lymphocytic leukemia were tested in vitro. The product was tested at the concentrations ranging from 15-75 micrograms/ml. The cell kill was observed as early as three hr after the treatment. The effects of acetylated oil on the biosynthesis of DNA, RNA and protein using labelled thymidine, uridine and leucine respectively showed that the product inhibited the biosynthesis of all the three. This was indicated by the inhibition of the incorporation of their precursors. The uptake of 3H-thymidine was inhibited 15 min after treatment; while that of 3H-uridine and 14C-leucine took 30 and 45 min respectively. Since the S. anacardium oil was unstable due to air-oxidation, the studies were confined to its acetylated product.