Overexpressed proteins may act as mops removing their ligands from the host cells: a case study of calf PNP.

Calf purine nucleoside phosphorylase (PNP) was overexpressed in Escherichia coli. The basic kinetic parameters of recombinant PNP were found to be similar to the values published previously for non-recombinant PNP from calf spleen. However, upon titration of the recombinant enzyme with the tight-binding multisubstrate analogue inhibitor DFPP-DG, endothermic as well as exothermic signals were obtained. This was not the case for PNP isolated from calf spleen for which only the endothermic process was observed. Further calorimetric titrations of the recombinant and non-recombinant enzyme with its potent and moderate ligands, and studied involving partial inactivation of the enzyme, lead to the conclusion that a part of the recombinant enzyme forms a complex with its product, hypoxanthine, although hypoxanthine was not present at any purification stage except for its natural occurrence in E. coli cells. Binding of hypoxanthine is accompanied with a large negative change of the free enthalpy, and therefore the replacement of this compound by DFPP-DG yields positive heat signal. Our data obtained with calf PNP indicate that similar processes--moping of ligands from the host cells--may take place in the case of other proteins with high overexpression yield.

Malignant mesothelioma in situ diagnosed by methylthioadenosine phosphorylase loss and homozygous deletion of CDKN2A: a case report.

Malignant pleural mesothelioma (MPM), associated with unfavorable outcomes, is closely associated with asbestos exposure. Early detection and treatment are critical to prolong survival of patients with MPM because of the rapid progression and resistance to treatment. The recently defined malignant mesothelioma in situ (MIS) has been gaining increasing attention with advances in genome-based methods including fluorescence in situ hybridization (FISH) as well as immunohistochemistry. We herein report the case of a MIS in a 73-year-old male with a history of asbestos exposure presenting with massive pleural effusion in the right thoracic cavity. Video-assisted thoracoscopic surgery with pleural biopsy of the right side revealed a single layer of atypical mesothelial cells without invasive lesions by hematoxylin and eosin staining. However, these mesothelial cells exhibited a loss of methylthioadenosine phosphorylase (MTAP) by immunohistochemistry and homozygous deletion of CDKN2A (p16) by FISH, leading to the diagnosis of MIS.

Efficient bacterial expression of fusion proteins and their selective processing by a recombinant Kex-1 protease.

A secreted, soluble variant of the Kex-1 endopeptidase from Kluyveromyces lactis has been produced and studied as a novel cleavage enzyme exhibiting high specificity for the Lys-Arg peptide. This highly selective, efficient enzyme is particularly adapted for use in manufacturing when a recombinant therapeutic protein, possessing its native N-terminus, has to be released in vitro from a bacterially-expressed fusion protein. In this paper, we describe the preparation of a Kex-1 variant using Saccharomyces cerevisiae and its application in the production of important therapeutic recombinant proteins such as human growth hormone, granulocyte colony-stimulating factor and interferon-alpha-2b.

Characterization of methylthioadenosin phosphorylase (MTAP) expression in colorectal cancer.

PURPOSE: Tumour seriously affects people's quality of life. Colorectal cancer is a refractory tumour in digestive tract tumors. In colorectal cancer, gene expression abnormalities is the main reason for its incidence, we mainly focus on the molecular mechanism of MTAP in the development of colorectal cancer. METHODS: The tumour tissue and its adjacent tissue samples of 50 patients with colorectal cancer were screened from July 2011 to February 2015, and the expression of MTAP was detected. Cell lines that overexpress MTAP and low expression of MTAP were constructed in colorectal cancer cell lines. The cell proliferation, invasion and migration was detected in the cells with different expression levels of MTAP. Immunohistochemistry was used to detect the expression of MTAP in liver metastasis and to investigate its clinical significance. And statistics of clinical significance. RESULTS: Q-PCR results showed that the expression of MTAP in colorectal cancer cell lines were significantly higher than that normal human colonic myofibroblasts cell line. Cell proliferation test results showed that cell proliferation was accelerated when MTAP was overexpression, cell invasion and migration were simultaneously accelerated. The expression of MTAP in primary liver was positively correlated with metastatic disease in patients with liver metastatic colorectal cancer via EMT. CONCLUSIONS: MTAP accelerates the growth and metastasis of colorectal cancer through EMT.

Expression of a cDNA sequence encoding human purine nucleoside phosphorylase in rodent and human cells.

A cDNA sequence which contains the entire coding region for human purine nucleoside phosphorylase (PNP) was recombined for selection and expression in mammalian cells. Plasmids containing either the simian virus 40 early promoter or the mouse metallothionein promoter positioned just upstream of the PNP coding sequence were constructed. These plasmids also contained the gene for a methotrexate-resistant dihydrofolate reductase, allowing for selection and amplification of positive transferrents after transfection of cells by the DNA-calcium phosphate coprecipitation technique. Expression of human PNP activity was readily detected in both mouse (L) and CHO cells by isoelectric focusing of cell extracts followed by histochemical staining for PNP activity. The simian virus 40 early promoter directed considerable expression of human PNP activity in CHO cells but only scant activity in mouse cells. The mouse metallothionein promoter was not successful in effecting human PNP expression in CHO cells but provided substantial human PNP activity in mouse cells and was inducible by incubation with zinc. HeLa cell transferrents were isolated and screened for the presence of transferred PNP cDNA sequences by Southern hybridization analysis. RNA transcripts derived from the transferred PNP cDNA were identified in one of these cell lines.

The effect of a novel transition state inhibitor of methylthioadenosine phosphorylase on pemetrexed activity.

Pemetrexed is a new-generation antifolate inhibitor of thymidylate synthase (TS) and a weaker inhibitor of glycinamide ribonucleotide transformylase (GARFT) required for de novo purine synthesis. Methylthioadenosine phosphorylase (MTAP) salvages purines by releasing adenine from methylthioadenosine and is often deleted in mesothelioma. The current study addresses the effect of MTAP on pemetrexed activity using a highly potent transition state inhibitor of MTAP, MT-DADMe-Immucillin A (ImmA; K(i) = 86 pmol/L) in the MTAP(+) NCI-H28 and MTAP(-) NCI-H2052 mesothelioma cell lines. Based on selective nucleoside protection, TS was found to be the primary pemetrexed target in both cell lines with GARFT inhibition requiring 20- to 30-fold higher pemetrexed concentrations. ImmA had no effect on pemetrexed activity but, when thymidine was added, the pemetrexed IC(50) decreased by a factor of approximately 3 in MTAP(+) H28 cells with no effect in MTAP(-) H2052 cells. Conversely, the transfection of MTAP into H2052 cells increased the pemetrexed IC(50) by nearly 3-fold but only in the presence of thymidine; this was reversed by ImmA. An MTAP-specific short interfering RNA produced a 2-fold decrease in pemetrexed IC(50) in MTAP(+) HeLa cells in the presence of thymidine. These data indicate that suppression of constitutive MTAP has no effect on pemetrexed activity when the primary target is TS. There is a modest salutary effect when the pemetrexed target is GARFT alone.

Antitumor effects of a dual-specific lentiviral vector carrying the Escherichia coli purine nucleoside phosphorylase gene.

The Escherichia coli purine nucleoside phosphorylase/Fludarabine phosphate (ePNP/Fludara) suicide system has several drawbacks, such as side-effects and the low efficiency of ePNP expression. In this study, we evaluated the antitumor effects of the dual-specific 8HSEs-hTERTp-ePNP/Fludara suicide system under hyperthermia in vitro and in vivo. Luciferase activities from the 8HSEs­hTERT and CMV promoters were compared using the dual luciferase assay in SW480 (high hTERT expression) and MKN74 cells (hTERT-negative) in the presence and absence of hyperthermia. Then, we investigated the effects of overexpressing the suicide gene ePNP using 8HSEs­hTERT-driven lentiviral vectors with Fludara on in vitro cell viability, side-effects, apoptosis, cycle distribution, colony formation and in vivo xenograft tumor growth. At 43˚C, luciferase activity from the 8HSEs­hTERT promoter was significantly increased in SW480 cells, but not in MKN74 cells. Importantly, luciferase activities from the 8HSEs­hTERT promoter were much higher than from the CMV promoter in hTERT-expressing SW480 cells under heated conditions. The in vitro quantitative analysis showed a 4-fold higher ePNP protein expression from the 8HSEs­hTERT promoter at 43˚C than at 37˚C in SW480 cells and the ePNP mRNA expression in SW480 cells at 43˚C was also higher than at 37˚C. Conversely, ePNP mRNA and protein expression were low, almost absent, in hTERT-negative MKN74 cells with or without hyperthermia. After Fludara addition, cell cytotoxicity assays showed that the significant inhibitory effect of the 8HSEs­hTERTp-ePNP on SW480 cells was dose- and time-dependent with hyperthermia. The 8HSEs­hTERTp-ePNP/Fludara suicide system significantly inhibited SW480 cell viability, colony formation, cell cycle progression and induced apoptosis in vitro, but also induced significant bystander effects, especially under the heated conditions. At the protein level, the suicide system significantly promoted Bax, caspase-3 and p53 expression and suppressed Bcl-2 expression. In sections from mouse xenografts, TUNEL assays showed that the suicide system reduced xenograft growth and induced SW480 apoptosis. These results indicated that the combinatorial cancer- and heat-specific promoter system has great potential for improving the efficacy of cancer treatment with hyperthermia. The 8HSEs­hTERTp-ePNP/Fludara system may serve as a powerful strategy for cancer gene therapy combined with hyperthermia.

Differences in activities of the enzymes of nucleotide metabolism and its implications for cardiac xenotransplantation.

Xenotransplantation is one be possible solution for a severe shortage of human organs available for transplantation. However, only a few studies addressed metabolic compatibility of transplanted animal organs. Our aim was to compare activities of adenosine metabolizing enzymes in the heart of different species that are relevant to clinical or experimental xenotransplantation. We noted fundamental differences: ecto-5' nucleotidease (E5' N) activity was 4-fold lower in pig and baboon hearts compared to the human hearts while mouse activity was compatible with human and rat activity was three times higher than human. There also were significant differences in AMP-deaminase (AMPD), adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities. We conclude that differences in nucleotide metabolism may contribute to organ dysfunction after xenotransplantation.

Excellent in vivo bystander activity of fludarabine phosphate against human glioma xenografts that express the escherichia coli purine nucleoside phosphorylase gene.

Escherichia coli purine nucleoside phosphorylase (PNP) expressed in tumors converts relatively nontoxic prodrugs into membrane-permeant cytotoxic compounds with high bystander activity. In the present study, we examined tumor regressions resulting from treatment with E. coli PNP and fludarabine phosphate (F-araAMP), a clinically approved compound used in the treatment of hematologic malignancies. We tested bystander killing with an adenoviral construct expressing E. coli PNP and then more formally examined thresholds for the bystander effect, using both MuLv and lentiviral vectoring. Because of the importance of understanding the mechanism of bystander action and the limits to this anticancer strategy, we also evaluated in vivo variables related to the expression of E. coli PNP (level of E. coli PNP activity in tumors, ectopic expression in liver, percentage of tumor cells transduced in situ, and accumulation of active metabolites in tumors). Our results indicate that F-araAMP confers excellent in vivo dose-dependent inhibition of bystander tumor cells, including strong responses in subcutaneous human glioma xenografts when 95 to 97.5% of the tumor mass is composed of bystander cells. These findings define levels of E. coli PNP expression necessary for antitumor activity with F-araAMP and demonstrate new potential for a clinically approved compound in solid tumor therapy.

A methylthioadenosine phosphorylase (MTAP) fusion transcript identifies a new gene on chromosome 9p21 that is frequently deleted in cancer.

Homozygous deletions of human chromosome 9p21 occur frequently in malignant cell lines, and are also common in primary gliomas, lung cancers, and leukemias. Moving from the centromere to the telomere, this complex region encodes the tumor suppressor genes p15INK4B (CDKN2B), p14ARF, p16INK4A (CDKN2A), and the housekeeping gene methylthioadenosine phosphorylase (MTAP). However, not all chromosome 9p21 deletions in tumors include these tumor suppressor genes. Here we describe the partial sequence and the exact localization of a new gene on chromosome 9p21 centromeric of p15INK4B, that formed an in frame fusion transcript with MTAP in a glioma xenograft, and that is homozygously deleted in various malignant cell lines. Northern blot revealed corresponding 1.5 kb transcript in non-deleted cell lines as well as in normal lymphocytes. Using a RNA master blot membrane including 50 different tissues, we could show that this new transcript is expressed in all tissues of the adult but not or only at very low levels in most of the fetal tissues tested. The expression pattern is similar to that of p16INK4A. The localization as well as the deletion pattern makes this transcript a candidate for a new tumor suppressor gene.

R-region cDNA inserts in retroviral vectors are compatible with virus replication and high-level protein synthesis from the insert.

Protein expression from retroviral vectors is often highest when the expressed cDNA is driven by the retroviral promoter. However, the typical retroviral vector design places the cDNA downstream of the retroviral packaging signal and far from the retroviral promoter. In an attempt to improve protein production levels from cDNAs expressed in retroviral vectors, we inserted the MyoD or the purine nucleoside phosphorylase (PNP) cDNAs into the R regions of both retroviral LTRs, close to the retroviral promoter and just upstream of the polyadenylation signal present in each long terminal repeat (LTR). These R-region double-copy vectors could be produced in unrearranged form, although the titer was about seven-fold lower than that of typical vectors. R-region positioning of the MyoD cDNA resulted in five-fold higher MyoD expression compared to MyoD expression in a typical vector, whereas PNP expression was not improved. Thus, R-region double-copy vectors provide an alternative vector design that can improve protein expression from some cDNAs.

Molecular cloning of the human methylthioadenosine phosphorylase processed pseudogene and localization to 3q28.

Human methylthioadenosine phosphorylase (MTAP) is a purine and methionine metabolic enzyme present ubiquitously in all normal tissues, but often deleted in many types of cancer. The gene for this enzyme maps to chromosome 9 at band p21 where the cyclin-dependent kinase inhibitor genes for p16 and p15 also reside. During our efforts to clone this gene we also isolated a phage clone containing a processed pseudogene of MTAP. The sequence is 92% homologous to the MTAP cDNA, is flanked at its 3' end by a repetitive element, but does not possess a poly(A) stretch. We localized this processed pseudogene to band 28 on the long arm of chromosome 3 by fluorescence in situ hybridization. All 22 malignant cell lines with deletions at 9p21 screened possessed the pseudogene.

Status of methylthioadenosine phosphorylase and its impact on cellular response to L-alanosine and methylmercaptopurine riboside in human soft tissue sarcoma cells.

The aim of this study was to examine the expression of methylthioadenosine phosphorylase (MTAP) in 21 fresh tumor samples from patients with soft tissue sarcomas (STS) and 11 human soft tissue sarcoma cell lines, and to determine if loss of expression of this enzyme was correlated with increased sensitivity to L-alanosine and/or 6-methylmercaptopurine. We used a polyclonal antibody to measure the expression of MTAP in soft tissue sarcoma cell lines and in fresh tumor samples. Transfection of the HT-1080 cell line with a plasmid containing the cDNA for the MTAP gene was also performed to generate cell lines for in vitro and in vivo comparative sensitivity studies. MTAP was not expressed in 8 of 21 fresh STS tumors. The expression of MTAP was also not detectable in 3 of the 11 soft tissue sarcoma cell lines (HT-1080, HS42, and M-9 110). These three cell lines were more sensitive to L-alanosine, a potent inhibitor of de novo AMP synthesis, and to an inhibitor of de novo purine nucleotide synthesis, 6-methylmercaptopurine riboside (MMPR). The IC50 values for L-alanosine and MMPR were >20-fold lower in MTAP-deficient cells than in MTAP-positive cells. Restoration of MTAP into HT-1080 MTAP-deficient cells also led to decreased sensitivity to L-alanosine and MMPR. An in vivo study using HT-1080 cell tumors with and without MTAP expression confirmed that tumors lacking MTAP were more sensitive to L-alanosine than tumors expressing MTAP. These results provide the basis for selective therapy using inhibitors of de novo purine nucleotide synthesis such as L-alanosine or MMPR to treat patients with STS lacking this enzyme.

Acute myeloid leukemia: correlation between purine metabolism enzyme activities and membrane immunophenotype.

A total of 34 AML patients with heterogenous age distribution (from 2 years up to 82 years) were observed. Purine metabolism enzyme activities were compared and correlated with membrane immunophenotype. Analysis of bone marrow and peripheral blood samples based on FAB criteria and immunologic phenotyping of acute myeloid leukemia (AML) provided useful--either confirmatory or contradictory-information on the distribution of M1-M6 patients demonstrating a predominance of M1+M2 and M4 groups (44% and 32.4%, respectively). In contrast, it was demonstrated that less frequent subtypes were M3 and M6 (5.9% and 2.9%, respectively). AML subtypes were correlated with expression of surface antigens detected by the following monoclonal antibodies: CD13, CD33, CDw65, CD11b, CD15, CD14, HLA-DR and CD34. On the basis of immunophenotyping we found the following characteristic markers: M1, M2-CD34, HLA-DR, CD13, CD33, CDw65; M3-CD13, CD33, HLA-DR (negative); M4, M5-CDw65, CD14, CD13, CD33 and HLA-DR. CD14 was confirmed to be a typical marker for discriminating myeloid from monocytoid FAB AML subtypes. Analysis of purine metabolism enzyme activities showed that there is a correlation between the values of adenosine deaminase and purine nucleoside phosphorylase and various immunotypes of AML. High ADA/PNP ratio (> 1.0) was found in M1, M2, M3 subtypes. It was due to the increased level of ADA activity (> 100 pkat.10(-6) cells), though these activities overlapped to a certain extent. It was shown that PNP activity simultaneously decreased. With maturation of cells within AML lineage ADA activity decreased and PNP activity increased. This corresponded with ADA/PNP ratio that was < 1.0 in cells of more mature AML subtypes. We found that the enzymatic values were characteristic mainly in cells of M5 (monocytic) AML subtype and were characterized by decreased values of ADA activity with a simultaneous increase in PNP activity. It follows from our results that ADA/PNP ratio enables to discriminate between myeloid and monocytoid subtypes of AML.

[Expression and clinical significance of methylthioadenosine phosphorylase gene in patients with non-small cell lung cancer].

OBJECTIVE: To investigate the change of expression of methylthioadenosine phosphorylase (MTAP) gene in patients with non-small cell lung cancer and its clinical significance. METHODS: Thirty fresh samples of cancer with adjacent tissues were collected from 15 patients with lung cancer, 12 males and 8 females, aged 53.6 (38 approximately 72), 8 with squamous cell carcinoma and 7 with adenocarcinoma. The expressions of MTAP mRNA and of its protein were analyzed by RT-PCR and Western blotting. RESULTS: In 11 out of the 15 tumor samples (73.3%) MTAP mRNA was not expressed or expressed only after the additional 5 circulations. However, the MTAP mRNA expression rate was 93.3% (14/15) in the adjacent tissues. The result in the 11 samples with none or low MTAP mRNA expression was confirmed by Western blot analysis. The low expression rates were not significantly different between lung adenocarcinoma and squamous cell carcinoma. But the low expression rate of MTAP in intermediate and poorly differentiated lung cancer was significantly higher than that in well-differentiated cancer. CONCLUSION: The low expression or loss of MTAP gene may be relevant closely to the differentiation degree in lung cancer.

[Regulator mutants for the synthesis of a 2d purine nucleoside phosphorylase in Escherichia coli K-12. II. Mapping and study of the dominance of pndR mutations]. / Reguliatornye mutanty po sintezu vtoroi purinnukleozidfosforilazy u Escherichia coli K-12. Soobshchenie II. Kartirovanie i izuchenie dominantnosti mutatsii pndR.

The synthesis of a second purine nucleoside phosphorylase (PNPII) in the wild type strains of Escherichia coli K-12 is induced by xanthosine. Three types of pndR mutants were studied, which are altered in regulation of PNPII synthesis: 1) constitutive, 2) inducible by nucleosides of hypoxantine and adenine as much as by xanthosine and 3) defective in synthesis of PNPII. All pndR mutations are located in transductional crosses on 51 min of E. coli genetic map. The order of genes established is as follows: pndR-ptsH-cysA. Mutations of the first and second type are dominant, while pndR21 mutation of the third type is recessive to the pndR+ allele on F' episome. The data obtained support the suggestion that the product of pndR regulatory gene is an activator protein necessary for the expression of the PNPII structural gene.

[Mutations in the purine nucleoside phosphorylase (pup) gene of Escherichia coli K-12 characterized by leaky damage to enzymatic activity and a pleiotropic effect]. / Mutatsii po purinnukleozidfosforilaznomu (pup) genu Escherichia coli K-12, kharakterizuiushchiesia nepolnym povrezhdeniem aktivnosti fermenta i pleiotropnym éffektom.

The phenotype of earlier obtained mutants AIR38 and AIR6 is caused by leaky mutations of the structural gene for purine nucleoside phosphorylase (pup). These mutants are unable to grow on the medium with inosine as the only carbon source in the presence of thymidine. In contrast to ordinary leaky mutations, AIR38 and AIR6 are dominant in heterozygotes. When the strain F' with mutations AIR38 or AIR6 on episomes was used for conjugational matings with F- recA (pup+), recombinants with unexpected phenotype were observed: about 20% of recombinants F' became pup+ and thus the convertion of AIR38 and AIR6 alleles to pup+ took place. AIR38 mutation, unlike AIR6, is mapped in the proximal region of the pup gene and is characterized by pleyotropic effect on deo-genes: the inosine-induction of purine nucleoside phosphorylase in AIR38 mutant is absent and the induction of thymidine phosphorylase by exogenous thymidine is decreased. A speculation was made that the mutation AIR38 altered the structure of purine nucleoside phosphorylase in the site responsible for its interaction with membrane.

[Regulatory mutants for the synthesis of a 2d purine nucleoside phosphorylase in Escherichia coli K-12. I. Synthesis inducers and the substrate specificity of purine nucleoside phosphorylase in pndR mutants]. / Reguliatornye mutanty po sintezu vtoroi purinnukleozidfosforilazy u Escherichia coli K-12. Soobshchenie I. Induktory sinteza i substratnaia spetsifichnost' purinnukleozidfosforilazy u mutantov pndR.

Restoration of the ability to catabolise the purine nucleosides in phenotypic revertants of Escherichia coli K-12 mutants defective in deoD encoded purine nucleoside phosphorylase (PNPase 1) is the result of regulatory pndR mutations for synthesis of a second purine nucleoside phosphorylase (PNPase 2). In pndR+ strains synthesis of PNPase 2 is induced by xanthosine; in pndR mutants catabolising all purine nucleosides synthesis of this enzyme is constitutive; in other pndR mutants only catabolising some of purine nucleosides, this catabolisible nucleosides, namely, deoxyinosine, deoxyadenosine as well as, in some cases, inosine and adenosine, act as inducers of PNPase 2 synthesis. In some pndR mutants with inducible PNPase 2, xanthosine is a stronger inducer, in others it is weaker, in comparison with pndR+ strains. In bacterial cells PNPase 2 catalyses the phosphorolytic cleavage of adenosine, inosine, deoxyinosine, guanosine, deoxyguanosine and xanthosine, though in crude extracts adenosine and deoxyadenosine phosphorylase activities of the enzyme are not expressed.

Lack of expression of MTAP in uncommon T-cell lymphomas.

UNLABELLED: The majority of peripheral T-cell lymphomas were found to lack methylthioadenosine phosphorylase, an enzyme that is essential for the salvage of adenine from methylthioadenosine, a product of polyamine synthesis. Importantly, tumors that lack this enzyme have been shown to be more sensitive to inhibitors of de novo purine synthesis (6-thioguanine, methotrexate). BACKGROUND: T-cell lymphomas, in particular peripheral T-cell lymphoma (PTCL), angioimmunoblastic T-cell lymphoma (AITL), and anaplastic large cell lymphoma (ALCL), have only limited and noncurative treatment options. PATIENTS AND METHODS: We report here that a high percentage of PTCL, AITL, and ALCL lack the enzyme methylthioadenosine phosphorylase (MTAP), as do T-cell leukemia and T-cell lymphoblastic leukemia. MTAP-deficient cells cannot cleave endogenous methylthioadenosine to adenine and 5-methylthioribose-1-phosphate, a precursor of methionine, and as a result have enhanced sensitivity to inhibitors of de novo purine biosynthesis. A recently introduced antifolate, pralatrexate, which has been shown to inhibit de novo purine biosynthesis, has been approved for treatment of PTCL and may have an increasing role in therapy. An alternative strategy involving coadministration of methylthioadenosine and high-dose 6-thioguanine has been proposed and may prove to be selectively toxic to MTAP-deficient uncommon lymphomas. CONCLUSION: Thus the consequences of MTAP deficiency suggest that new therapeutic interventions for T-cell lymphoma may be feasible.