Concurrent acute lymphoblastic leukemia and juvenile pilocytic astrocytoma in a pediatric patient.

The concurrence of acute lymphoblastic leukemia (ALL) and an asymptomatic juvenile pilocytic astrocytoma is described. A 6-year-old boy without clinical evidence of neurofibromatosis had a juvenile pilocytic astrocytoma diagnosed on radiologic examination and before treatment of acute pre-B cell lymphoblastic leukemia. The patient has had a partial resection of the astrocytoma and is 9 months into treatment of his ALL, which is in complete remission. p53 gene mutation was not identified in this patient. The concurrent diagnosis before treatment of ALL and juvenile pilocytic astrocytoma, the latter normally an indolent tumor, suggests that some cases of astrocytoma previously ascribed to radiotherapy or other treatment may in fact be caused by other factors.

Patients with both pancreatic adenocarcinoma and melanoma may harbor germline CDKN2A mutations.

Germline mutations of the CDKN2A tumor suppressor gene have been identified in melanoma kindreds linked to 9p21, and pancreatic adenocarcinoma is the second most common malignancy in some of these families. We hypothesized that unselected patients with both primary cancers, i.e., pancreatic cancer and malignant melanoma, have a genetic predisposition to tumor development, and that this susceptibility may be due to germline CDKN2A mutations. Fourteen patients, with both pathologically verified pancreatic adenocarcinoma and melanoma, were assessed for germline CDKN2A mutations by polymerase chain reaction amplification and sequencing of six overlapping fragments encompassing exons 1alpha and 2. A yeast two-hybrid assay was used to assess the functional consequences of CDKN2A variants. Germline CDKN2A mutations were identified in 2/14 patients: I49S, a novel substitution in exon 1alpha, and M53I, a previously reported missense mutation in exon 2. Both variants lead to compromised CDKN2A function. We conclude that the occurrence of both pancreatic cancer and melanoma, in the same patient, signals an inherited susceptibility to cancer, and that this predisposition is, in some cases, due to germline CDKN2A mutations. This finding has important implications not only for the proband, but also for other family members.

Adenovector-mediated gene delivery of interleukin-2 in metastatic breast cancer and melanoma: results of a phase 1 clinical trial.

We conducted a phase 1 trial of direct injection of an E1, E3-deleted adenovirus encoding interleukin-2 (AdCAIL-2) into subcutaneous deposits of melanoma or breast cancer. Twenty-three patients were injected at seven dose levels (10(7)-10(10) p.f.u). Local inflammation was observed at the site of injection in 60% of patients, but side-effects were otherwise minor. Incomplete local tumor regression occurred at the site of injection in 24% of patients, but no conventional clinical responses were seen. Circulating CD4 and CD8 counts fell significantly 24 h after injection. Post-injection biopsies demonstrated tumor necrosis and lymphocytic infiltration with the predominant tumor-infiltrating cells both CD3- and CD8-positive. Vector-derived sequences were detected in 14 of 18 biopsies examined 7 days after injection and vector-derived hIL-2 mRNA was detected in 80% of 7-day biopsies processed after injection of 10(8) p.f.u. of AdCAIL-2 or higher. While IL-2 was detectable by ELISA in tumor biopsies at 48 h, no protein was detectable in injected tumors after 7 days and no circulating IL-2 was detectable at any time-point. No Ad5E1 sequences were detected either before or after injection indicating absence of replication-competent virus or endogenous E1-like sequence; furthermore, only rare vector shedding was detected. Anti-adenovirus and neutralizing antibody titers were elevated 1 month after injection in all patients. This trial therefore confirms the safety of use of adenoviral vectors for gene delivery in humans and demonstrates successful transgene expression even in the face of pre-existing immunity to adenovirus.

Role of the cyclin-dependent kinase inhibitor CDKN2A in familial melanoma.

BACKGROUND: Approximately 8 to 12% of melanoma appears to be inherited in an autosomal dominant form. Although most early stage melanomas can be treated successfully by simple surgical excision, patients with advanced disease are rarely cured even with aggressive chemotherapy and/or immunotherapy. OBJECTIVE: There is now compelling evidence that germline mutations of the CDKN2A gene on chromosome 9p21 predispose to melanoma in a subset of melanoma-prone families. In this article the evidence for the role of CDKN2A in the genesis of familial melanoma is reviewed and the implications of genetic testing in families with this disease are discussed. CONCLUSION: The identification and subsequent surveillance of unaffected individuals who have a genetic predisposition to melanoma may lead to the detection of early (curable) melanomas and to a reduction in mortality.

CDKN2A mutations in multiple primary melanomas.

BACKGROUND: Germ-line mutations in the CDKN2A tumor-suppressor gene (also known as p16, p16INK4a, and MTS1) have been linked to the development of melanoma in some families with inherited melanoma. Whether mutations in CDKN2A confer a predisposition to sporadic (nonfamilial) melanoma is not known. In some patients with sporadic melanoma, one or more additional primary lesions develop, suggesting that some of these patients have an underlying genetic susceptibility to the cancer. We hypothesized that this predisposition might be due to germ-line CDKN2A mutations. METHODS: We used the polymerase chain reaction, single-strand conformation polymorphism analysis, and direct DNA sequencing to identify germ-line mutations in the CDKN2A gene in patients with multiple primary melanomas who did not have family histories of the disease. A quantitative yeast two-hybrid assay was used to evaluate the functional importance of the CDKN2A variants. RESULTS: Of 33 patients with multiple primary melanomas, 5 (15 percent; 95 percent confidence interval, 4 percent to 27 percent) had germ-line CDKN2A mutations. These included a 24-bp insertion at the 5' end of the coding sequence, three missense mutations (Arg24Pro, Met53Ile, and Ser56Ile), and a 2-bp deletion at position 307 to 308 (resulting in a truncated CDKN2A protein). In three families, CDKN2A mutations identical to those in the probands were found in other family members. In two families with mutations, we uncovered previously unknown evidence of family histories of melanoma. CONCLUSIONS: Some patients with multiple primary melanomas but without family histories of the disease have germ-line mutations of the CDKN2A gene. The presence of multiple primary melanomas may signal a genetic susceptibility to melanoma not only in the index patient but also in family members, who may benefit from melanoma-surveillance programs.

CDKN2A mutation in a non-FAMMM kindred with cancers at multiple sites results in a functionally abnormal protein.

The CDKN2A gene encodes p16 (CDKN2A), a cell-cycle inhibitor protein which prevents inappropriate cell cycling and, hence, proliferation. Germ-line mutations in CDKN2A predispose to the familial atypical multiple-mole melanoma (FAMMM) syndrome but also have been seen in rare families in which only 1 or 2 individuals are affected by cutaneous malignant melanoma (CMM). We therefore sequenced exons 1alpha and 2 of CDKN2A using lymphocyte DNA isolated from index cases from 67 families with cancers at multiple sites, where the patterns of cancer did not resemble those attributable to known genes such as hMLH1, hMLH2, BRCA1, BRCA2, TP53 or other cancer susceptibility genes. We found one mutation, a mis-sense mutation resulting in a methionine to isoleucine change at codon 53 (M531) of exon 2. The individual tested had developed 2 CMMs but had no dysplastic nevi and lacked a family history of dysplastic nevi or CMM. Other family members had been diagnosed with oral cancer (2 persons), bladder cancer (1 person) and possibly gall-bladder cancer. While this mutation has been reported in Australian and North American melanoma kindreds, we did not observe it in 618 chromosomes from Scottish and Canadian controls. Functional studies revealed that the CDKN2A variant carrying the M531 change was unable to bind effectively to CDK4, showing that this mutation is of pathological significance. Our results have confirmed that CDKN2A mutations are not limited to FAMMM kindreds but also demonstrate that multi-site cancer families without melanoma are very unlikely to contain CDKN2A mutations.

Fine mapping of the MLK-3 gene within 11q13 and its exclusion as the MEN1 susceptibility gene.

MLK-3 kinase is a widely expressed serine/ threonine kinase that bears multiple protein interaction domains and regulates signals mediated by the stress-responsive pathway. Thus, MLK-3 signaling affects numerous cellular processes, raising the possibility that MLK-3 might play a role in oncogenesis. In this report, we describe the fine mapping of the MLK-3 gene within the 11q13.1 chromosomal region. By integrating data from somatic cell hybrids and double color fluorescence in situ hybridization on metaphase chromosomes and DNA fibers. MLK-3 has been assigned approximately 1 Mb telomeric of PYGM, close to the D11S546 locus. Since the MEN1 susceptibility locus is also located within the 11q13.1 region, we have carried out Southern and Northern blot analyses, as well as protein truncation assays to establish whether abnormalities in MLK-3 lead to the development of this familial cancer syndrome. Our observations exclude MLK-3 as the MEN1 gene.

Lack of germ-line mutations of CDK4, p16(INK4A), and p15(INK4B) in families with glioma.

Gliomas are tumors of the central nervous system that may be inherited in some patients. The gene(s) responsible for the clustering of gliomas in families have not yet been identified. Molecular studies of sporadic high-grade gliomas have revealed mutations or deletions of the genes encoding the protein kinase inhibitors p16(INK4A) and p15(INK4B) in a large proportion of tumors. Moreover, those tumors without deletions frequently display gene amplification and/or over-expression of mRNA encoding the protein kinase cdk4. We hypothesized that germ-line mutations in the p16(INK4A), p15(INK4B), or CDK4 genes might contribute to some cases of familial gliomas. To address this issue, we analyzed 36 kindreds with a predisposition to glial tumors. Genomic DNA from index members of these families was screened by PCR-single-strand conformational polymorphism analysis. We did not detect any functional mutations in the p16(INK4A), p15(INK4B), or CDK4 genes, although two individuals did have a previously described A140T polymorphism in p16(INK4A). Thus, despite the association between the sporadic forms of high-grade glioma and abnormalities of p16(INK4A), p15(INK4B), or CDK4, we found no evidence that germ-line mutations in the coding region of these three genes predispose to inherited glial tumors.

Affected members of melanoma-prone families with linkage to 9p21 but lacking mutations in CDKN2A do not harbor mutations in the coding regions of either CDKN2B or p19ARF.

Mutations in the gene encoding the cell cycle inhibitor CDKN2A have been identified in some melanoma kindreds linked to 9p21. However, many such families show no evidence of mutations in the coding regions of CDKN2A. In this study, we examined whether two other potential tumor suppressors, CDKN2B and p19ARF, which also map within the 9p21 region, play a role in the development of familial melanoma. We found no mutations in the coding regions of either gene in melanoma-prone families with evidence of linkage to 9p21. We conclude either that another melanoma susceptibility gene exists within this chromosomal area or that mutations in noncoding regions of CDKN2A, CDKN2B, or p19ARF predispose to melanoma.

MLK-3 activates the SAPK/JNK and p38/RK pathways via SEK1 and MKK3/6.

Mixed lineage kinase-3 (MLK-3) is a 97 kDa serine/threonine kinase with multiple interaction domains, including a Cdc42 binding motif, but unknown function. Cdc42 and the related small GTP binding protein Rac1 can activate the SAPK/JNK and p38/RK stress-responsive kinase cascades, suggesting that MLK-3 may have a role in upstream regulation of these pathways. In support of this role, we demonstrate that MLK-3 can specifically activate the SAPK/JNK and p38/RK pathways, but has no effect on the activation of ERKs. Immunoprecipitated MLK-3 catalyzed the phosphorylation of SEK1 in vitro, and co-transfected MLK-3 induced phosphorylation of SEK1 and MKK3 at sites required for activation, suggesting direct regulation of these protein kinases. Furthermore, interactions between MLK-3 and SEK and MLK-3 and MKK6 were observed in co-precipitation experiments. Finally, kinase-dead mutants of MLK-3 blocked activation of the SAPK pathway by a newly identified mammalian analog of Ste20, germinal center kinase, but not by MEKK, suggesting that MLK-3 functions to activate the SAPK/JNK and p38/RK cascades in response to stimuli transduced by Ste20-like kinases.

Germline p16INK4A mutation and protein dysfunction in a family with inherited melanoma.

The gene encoding the cell cycle inhibitor p16INK4A (also known as p16, MTS1, CDKN2 and INK4) has been mapped to human chromosome band 9p21, a region that also contains a putative melanoma susceptibility gene. Although germline mutations in the coding region of the p16INK4A gene have been detected in some families with inherited melanoma, many other families show no evidence of such mutations and hence the role of p16INK4A in the development of this tumor is still unclear. In this report, we describe a family with inherited melanoma in which a novel mutation in exon 2 of the p16INK4A gene segregates with the disease. The mutant gene encodes a protein with an in-frame deletion of two amino acids (Asp96 and Leu97). We show that the mutant protein is functionally abnormal: it is unable to bind cdk4 in vitro and does not inhibit colony formation in tertiary passage rat embryo fibroblasts. Moreover, in a metastatic lesion from one patient the wild type p16INK4A allele was deleted and the mutant allele retained. We conclude that family members carrying this germline mutation in the p16INK4A gene are predisposed to melanoma. By extension, these findings implicate the p16INK4A gene in the development of some cases of familial melanoma.

Reversible thrombocytopenia with levamisole.

Levamisole is an immunomodulatory agent which is used in the adjuvant therapy of certain malignancies. Agranulocytosis is the most commonly reported hematologic side effect associated with this drug. We report here a patient who developed thrombocytopenia nearly 2 years after starting adjuvant levamisole therapy for malignant melanoma. In this case, thrombocytopenia was shown to be levamisole-related by rechallenge with the drug. Levamisole-induced thrombocytopenia (LIT) has been rarely diagnosed, but may be seen more frequently as increasing numbers of patients receive adjuvant therapy for colon cancer and melanoma.

Protein kinases in normal and transformed melanocytes.

Aberrant function of protein kinases has been implicated in the development of melanoma. In an effort to define the molecular events involved in initiation and progression of this malignancy, we used RT-PCR to identify protein kinases in both normal and transformed melanocytes. Collectively, we identified seven clones corresponding to previously characterized protein kinases (JAK-1, TYK02, AXL/UFO, IGF1-R, KDR and FER) as well as the recently identified MLK-3/PTK1 protein kinase. Northern analysis was used to determine the expression pattern of each protein kinase in both normal melanocytes and a variety of melanoma cell lines. Relatively abundant levels of UFO/AXL and KDR mRNAs were observed in a subset of the melanoma cell lines whereas most of the remaining protein kinases were expressed at similar levels in both normal and transformed melanocytes.

MLK-3: identification of a widely-expressed protein kinase bearing an SH3 domain and a leucine zipper-basic region domain.

We have identified a novel protein kinase, designated MLK-3, from human thymus using RT-PCR and cDNA library screening. The deduced open reading frame, derived from sequencing a 3.5 kb MLK-3 cDNA, encodes a protein of 847 amino acids with several interesting structural features. These include an SH3 domain in the absence of an SH2 domain, a region containing two leucine zippers with an adjacent carboxy-terminal basic region, and a proline rich region. This kinase shows homology with the mixed-lineage family of protein kinases (MLK) and shares the unusual leucine zipper-basic motif found in previously identified MLK kinases. By northern analysis, MLK-3 mRNA was detected in a wide variety of normal and transformed human cell lines and tissue specimens. The gene encoding MLK-3 has been mapped using fluorescence in situ hybridization to human chromosome 11 q13.1-13.3, a region frequently altered in human malignancies.

VEGF receptor subtypes KDR and FLT1 show different sensitivities to heparin and placenta growth factor.

Vascular endothelial growth factor (VEGF) is an angiogenic growth factor which binds to two structurally related tyrosine kinase receptors denoted KDR and FLT1. To compare the interaction of VEGF with each receptor, cell lines which express individual receptor subtypes were identified using Northern blot hybridization. Bovine aortic endothelial (ABAE) cells and WM35 melanoma cells were found to express KDR, while FLT1 was primarily expressed on SK-MEL-37. Both receptor subtypes were detected on another melanoma cell line (WM9). Heparin augmented VEGF binding to KDR-expressing cells (ABAE and WM35), but inhibited VEGF binding to FLT1-expressing cells (SK-MEL-37 and WM9). The concentration of heparin required for half maximal stimulation of VEGF binding to KDR-expressing cells (500 ng/ml) was 25 times greater than that required for half maximal inhibition of binding to FLT1-expressing cells (20 ng/ml). In WM9 cells, the effect of heparin was bimodal; low concentration inhibited, while higher concentrations stimulated binding of 125I-VEGF. Placenta growth factor (PIGF-1) is a recently described growth factor structurally similar to VEGF. PIGF-1 had a negligible or no effect on 125I-VEGF binding to KDR-expressing cells (ABAE, WM35), but did complete for binding to FLT1-expressing cells (SK-MEL-37 and WM9). Addition of heparin had no effect on its ability to compete for binding with 125I-VEGF. The data indicate differential regulation of the two VEGF receptors by heparin and extended specificity of FLT1 receptor, but not KDR, for binding PIGF-1 growth factor.

Overexpression of p53 is a late event in the development of malignant melanoma.

Overexpression of the p53 gene product has been observed in a high percentage of malignant melanomas. To evaluate the role of this protein in the development of melanoma, we examined p53 expression in benign, premalignant, and malignant melanocytic lesions. Using the antibodies DO-7 and 1801, which recognize both wild-type and most mutant forms of the p53 protein, we analyzed by immunohistochemical staining 26 benign nevi, 34 dysplastic nevi from patients at low risk for the development of melanoma, 22 dysplastic nevi from patients at high risk for the development of melanoma, 61 primary melanomas (including 15 that arose from dysplastic nevi), and 10 metastatic melanomas. Expression of the p53 protein was not observed in any of the benign or dysplastic nevi. Of the primary melanomas only 3 (5%) demonstrated nuclear staining, whereas 70% of the metastatic melanomas showed a positive reaction for p53. These data suggest that overexpression of the p53 gene product is a late event in the progression of melanoma and consequently indicate that expression of this protein cannot be used as a marker to identify patients at high risk for the subsequent development of melanoma.

Protein kinase activity immunoprecipitated from adenovirus infected cells by sera from tumor-bearing hamsters.

Earlier, we reported (N. J. Lassam, S. T. Bayley, F. L. Graham, and P. E. Branton, Nature (London) 277:241-243, 1979) detecting protein kinase activity when cytoplasmic extracts of human adenovirus type 5 (Ad5)-infected KB cells immunoprecipitated with 14b antitumor serum directed against the transforming proteins of Ad5, were incubated with [gamma-32P]ATP. Here we show that in the in vitro assay this kinase phosphorylated both the heavy chain of immunoglobulin G and polypeptide than comigrated on sodium dodecyl sulfate gels with the 58,000-dalton Ad5 antigen. It also phosphorylated added histone H3. Evidence is presented that the protein kinase activity found with extracts from Ad5-infected cells is not due to nonspecific trapping of cellular enzymes in immune complexes, but to an enzyme which is distinct from kinases detected at background levels in controls. Serine and threonine were the major phosphorylated amino acids, and essentially no phosphotyrosine was detected. Protein kinase activity detected in Ad12-infected cells immunoprecipitated by an antiserum derived from hamsters bearing Ad12-induced tumors appeared to be immunologically distinct from that immunoprecipitated from Ad5-infected cells by 14b serum.