Histiocytes and histiocytosis.

The term histiocyte refers to cells of either the macrophage or Langerhans cell lineages. The histiocytic disorders are characterized by the proliferation of cells of these lineages. With recent advances in knowledge of the developmental biology of histiocytic cells, it is now possible to formulate a reasonable catalogue of histiocytic diseases based on ultra-structural and phenotypic markers of cellular origins and molecular or chromosomal markers of malignancy. The catalogue includes the following groups of diseases. Nonmalignant reactive macrophage disorders include (1) macrophage storage diseases, (2) several benign proliferative macrophage disorders that predominantly involve skin and bone, and (3) several hemophagocytic syndromes that vary from indolent and benign to fulminant and fatal. In some of the latter disorders, viruses have been identified as the inciting stimulus. The malignant macrophage disorders include (1) acute monocytic leukemia and (2) chronic myelomonocytic leukemia. A rare disorder that gave rise to a permanent cell line with an anomaly of chromosomal segment 5q35 may also be an example of a histiocytic malignancy. The existence of a separate category of true histiocytic lymphoma of macrophage type is uncertain. Reactive Langerhans cell disorders include (1) congenital self-healing histiocytosis, (2) the many variants of eosinophilic granuloma, and (3) a related disorder designated as relapsing Langerhans cell histiocytosis that is characterized by a relapsing course and infiltration of bone and soft tissues by Langerhans cells. Presumptively neoplastic diseases of Langerhans and dendritic cells include (1) progressive Langerhans cell histiocytosis, a disease with prominent involvement of blood and BM as well as skin and viscera; (2) Langerhans cell lymphoma, and (3) dendritic cell lymphoma. However, clonality as a marker of malignancy has not been proven in these disorders.

Sequential relapses of blastic crisis may involve different clones of cells with different molecular abnormalities.

A patient with typical Ph1-positive CML was studied during sequential phases: (1) initial chronic phase, (2) first myeloid blast crisis, (3) second chronic phase, and (4) accelerated disease leading to a second blast crisis. A point mutation in codon 239 of the p53 gene and a novel chromosome 17 alteration appeared concomitantly with the first blast crisis and then disappeared with re-establishment of a second chronic phase. They did not reappear with the second acute phase, indicating that the clone responsible for the original blast crisis had been suppressed and supplanted by another clone of malignant cells. This observation suggests that in at least some CML patients drug therapy can suppress or eliminate an aggressive malignant cell clone, but that the underlying molecular defect in haemopoietic cells (in this case the c-ABL translocation) persists and other aggressive clones with different molecular lesions eventually arise. Our observations and inferences are consistent with the hypothesis advanced by Fialkow et al (1991) to explain clonal remissions in acute non-lymphocytic leukaemia.

Molecular mechanisms in the evolution of chronic myelocytic leukemia.

Chronic myelocytic or Ph1-positive acute lymphoblastic leukemias have been analyzed for alterations in a variety of proto-oncogenes and anti-oncogenes implicated in the progression of chronic myeloid leukemia (CML) from its chronic phase to blast crisis. The most frequent genetic change found in disease evolution is an alteration of the p53 gene involving a point mutation, a rearrangement or a deletion. These gene changes are common in myeloid and undifferentiated variants of blast crisis but are usually undetectable in lymphoid leukemic transformants. Other molecular changes also occur in the clonal evolution of CML. The retinoblastoma-susceptibility (Rb) gene is an anti-oncogene. Structural abnormalities of Rb are frequent in all types of human acute leukemia, but are particularly common in Ph1-positive leukemia of lymphoid phenotype including both Ph1-positive ALL and lymphoid blast crisis of CML. Changes in Rb occur early in the transition to blast crisis with loss of Rb protein being the common factor. Mutations in the N-RAS gene also occur, but are rare in typical blast crisis. They are sometimes seen in Ph1-negative myeloid blast crisis. Since changes in the p53 gene are generally associated with progression of disease of a myeloid phenotype and changes in the Rb gene occur more often with a lymphoid phenotype, a particular molecular alteration may influence the character of disease evolution in CML.

Abnormalities of the retinoblastoma gene in the pathogenesis of acute leukemia.

The retinoblastoma-susceptibility (Rb) gene is an antioncogene that is frequently altered in retinoblastomas, sarcomas, and some epithelial tumors. We examined the structure of the Rb gene by Southern blotting in 215 cases of leukemias and lymphomas of diverse phenotype and in 15 leukemic cell lines. In selected cases Rb protein expression was examined with specific monoclonal antibodies. Structural abnormalities of the Rb gene with absent protein expression were frequent in all types of human acute leukemia, but were particularly common (27% incidence) in M4 and M5 myeloid leukemia with monocytic differentiation and in Philadelphia chromosome (Ph1)-positive leukemia of lymphoid phenotype (11% to 29% incidence). Changes in Rb were observed early in the transition to acute leukemia in cases of myelodysplastic syndrome and in the accelerated phase of chronic myelocytic leukemia in transition to blast crisis. In one case, molecular changes in Rb could be correlated with leukemia remission and relapse. We conclude that the Rb antioncogene is commonly involved in the evolution of human acute leukemias, particularly in those of a monocytic phenotype and in lymphoid leukemia in which there is an antecedent alteration of the Ph1 chromosome.

Correlation between molecular and clinical events in the evolution of chronic myelocytic leukemia to blast crisis.

A patient with typical Philadelphia chromosome (Ph1)-positive chronic myelocytic leukemia (CML) was studied during sequential phases of disease: (1) initial chronic phase; (2) myeloid blast crisis; (3) second chronic phase; and (4) accelerated disease. A point mutation in the coding sequence of the p53 gene first appeared concomitantly with the blast crisis and then disappeared with the re-establishment of a second chronic phase. The chromosomal concomitant of the molecular alteration was a deletion of 17p. These observations suggest that abnormalities of the p53 anti-oncogene are temporally related to the clinical progression of some cases of CML and are probably responsible for the development of blast crisis in these cases.

A splicing mutation accounts for the lack of p53 gene expression in a CML blast crisis cell line: a novel mechanism of p53 gene inactivation.

Alterations of the p53 anti-oncogene have recently been found to occur frequently in the blast crisis of chronic myelocytic leukaemia. The p53 gene may be altered by gross structural alterations or by point mutations in the coding sequence. We now report a novel mechanism of gene inactivation in a blast crisis cell line where a mutation in a splice donor site at the 5' end of the fifth intron of the gene interrupts RNA processing and gene expression.

Variation in the protein coding region of the human p53 gene.

This report describes a restriction fragment length polymorphism for the enzyme BglII caused by a conserved C to T change at residue 21 of the human p53 gene. This RFLP could potentially be misinterpreted as a rearrangement or a point mutation if paired constitutional tissue is not simultaneously analyzed.

The pattern of mutational involvement of RAS genes in human hematologic malignancies determined by DNA amplification and direct sequencing.

DNA from 161 patients with various forms of hematologic malignancies were investigated for mutations in exons 1 and 2 of the N-RAS, K-RAS and Ha-RAS gene by direct sequencing of DNA amplified in vitro by the polymerase chain reaction. Mutations involving either codons 11, 12, or 13 of the N-RAS gene were identified in 18 of the 161 patients. The relative frequencies of N-RAS gene mutations in these hematologic disorders was as follows: acute myelogenous leukemia (AML), 15%; acute lymphoblastic leukemia (ALL), 14%; myelodysplastic syndromes, 24%; and myeloid and lymphoid blast crisis of chronic myelogenous leukemia (CML), 3%. No correlation was observed between the presence of mutations and cytologic features or immunophenotype of these malignancies. Mutations involving codons 12 or 13 were equally prevalent, with a glycine to aspartic acid substitution being the most frequently encountered change. A single T-ALL case had a codon 11 mutation resulting in substitution of alanine with threonine. We failed to find mutations in exons 1 and 2 of the K-RAS or Ha-RAS genes in any case except a single AML with a mutation in codon 61 of the K-RAS gene. Also, no mutations were identified in chronic phase of CML, chronic lymphocytic leukemia. Ph1 positive ALL, non-Hodgkin's lymphoma, Hodgkin's disease, or multiple myeloma. These results indicate that RAS mutations, especially those involving exon 1 of the N-RAS gene, are frequent only in a subset of hematologic malignancies.

Nerve growth factor acts through cAMP-dependent protein kinase to increase the number of sodium channels in PC12 cells.

cAMP-dependent protein kinase (PKA) and phospholipid-dependent protein kinase (PKC) play a role in nerve growth factor (NGF)-mediated differentiation. In PC12 cells, NGF causes neurite outgrowth and increases the number of voltage-gated Na+ channels. Neurite outgrowth involves in part activation of PKC. How NGF regulates Na+ channel number is unknown. Using patch-clamp techniques, we find that agents activating PKC, including phorbol esters and a ras oncogene product (p21) that induces neurites, caused little increase in channel number. In contrast, agents increasing intracellular cAMP were as effective as NGF. A specific protein inhibitor of the PKA catalytic subunit blocked increases by NGF or cAMP. Thus, NGF increases Na+ channel number in PC12 cells in part by activating PKA but apparently not PKC.

Analysis of proto-oncogenes in acute myeloid leukemia: loss of heterozygosity for the Ha-ras gene.

At least 13 of 34 patients with acute myeloid leukemia (AML) of varying FAB types were heterozygous for a BamHI restriction fragment length polymorphism (RFLP) of the Ha-ras gene on chromosome 11. In 4 of these 13 patients, one allele of the Ha-ras gene was deleted. Two of these cases had an informative heterozygosity for an RFLP on the long arm of chromosome 11. Analysis of these cases indicated that loss of genes from chromosome 11 was restricted to the short arm. In three cases with loss of one Ha-ras gene, the remaining gene had no mutations in critical areas of exons 1 and 2. With the exception of one AML case with amplification of MYC, no gross structural abnormalities in 12 other oncogenes were detected.

Direct sequencing analysis of exon 1 of the c-K-ras gene shows a low frequency of mutations in human pancreatic adenocarcinomas.

We have applied direct dideoxy sequencing of DNA fragments amplified in vitro by the polymerase chain reaction to the detection of mutations in exon 1 of the c-K-ras gene in human pancreatic adenocarcinomas. Four fresh frozen primary tumors, one metastatic tumor, and twelve formalin-fixed paraffin embedded tumors were analysed. Only three cases showed a possible mutation in codon 12 in a small population of cells, in contrast to the high frequency reported for this alteration with the RNAase A protection assay and allele-specific oligoxynucleotide hybridization. No major difference in sensitivity was found between DNA sequence analysis, and the latter method. Our results suggest that if c-K-ras mutations are indeed present in pancreatic adenocarcinomas at the high frequency reported by others, they must be confined to a small fraction of the cell population to escape detection by direct sequencing. Such a phenomenon would have implications for c-K-ras mutations in the pathogenesis of pancreatic adenocarcinomas.

Alterations in the p53 gene and the clonal evolution of the blast crisis of chronic myelocytic leukemia.

Molecular mechanisms responsible for the clinical progression of chronic myelocytic leukemia to its accelerated phase or to blast crisis have not been defined. We found alterations of the p53 gene (p53 is a 53-kDa nuclear protein) including deletions and rearrangements in 8 of 34 patients in blast crisis and 1 of 4 patients in the accelerated phase, but in only 1 of 38 patients in the chronic phase of chronic myelocytic leukemia. Only two other examples of p53 gene alterations were found among 203 patients with hematologic malignancies and solid tumors. Transcripts of the p53 gene were uniformly found in chronic-phase cells, but gene expression was variable in blast crisis, and transcripts were reduced or undetectable in 10 of 16 patients. Heterogeneous alterations in the structure and expression of the p53 gene appear to be relatively frequent in blast crisis and may be involved in the evolution of disease.

Oncogenes in hematologic neoplasia.

Proto-oncogenes are normal genes involved in cellular proliferation and differentiation. Structural alterations in these genes can convert them to oncogenes involved in the initiation or progression of malignancy. About 50 proto-oncogenes have been described and four different activation mechanisms are known. Proto-oncogene alterations specific for human hematologic malignancies are well characterized.

N-RAS mutations in T-cell acute lymphocytic leukaemia: analysis by direct sequencing detects a novel mutation.

A novel mutation of the N-RAS gene of T-ALL blast cells was detected by a direct sequencing of in vitro amplified exon-1 of the N-RAS gene. Threonine (ACA) was substituted for alanine (GCA) at codon 11. This mutation would have been overlooked by conventional probe hybridization techniques. A search for other mutations in N-RAS exon-1 in T-ALL revealed a codon 13 mutation substituting aspartic acid (GAT) for glycine (GGT) in one of 18 patients. No mutations at codon 12 were detected.

Analysis of N-RAS exon-1 mutations in myelodysplastic syndromes by polymerase chain reaction and direct sequencing.

Mutations in codons 12 or 13 of the first exon of the N-RAS gene have been reported in myelodysplastic syndromes (MDS) in frequencies that vary between 9% and 40% depending on the techniques used in analysis. Gene amplification and direct sequencing provides the only unambiguous method of detecting those mutations that induce amino acid alterations. Using this technique, we analyzed 21 MDS patients for mutations in exon-1 of N-RAS. Codon 12 mutations substituting aspartic acid (GAT) for glycine (GGT) were found in four cases, and a codon 13 mutation substituting alanine (GCT) for glycine (GGT) was detected in one patient. We conclude that N-RAS exon-1 mutations in one patient. We conclude that N-RAS exon-1 mutations producing amino acid changes occur in about 20% to 25% of MDS cases.

Proto-oncogene abnormalities in human breast cancer: c-ERBB-2 amplification does not correlate with recurrence of disease.

The incidence of c-ERBB-2 amplification in breast cancers and its usefulness as a predictor of tumor recurrence after treatment have been subjects of controversy (Ali et al., 1988, Slamon et al., 1987). We re-examined this subject by analysing 157 primary and 14 metastatic breast cancers with c-ERBB-2 and 18 other molecular probes as controls. Five proto-oncogenes were found to be occasionally amplified in primary breast cancers: c-ERBB-2 (11%), c-MYB (3%), c-RAS-Ki (3%), INT-2 (4%) and c-MYC (6%). No statistically significant correlation between amplification of c-ERBB-2 and recurrence of tumors was observed. The only significant correlation observed was between early recurrence of advanced (stage III) tumors and amplification of one or another of the above five proto-oncogenes. We conclude that breast cancer utilize multiple genetic mechanisms in their progression and metastasis, and that analysis of c-ERBB-2 alone is not a useful guide.

A unique pattern of proto-oncogene abnormalities in ovarian adenocarcinomas.

Twelve cases of ovarian adenocarcinoma were studied for alterations in proto-oncogenes, and a unique pattern of altered ras proto-oncogenes was observed. Amplification of ras-Ki was found in three of seven ovarian tumors and amplification of ras-Ha in one of 12. In contrast, ras-Ha amplification was not found in any of the 334 other tumors and ras-Ki amplification was only seen in breast cancer at a frequency of 3%. Other proto-oncogenes altered in ovarian adenocarcinomas included c-myc and c-erbb-2. Proto-oncogene abnormalities were more frequent in aggressive tumors of high histologic grade.