Histopathologic, Immunologic, and Cytogenetic Characteristics of LCH
Cell of Origin and Biologic Correlates
Modern classification of the histiocytic diseases subdivides them into dendritic cell–related, monocyte/macrophage-related, or true malignancies. Langerhans cell histiocytosis (LCH) is a dendritic cell disease.[1,2] The Langerhans cells (LCH cells) in LCH lesions are immature dendritic cells making up less than 10% of the cells present in LCH lesions.[3,4] Comprehensive gene expression array data analysis on LCH cells is consistent with the concept that the skin LC is not the cell of origin for LCH. Rather it is likely to be a myeloid dendritic cell, which expresses the same antigens (CD1a and CD207) as the skin LC. This concept was further supported by a study reporting that the transcription profile of LCH cells was distinct from myeloid and plasmacytoid dendritic cells, as well as epidermal LCs.[6,7]
LCH lesions also contain lymphocytes, macrophages, neutrophils, eosinophils, fibroblasts, and sometimes multinucleated giant cells. In the brain, the following three types of histopathologic findings have been described in LCH:
- Mass lesions in meninges or choroid plexus with CD1a-positive LCH cells and predominantly CD8-positive lymphocytes.
- Mass lesions in connective tissue spaces with CD1a-positive LCH cells and predominantly CD8-positive lymphocytes causing an inflammatory response and neuronal loss.
- Predominantly CD8+ lymphocyte infiltration with neuronal degeneration, microglial activation, and gliosis.
Normally, the LC is a primary presenter of antigen to naïve T-lymphocytes. However, in LCH, the pathologic dendritic cell does not efficiently stimulate primary T-lymphocyte responses. Antibody staining for the dendritic cell markers, CD80, CD86, and class II antigens, has been used to show that in LCH, the abnormal cells are immature dendritic cells that present antigen poorly and are proliferating at a low rate.[3,9,10] Transforming growth factor-beta (TGF-beta) and interleukin (IL)-10 are possibly responsible for preventing LCH cell maturation in LCH. The expansion of regulatory T cells in patients with LCH has been reported. The population of CD4-positive CD25(high) FoxP3(high) cells was reported to comprise 20% of T cells and appeared to be in contact with LCH cells in the lesions. These T cells were present in higher numbers in the peripheral blood of patients with LCH than in controls and returned to a normal level when patients were in remission.
Cytogenetic and Genomic Studies
Studies showing clonality in LCH using polymorphisms of methylation-specific restriction enzyme sites on the X-chromosome regions coding for the human androgen receptor, DXS255, PGK, and HPRT were published in 1994.[14,15] Biopsies of lesions with single-system or multisystem disease were found to have a proliferation of LCH cells from a single clone. Pulmonary LCH in adults is usually nonclonal and it is possible that this group represents a reactive process to smoking. However, a subset appeared to be clonal. An analysis of BRAF mutations showed that a significant proportion (25%–30%) of patients have evidence for mutant BRAF V600E, which has a statistically significant association with cigarette smoking. Cytogenetic abnormalities in LCH have rarely been reported. One study described an abnormal clone t(7;12)(q11.2;p13) from a vertebral lesion of one patient. This study also reported nonclonal karyotypic abnormalities in three patients. An increase in chromosomal breakage was also noted.
An activating mutation of the BRAF oncogene (V600E) was detected in 35 of 61 (57%) LCH biopsy samples, with mutations being more common in patients younger than 10 years (76%) than in patients aged 10 years and older (44%). A subsequent study with a larger sample size did not confirm this association. The RAS signaling pathway (Figure) transmits signals from a cell surface receptor (e.g., a growth factor) through the RAS pathway (via one of the RAF proteins [A, B, or C]) to phosphorylate MEK and then extracellular signal-regulated kinase (ERK), which leads to nuclear signals affecting cell cycle and transcription regulation. The V600E mutation of BRAF leads to continuous phosphorylation, and thus activation, of MEK and ERK without the need for an external signal.
The RAS pathway was activated in a few samples that were tested for MEK and ERK expression, whether or not the BRAF V600E mutation was present. The BRAF V600E mutation in LCH has been demonstrated in flow-sorted CD1a-positive LCH cells from fresh lesions in 11 of 16 samples. Another BRAF mutation (BRAF 600DLAT) was identified that resulted in the insertion of four amino acids and that also appeared to activate MAPK pathway signaling. No clinical characteristics associated with the BRAF mutation have been identified.[19-21]
A series of 135 biopsies from 100 patients were tested for the BRAF V600E mutation by a sensitive quantitative polymerase chain reaction technique and found the mutation in 65% of patients. Circulating cells with the BRAF V600E mutation could be detected in all high-risk patients and in a subset of low-risk multisystem patients. Presence of the circulating cells with the mutation conferred a twofold increased risk of relapse. The myeloid dendritic cell origin of LCH was confirmed by finding CD34+ stem cells with the mutation in the bone marrow of high-risk patients. Those with low-risk disease had more mature myeloid dendritic cells with the mutation, but not the stem cells suggesting the stage of cell development is critical in defining the clinical characteristics of LCH, which can now be considered a myeloid neoplasia.
Whole-exome sequencing of 41 biopsy samples revealed that 2 of 7 patients with MAP2K1 mutant alleles relapsed, while 17 of 26 patients with the wild type allele and 16 of 20 patients with the BRAF V600E mutation relapsed. An in vitro assay showed that these mutations activated ERK phosphorylation. Another study reported mutations involving MAPK1 exon 2 (45%) and exon 3 (27%) in samples without BRAF V600E mutations. Thus, it is likely that ERK activation defines a common pathophysiology of LCH.
Cytokine Analysis by Immunohistochemical Staining and Gene Expression Array Studies
Immunohistochemical staining of LCH lesions has shown apparent upregulation of the chemokines CCR6 and possibly CCR7.[24,25] In an analysis of gene expression in LCH by gene array techniques, 2,000 differentially expressed genes were identified. Of 65 genes previously reported to be associated with LCH, only 11 were found to be upregulated in the array results. The most highly upregulated gene in both CD207 and CD3-positive cells was osteopontin; other genes that activate and recruit T cells to sites of inflammation are also upregulated. The expression profile of the T cells was that of an activated regulatory T-cell phenotype with increased expression of FOXP3, CTLA4, and osteopontin. These findings support a previous report on the expansion of regulatory T cells in LCH. There was pronounced expression of genes associated with early myeloid progenitors including CD33 and CD44, which is consistent with an earlier report of elevated myeloid dendritic cells in the blood of patients with LCH. A model of "Misguided Myeloid Dendritic Cell Precursors" has been proposed, whereby myeloid dendritic cell precursors are recruited to sites of LCH by an unknown mechanism and the dendritic cells in turn recruit lymphocytes by excretion of osteopontin, neuropilin-1, and vannin-1.
Several investigators have published studies evaluating the level of various cytokines or growth factors in the blood of patients with LCH that have included many of the genes found not to be upregulated by the gene expression results discussed above. One explanation for elevated levels of these proteins is a systemic inflammatory response with the cytokines/growth factors being produced by cells outside the LCH lesions. A second possible explanation is that macrophages in the LCH lesions produce the cytokines measured in the blood or are concentrated in lesions.
IL-1 beta and prostaglandin GE2 levels were measured in the saliva of patients with oral LCH lesions or multisystem high-risk patients with and without oral lesions; levels of both were higher in patients with active disease and decreased after successful therapy.
Human Leukocyte Antigen (HLA) Type and Association With LCH
Specific associations of LCH with distinct HLA types and extent of disease have been reported. In a study of 84 Nordic patients, those with only skin or bone involvement more frequently had HLA-DRB1*03 type than those with multisystem disease. In 29 patients and 37 family members in the United States, the Cw7 and DR4 types were significantly more prevalent in Caucasians with single-bone lesions.
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