Tertiary lymphoid structures are confined to patients presenting with unifocal Langerhans Cell Histiocytosis.

Langerhans cell histiocytosis (LCH) is a neoplastic myeloid disorder with a thus far poorly understood immune component. Tertiary lymphoid structures (TLS) are lymph node-like entities which create an immune-promoting microenvironment at tumor sites. We analyzed the presence and clinical relevance of TLS in n = 104 H&E-stained, therapy-naive LCH lesions of non-lymphoid origin and applied immunohistochemistry to a smaller series. Lymphoid-follicular aggregates were detected in 34/104 (33%) lesions. In line with the lymphocyte recruitment capacity of MECA-79(+) high endothelial venules (HEVs), MECA-79(+)-expressing-LCH lesions (37/77, 48%) contained the most CD3(+) T-lymphocytes (p = 0.003). TLS were identified in 8/15 lesions and contained T-and B-lymphocytes, Follicular Dendritic Cells (FDC), HEVs and the chemokines CXCL13 and CCL21 representing key cellular components and TLS-inducing factors in conventional lymph nodes (LN). Lymphoid-follicular aggregates were most frequently detected in patients presenting with unifocal LCH (24/70, 34%) as compared to patients with poly-ostotic or multi-system LCH (7/30, 23%, p = 0.03). In addition, patients with lymphoid-follicular aggregates-containing lesions had the lowest risk to develop new LCH lesions (p = 0.04). The identification of various stages of TLS formation within LCH lesions may indicate a key role for the immune system in controlling aberrant histiocytes which arise in peripheral tissues.

The presence of CXCR4+ CD1a+ cells at onset of Langerhans cell histiocytosis is associated with a less favorable outcome.

PURPOSE: Langerhans Cell Histiocytosis (LCH) is a neoplastic disorder characterized by tissue accumulating CD1a+ histiocytes which frequently carry somatic mutations. Irrespective of mutation status, these LCH-cells display constitutively active kinases belonging to the MAPK pathway. We evaluated, in retrospect, the contribution of individual components of the MAPK-activating and chemotaxis-promoting TNF-CXCR4-CXCL12 axis to LCH manifestation and outcome. EXPERIMENTAL DESIGN: CXCR4, CXCL12 and TNF protein expression was immunohistochemically analyzed in 70 LCH-affected biopsies. The presence of CXCR4+CD1a+ cells in peripheral blood (PB) and/or bone marrow (BM) samples was evaluated by flowcytometry in 13 therapy-naive LCH-patients. RESULTS: CXCL12 was detected in 68/70 (97%) biopsies. CXCR4+LCH-cells were present in 50/70 (71%) biopsies; their presence was associated with higher levels of intralesional TNF. Circulating CD1a+CXCR4+ cells were detected in 4/13 (31%) therapy-naïve LCH-patients which displayed BRAFV600E (2/4), MAP2K1 (1/4) or no (1/4) mutations in their tissues. These CD11c co-expressing CD1a+CXCR4+cells migrated to CXCL12 in chemotaxis assays. Lesional CXCR4+LCH-cells were detected in 18/20 cases who presented with LCH manifestation at multiple sites and in 5/23 (22%) patients who developed additional lesions after initially presenting with a single lesion. The CXCR4 status at onset proved to be an independent risk factor for LCH reactivation in multivariate analysis (odds ratio 10.4, p = 0.034). CONCLUSIONS: This study provides the first evidence that CXCR4 is involved in the homing and retention of LCH-cells in CXCL12-expressing tissues and qualifies CXCR4 as a candidate prognostic marker for less favorable disease outcome.

Activated Conventional T-Cells Are Present in Langerhans Cell Histiocytosis Lesions Despite the Presence of Immune Suppressive Cytokines.

Langerhans cell histiocytosis (LCH) lesions are characterized by neoplastic CD1a(+)/Langerin(+) histiocytes (LCH-cells) and display many features of chronic inflammation. Cancer cells can escape immune-surveillance through intra-tumoral secretion of immune-suppressive cytokines. We therefore studied by immunohistochemistry the local cytokine milieu and phenotypic characteristics of T-cells and LCH-cells present in LCH lesions collected from 25 therapy naïve patients. LCH biopsies predominantly expressed interleukin-10 (IL-10) (10/25), transforming growth factor-beta (TGF-ß) (9/25), or both cytokines (6/25). The absolute number of CD3(+)T-cells and the CD3(+)FOXP3(-) conventional cell (T-CONV) versus the CD3(+)FOXP3(+) regulatory T-cell (T-REG) was comparable for each suppressive cytokine profile (5:1). IL-10-expressing lesions contained, however, a higher proportion of T-CONV expressing the activation markers CD25 98% (38%-100%) and inducible costimulatory molecule (ICOS) 86% (47%-100%) than lesions wherein solely TGF-ß was detected (CD25(+) 20% (6%-54%); ICOS(+) 29% (7%-51%)). Virtually all T-REG expressed CD25 and ICOS in IL-10 lesions, whereas TGF-ß(+) lesions contained a lower proportion of ICOS(+) T-REG (P=0.05). IL-10(+) lesions contained more LCH-cells expressing high intensity of ICOS ligand (ICOSL) compared with TGF-ß(+) lesions (P=0.03). ICOS expression by lesion-infiltrating T-CONV and T-REG positively correlated to the extent of ICOSL expression by LCH-cells (P=0.004). Our study points out that the combined detection of interlesional IL-10 and ICOSL expression by LCH-cells is associated with the highest prevalence of activated T-CONV. Immune profiling of LCH-affected tissues obtained at the time of diagnosis may set the stage for the development of new types of therapies, which aim at local boosting of immune cells that recognize and eliminate neoplastic LCH-cells.

MAP2K1 and MAP3K1 mutations in Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is now understood to be a neoplastic disease in which over 50% of cases have somatic activating mutations of BRAF. However, the extracellular signal-related (ERK) pathway is activated in all cases including those with wild type BRAF alleles. Here, we applied a targeted massively parallel sequencing panel to 30 LCH samples to test for the presence of additional genetic alterations that might cause ERK pathway activation. In 20 BRAF wild type samples, we found 3 somatic mutations in MAP2K1 (MEK1) including C121S and C121S/G128D in the kinase domain, and 56_61QKQKVG>R, an in-frame deletion in the N-terminal regulatory domain. All three variant proteins constitutively phosphorylated ERK in in vitro kinase assays. The C121S/G128D and 56_61QKQKVG>R variants were resistant to the mitogen-activated protein kinase kinase (MEK) inhibitor trametinib in vitro. Within the entire sample set, we found 3 specimens with mutations in MAP3K1 (MEKK1), including two truncation mutants, T779fs and T1481fs; T1481fs encoded an unstable and nonfunctional protein when expressed in vitro. T779fs was present in a specimen carrying BRAF V600E. The third variant was a single nucleotide substitution, E1286V, which was fully functional and is likely a germline polymorphism. These results indicate that LCH cells can harbor additional genetic alterations in the RAS-RAF-MEK pathway which, in the case of MAP2K1, may be responsible for ERK activation in a wild type BRAF setting. The resistance of some of these variants to trametinib may also have clinical implications for the combined use of RAF and MEK inhibitors in LCH.

Intact IFN-γR1 expression and function distinguishes Langerhans cell histiocytosis from mendelian susceptibility to mycobacterial disease.

PURPOSE: Poly-ostotic Langerhans Cell Histiocytosis (LCH) can be difficult to distinguish clinically and histologically from disseminated infection in manifesting specific subtypes of Mendelian Susceptibility to Mycobacterial Disease (MSMD). In MSMD-patients, dominant negative germline mutations in the IFN-γR1 gene, in particular in exon 6, lead to autosomal dominant IFN-γ receptor 1 deficiency (ADIFNGR1) and can mimic LCH. We hypothesized that similar defects might underlie the pathogenesis of LCH. METHODS: IFN-γR1 expression was immunohistochemically determined at disease onset in biopsies from 11 LCH-patients and four ADIFNGR1-patients. IFN-γR1 function was analyzed in 18 LCH-patients and 13 healthy controls by assessing the IFN-γ-induced upregulation of Fc-gamma-receptor I (FcγRI) expression on monocytes. Pro-inflammatory cytokine production was measured after stimulation of whole blood with LPS and IFN-γ. Exon 6 of the IFN-γR1 gene was sequenced in 67 LCH-patients to determine whether mutations were present. RESULTS: IFN-γR1 expression was high in three LCH-affected biopsies, similar to ADIFNGR1-affected biopsies, but varied from negative to moderate in eight other LCH-affected biopsies. No functional differences in IFN-γ signaling were detected between LCH-patients with active or non-active disease and healthy controls. No germline mutations in exon 6 of the IFN-γR1 gene were detected in any of the 67 LCH-patients. CONCLUSIONS: In contrast to ADIFNGR1-patients, IFN-γ signaling is fully functional in LCH-patients. Either performed before, during or after treatment, these non-invasive functional assays can distinguish LCH-patients from ADIFNGR1-patients and thereby facilitate correct therapy regimens for patients with recurrent osteolytic lesions.