Is Cutaneous T-Cell Lymphoma Caused by Ultraviolet Radiation? A Comparison of UV Mutational Signatures in Malignant Melanoma and Mycosis Fungoides.

Ultraviolet (UV) radiation is a strong environmental carcinogen responsible for the pathogenesis of most skin cancers, including malignant melanoma (MM) and non-melanoma (keratinocyte) skin cancers. The carcinogenic role of UV was firmly established based on epidemiological evidence and molecular findings of the characteristic mutation signatures which occur during the excision repair of cyclobutane pyrimidine dimers and 6,4-photoproducts. The role of UV in the pathogenesis of mycosis fungoides (MF), the most common type of primary cutaneous T-cell lymphoma, remains controversial. Here, we performed whole-exome sequencing of 61 samples of MF cells microdissected from cutaneous lesions, and compared their mutational signatures to 340 MMs. The vast majority of MM mutations had a typical UV mutational signature (SBS 7, SBS 38, or DSB 1), underscoring the key role of ultraviolet as a mutagen. In contrast, the SBS 7 signature in MF comprised < 5% of all mutations. SBS 7 was higher in the intraepidermal MF cells (when compared to the dermal cells) and in the cells from tumors as compared to that in early-stage plaques. In conclusion, our data do not support the pathogenic role of UV in the pathogenesis of MF and suggest that the UV mutations are the result of the cumulative environmental ultraviolet exposure of cutaneous lesions rather than an early mutagenic event.

Genomic instability in early systemic sclerosis.

OBJECTIVES: Systemic sclerosis (SSc) is associated with secondary malignancies. Previous studies have suggested that mutated cancer proteins, such as RNA polymerase III, are autoantigens promoting an inflammatory response in SSc. However, it has never been previously investigated whether non-neoplastic tissue in SSc harbors mutations which may play a role in SSc pathogenesis. METHODS: Skin biopsies were obtained from 8 sequential patients with a progressive form of early stage SSc (with severe skin and/or lung involvement). Areas of dermal fibrosis were microdissected and analyzed with deep, whole exome sequencing. Gene mutation patterns were compared to autologous buccal mucosal cells as a control. RESULTS: SSc skin biopsies were hypermutated with an average of 58 mutations/106 base pairs. The mutational pattern in all samples exhibited a clock-like signature, which is ubiquitous in cancers and in senescent cells. Of the 1997 genes we identified which were mutated in at least two SSc patients, 39 genes represented cancer drivers (i.e. tumor suppressor genes or oncogenes) which are commonly found in gynecological, squamous and gastrointestinal cancer signatures. Of all the mutations, the most common mutated genes were important in regulating pathways related to epigenetic histone modifications, DNA repair and genome integrity. CONCLUSIONS: Somatic hypermutation occurs in fibrotic skin in patients with early progressive SSc. Cancer driver gene mutations may potentially play a fundamental role in the pathogenesis of SSc.

Clonotype pattern in T-cell lymphomas map the cell of origin to immature lymphoid precursors.

Mature T-cell lymphomas (TCLs) are rare, clinically heterogeneous hematologic cancers with high medical need. TCLs have an inferior prognosis which is attributed to poor understanding of their pathogenesis. On the basis of phenotypic similarities between normal and neoplastic lymphocytes, it has been assumed that TCLs develop in the periphery, directly from various subtypes of normal T cells. To address the debated question of the cell of origin in TCLs, we attempted to identify the highly variable complementarity-determining regions (CDRs) of T-cell receptors (TCRs) to trace the clonal history of the T cells. We have collected previously published whole-genome, whole-exome, and whole-transcriptome sequencing data from 574 patients with TCL. TCR clonotypes were identified by de novo assembly of CDR3 regions of TCRα, TCRß, and TCRγ. We have found that the vast majority of TCLs are clonotypically oligoclonal, although the pattern of oligoclonality varied. Anaplastic large-cell lymphoma was the most diverse comprising multiple clonotypes of TCRα, TCRß, and TCRγ, whereas adult TCL or leukemia and peripheral TCLs often showed monoclonality for TCRß and TCRγ but had diverse TCRα clonotypes. These patterns of rearrangements indicated that TCLs are initiated at the level of the lymphoid precursor. In keeping with this hypothesis, TCR rearrangements in TCLs resembled the pattern seen in the human thymus, which showed biased usage of V (variable) and J (joining) segments of high combinatorial probability resulting in recurrent public CDR3 sequences shared across unrelated patients and different clinical TCL entities. Clonotypically diverse initiating cells may seed target tissues that are then responsible for disease relapses after therapy.

The Neoantigen Landscape of Mycosis Fungoides.

Background: Mycosis fungoides (MF) is the most common cutaneous T-cell lymphoma, for which there is no cure. Immune checkpoint inhibitors have been tried in MF but the results have been inconsistent. To gain insight into the immunogenicity of MF we characterized the neoantigen landscape of this lymphoma, focusing on the known predictors of responses to immunotherapy: the quantity, HLA-binding strength and subclonality of neoantigens. Methods: Whole exome and whole transcriptome sequences were obtained from 24 MF samples (16 plaques, 8 tumors) from 13 patients. Bioinformatic pipelines (Mutect2, OptiType, MuPeXi) were used for mutation calling, HLA typing, and neoantigen prediction. PhyloWGS was used to subdivide malignant cells into stem and clades, to which neoantigens were matched to determine their clonality. Results: MF has a high mutational load (median 3,217 non synonymous mutations), resulting in a significant number of total neoantigens (median 1,309 per sample) and high-affinity neoantigens (median 328). In stage I disease most neoantigens were clonal but with stage progression, 75% of lesions had >50% subclonal antigens and 53% lesions had CSiN scores <1. There was very little overlap in neoantigens across patients or between different lesions on the same patient, indicating a high degree of heterogeneity. Conclusions: The neoantigen landscape of MF is characterized by high neoantigen load and significant subclonality which could indicate potential challenges for immunotherapy in patients with advanced-stage disease.

Independent evolution of cutaneous lymphoma subclones in different microenvironments of the skin.

Mycosis fungoides (MF) is the most common cutaneous T-cell lymphoma. Lesions of MF are formed by hematogenous seeding the skin with polyclonal (clonotypically diverse) neoplastic T-cells which accumulate numerous mutations and display a high degree of mutational, intratumoral heterogeneity (ITH). A characteristic but poorly studied feature of MF is epidermotropism, the tendency to infiltrate skin epithelial layer (epidermis) in addition to the vascularized dermis. By sequencing the exomes of the microdissected clusters of lymphoma cells from the epidermis and the dermis, we found that those microenvironments comprised different malignant clonotypes. Subclonal structure witnessed the independent mutational evolution in the epidermis and dermis. Thus, the epidermal involvement in MF could not be explained by gradual infiltration from the dermis but was caused by a separate seeding process followed by a quasi-neutral, branched evolution. In conclusion, tissue microenvironments shape the subclonal architecture in MF leading to "ecological heterogeneity" which contributes to the total ITH. Since ITH adversely affects cancer prognosis, targeting the microenvironment may present therapeutic opportunities in MF and other cancers.

Branched evolution and genomic intratumor heterogeneity in the pathogenesis of cutaneous T-cell lymphoma.

Mycosis fungoides (MF) is a slowly progressive cutaneous T-cell lymphoma (CTCL) for which there is no cure. In the early plaque stage, the disease is indolent, but development of tumors heralds an increased risk of metastasis and death. Previous research into the genomic landscape of CTCL revealed a complex pattern of >50 driver mutations implicated in more than a dozen signaling pathways. However, the genomic mechanisms governing disease progression and treatment resistance remain unknown. Building on our previous discovery of the clonotypic heterogeneity of MF, we hypothesized that this lymphoma does not progress in a linear fashion as currently thought but comprises heterogeneous mutational subclones. We sequenced exomes of 49 cases of MF and identified 28 previously unreported putative driver genes. MF exhibited extensive intratumoral heterogeneity (ITH) of a median of 6 subclones showing a branched phylogenetic relationship pattern. Stage progression was correlated with an increase in ITH and redistribution of mutations from stem to clades. The pattern of clonal driver mutations was highly variable, with no consistent mutations among patients. Similar intratumoral heterogeneity was detected in leukemic CTCL (Sézary syndrome). Based on these findings, we propose a model of MF pathogenesis comprising divergent evolution of cancer subclones and discuss how ITH affects the efficacy of targeted drug therapies and immunotherapies for CTCL.

Skin colonization by circulating neoplastic clones in cutaneous T-cell lymphoma.

Mycosis fungoides (MF) is a mature T-cell lymphoma currently thought to develop primarily in the skin by a clonal expansion of a transformed, resident memory T cell. However, this concept does not explain the key characteristics of MF, such as the debut with multiple, widespread skin lesions or inability of skin-directed therapies to provide cure. The testable inference of the mature T-cell theory is the clonality of MF with respect to all rearranged T-cell receptor (TCR) genes. Here, we used a whole-exome sequencing approach to detect and quantify TCR-α, ß, and γ clonotypes in tumor cell clusters microdissected from MF lesions. This method allowed us to calculate the tumor cell fraction of the sample and therefore an unequivocal identification of the TCR clonotypes as neoplastic. Analysis of TCR sequences from 29 patients with MF stage I to IV proved the existence of multiple T-cell clones within the tumor cell fraction, with a considerable variation between patients and between lesions from the same patient (median, 11 clones; range, 2-80 clones/sample). We have also detected multiple neoplastic clones in the peripheral blood in all examined patients. Based on these findings, we propose that circulating neoplastic T-cell clones continuously replenish the lesions of MF, thus increasing their heterogeneity by a mechanism analogous to the consecutive tumor seeding. We hypothesize that circulating neoplastic clones might be a promising target for therapy and could be exploited as a potential biomarker in MF.

Clonotypic heterogeneity in cutaneous T-cell lymphoma (mycosis fungoides) revealed by comprehensive whole-exome sequencing.

Mycosis fungoides (MF), the most common type of cutaneous T-cell lymphoma, is believed to represent a clonal expansion of a transformed skin-resident memory T cell. T-cell receptor (TCR) clonality (ie, identical sequences of rearranged TCRα, TCRß, and TCRγ), the key premise of this hypothesis, has been difficult to document conclusively because malignant cells are not readily distinguishable from the tumor-infiltrating reactive lymphocytes that contribute to the TCR clonotypic repertoire of MF. Here, we have successfully adopted targeted whole-exome sequencing (WES) to identify the repertoire of rearranged TCR genes in tumor-enriched samples from patients with MF. Although some of the investigated MF biopsies had the expected frequency of monoclonal rearrangements of TCRγ corresponding to that of tumor cells, the majority of the samples presented multiple TCRγ, TCRα, and TCRß clonotypes by WES. Our findings are compatible with the model in which the initial malignant transformation in MF does not occur in mature memory T cells but rather at the level of T-lymphocyte progenitors before TCRß or TCRα rearrangements. We have also shown that WES can be combined with whole-transcriptome sequencing in the same sample, which enables comprehensive characterization of the TCR repertoire in relation to tumor content. WES/whole-transcriptome sequencing might be applicable to other types of T-cell lymphomas to determine clonal dominance and clonotypic heterogeneity in these malignancies.

Predicting bond strength from a single Hartree-Fock ground state using the localized pair model.

We present an application of the recently introduced Localized Pair Model (LPM) [Z. A. Zielinksi and J. K. Pearson, Comput. Theor. Chem., 2013, 1003, 7990] to characterize and quantify properties of the chemical bond in a series of substituted benzoic acid molecules. By computing interelectronic distribution functions for doubly-occupied Edmiston-Ruedenberg localized molecular orbitals (LMOs), we show that chemically intuitive electron pairs may be uniquely classified and bond strength may be predicted with remarkable accuracy. Specifically, the HF/u6-311G(d,p) level (where u denotes a complete uncontraction of the basis set) is used to generate the relevant LMOs and their respective interelectronic distribution functions can be linearly correlated to the well-known Hammett σp or σm parameters with near-unity correlation coefficients.