HTLV-1 is estimated to affect ~20 million people worldwide and in ~5% of carriers it produces Adult T-Cell Leukemia/Lymphoma (ATLL), which can often masquerade and present with classic erythematous pruritic patches and plaques that are typically seen in Mycosis Fungoides (MF) and Sézary Syndrome (SS), the most recognized variants of Cutaneous T-Cell Lymphomas (CTCL). For many years the role of HTLV-1 in the pathogenesis of MF/SS has been hotly debated. In this study we analyzed CTCL vs. HTLV-1+ leukemic cells. We performed G-banding/spectral karyotyping, extensive gene expression analysis, TP53 sequencing in the 11 patient-derived HTLV-1+ (MJ and Hut102) vs. HTLV-1- (Myla, Mac2a, PB2B, HH, H9, Hut78, SZ4, Sez4 and SeAx) CTCL cell lines. We further tested drug sensitivities to commonly used CTCL therapies and studied the ability of these cells to produce subcutaneous xenograft tumors in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice. Our work demonstrates that unlike classic advanced MF/SS cells that acquire many ongoing balanced and unbalanced chromosomal translocations, HTLV-1+ CTCL leukemia cells are diploid and exhibit only a minimal number of non-specific chromosomal alterations. Our results indicate that HTLV-1 virus is likely not involved in the pathogenesis of classic MF/SS since it drives a very different pathway of lymphomagenesis based on our findings in these cells. This study also provides for the first time a comprehensive characterization of the CTCL cells with respect to gene expression profiling, TP53 mutation status, ability to produce tumors in mice and response to commonly used therapies.

Genomic instability is a hallmark of cancer and an enabling factor for genetic alterations that drive cancer development and progression. The clashing of mitosis and aberrantly expressed meiosis machineries, which may contribute to genomic instability, has been coined cancer “meiomitosis”. LINE-1 retrotransposition, a process active in germ cells, acts outside of the meiotic machinery to create DNA double strand breaks (DNA DSBs) and has played an important role in the evolution of the human genome. We have previously demonstrated that in CTCL several cancer testis/meiotic genes are expressed. Furthermore, this cancer exhibits extensive and ongoing chromosomal/microsatellite instability. In this study we analyzed immortalized patient-derived cells and primary CTCL patient samples using RT-PCR, western blotting and confocal microscopy and found that proteins critically involved in meiosis and LINE-1 retrotransposition are expressed and are associated with chromosomal instability and DNA DSB formation. Using cell cycle synchronization, we show G1/S phase-transition-specific expression of meiosis proteins. Using the Alu retrotransposition assay, we demonstrate the functional activity of LINE-1 retrotransposon in CTCL. Histone acetyltransferase inhibition results in downregulation of the ectopic germ cell programs and concomitant decrease in DNA DSBs foci formation. Notably, LINE-1 and meiosis genes were expressed across a panel of other solid tumor cell lines. Taken together, our results indicate that malignant cells in culture undergo “cancer meiomitosis” rather than the classic mitosis division. The ectopic expression of meiosis genes and reactivation of LINE-1 may be contributing to genomic instability and represent novel targets for immunotherapy in this and other cancers.

Hypopigmented mycosis fungoides (HMF) is a form of cutaneous T-cell lymphoma (CTCL), a heterogeneous group of extranodal non-Hodgkin’s lymphomas. HMF has a unique set of defining features that include light colored to achromic lesions, a predilection for darker skin phototypes, an early onset of disease, and predominance of CD8+ T-cells, among others. In the current review, we detail the known pathways of molecular pathogenesis for this lymphoma and posit that an active Th1/cytotoxic antitumor immune response in part explains why this variant is primarily seen in children/adolescents and young adults, who do not exhibit signs of immunosenescence. As a result of this potent cytotoxic response, HMF patients experience mostly favorable overall prognosis, while hypopigmentation may in fact represent a useful surrogate marker of cytotoxic immunity targeting the malignant cells. Understanding the molecular processes behind the specific features that define HMF may lead to improved diagnostic accuracy, personalized prognosis by risk stratification, and improved management of HMF. Moreover, improving our knowledge of HMF may aid our further understanding of other cutaneous lymphomas.

Cancer testis (CT) antigens, under normal circumstances are uniquely expressed in testicular germ cells. Recent research has shown that meiosis-specific CT (meiCT) antigens are ectopically expressed in cutaneous T-cell lymphoma (CTCL) and may contribute to increased genomic instability. The aberrant activation of meiosis genes in a mitotic cell is now recognized as a distinctive process, “meiomitosis.” We have previously demonstrated the ectopic expression of several meiCT antigens in nine patient-derived CTCL cell lines and in expanded peripheral T lymphocytes isolated from Sézary Syndrome patients. In this study we analyzed the transcriptional expression of meiCT genes in Sézary Syndrome patients and healthy controls using publicly-available RNA sequencing (RNA-Seq) data. We corroborated our in silico analysis by examining the expression of 5 meiCT proteins in formalin-fixed, paraffin-embedded (FFPE) lesional samples from CTCL patients. Our results show significant differential gene expression of STAG3, SGO2, SYCP3 , and DMC1 in a cohort of Sézary Syndrome patients when compared to healthy controls. Additionally, our study demonstrates a heterogenous expression of meiCT genes involved in initiation ( STRA8 ), sister chromatin cohesion ( STAG3, SGO2 ), homologous chromosome synapsis ( SYCP3 ) and homologous recombination ( DMC1 ) in atypical lymphocytes in FFPE samples. Our results further confirm the ectopic expression of meiCT genes in CTCL which indicates that CTCL malignant cells likely undergo the process of cancer meiomitosis, as opposed to a typical mitotic division. The ectopic expression of meiCT genes together with investigations into the functional mechanisms of cancer meiomitosis will help provide a foundation to develop novel diagnostic tests to distinguish CTCL from benign inflammatory dermatoses and may enable us to develop additional targeted therapies for patients with this malignancy.

Immunostaining of non-adherent cells is commonly performed after adhesion of cells onto microscope slides either using cytocentrifugation or with the help of charged coating substrates. These techniques, however, require either specialized equipment or significant preparation time. Here, we describe a method for immunofluorescent staining of lymphocytes within multi-well culture plates, where cells suspended in phosphate buffered saline (PBS) are adhered to either the plastic well bottom or glass coverslips by gravity sedimentation. This technique requires only common laboratory materials, no coating steps, and allows for densely adherent cell coverage with 1 × 106 cells. Our data show that suspension of cells in PBS, but not serum-containing growth medium, allows for adhesion to plastic or glass after 30 min of gravity sedimentation. We show that this method is applicable for immunofluorescent staining of both primary human lymphocytes and immortalized lymphoma cells, and that it preserves cell morphology.