Mapping the Clinicopathologic and Molecular Genetic Evolution of Cutaneous T-Cell Lymphoma Peter Louis1, Michael Berger PhD1, Rose Brannon PhD1, Helen Won1, Christiane Querfeld MD PHD2, Steven Horwitz MD2, Patricia L. Myskowski MD2, Melissa Pulitzer MD1 Department of Pathology1 and Department of Medicine2 Memorial Sloan Kettering Cancer Center

Purpose The aim of this study is to determine if there is molecular genetic heterogeneity within chronologically and spatially distinct lesions of patients with Cutaneous T-Cell Lymphoma (CTCL), and to compare any such changes with clinical and pathologic characteristics.

Background CTCL is a rare but potentially lethal malignancy of skin-homing T-lymphocytes. The annual incidence of CTCL in the United States is between 4-8 per 1,000,000 persons Mycosis Fungoides (MF) is the most prevalent subset of CTCL, accounting for over 50% of cases While the overall mortality of MF is low, at approximately 100 deaths per year, patients with tumor stage disease have a 5-year disease specific survival of 30% Currently, there is no curative therapy for patients with advanced stage MF

Background Little is known in specific cancers, if genetic and epigenetic changes are conserved throughout the chronologic or spatial progression of a tumor Recent data suggests that mutational intratumoral heterogeneity may be greater than expected In this context, our objective is to evaluate sequential and spatially distinct specimens from select MF patients, in order to evaluate the molecular evolution of their tumors, and to relate genetic findings with clinical and pathologic features.

Background Patch stage lesions are typically thin, non-infiltrated, pink, and slightly scaly. The sites of predilections are the sun-protected areas such as the buttocks. Plaque stage lesions are generally flat, indurated and elevated above the skin surface. Any area of the body surface may be involved. Tumor stage MF most commonly presents as nodules. Erythrodermic MF presents as widespread erythema. Lymph node and visceral involvement, as well as large cell transformation, usually occur in the late stage of the disease.

Study Design We identified patients with a clinicopathologic diagnosis of advanced stage MF, confirmed by review of clinical records, and by examination of pathology slides and reports Seven patients were selected, who had in-house normal control tissue and sequential and/or synchronous biopsies of MF in skin and/or lymph node tissue. Clinical and pathologic data, including demographics, survival, histologic, immunophenotypic, and molecular findings were recorded. Formalin-fixed paraffin embedded (FFPE) tumor tissues and normal controls were obtained from the MSKCC pathology archive Genomic DNA was extracted from FFPE tissue sections using standard methods. Samples were analyzed for the presence of somatic point mutations, small indels and copy number alterations using publicly available algorithms

Table 1 . Patient and Biopsy Characteristics Case# Site of 1st Biopsy Stage at 1st Biopsy Clinical presentation at 1st Biopsy Age at 1st Biopsy Site of 2nd Biopsy Stage at 2nd Biopsy Clinical presentation at 2nd Biopsy Age at 2nd Biopsy Time Elapsed between Biopsies (Months) 1 Left cheek IIB Scaly patches, erythematous nodules, multiple violaceous tumors 59 Right upper abdomen Poikilodermatous patch, large erythematous plaques, and crusted ulcerations, extensive nodules on foot 60 6 2 Right abdomen IB Hypopigmented patches 66 Axillary lymph node IVA2 Hypopigmented patches, diffusely excoriated, thickened with scale 74 94 3 Left axilla Large ulcerated nodular tumors with necrosis Right axilla, soft tissue Multiple erythematous, weeping tumors 4 Left nasal bridge Hyperpigmented patches, eroded nodule 62 5 Right lower leg Multiple patches and thick plaques 73 Right dorsum tongue Multiple patches and plaques 75 18 Right lateral low back Multiple large erythematous patches and plaques Right middle low back 7 Left leg Diffuse erythema over face, erythrematous plaques 57 Left lower back Multiple slightly erythematous plaques and nodules 23 Age, Stage and Survival correlate with time at diagnosis. Anatomic site refers to skin, unless otherwise specified. "1st biopsy" indicates first biopsy analyzed for this study. "2nd biopsy" indicates second biopsy analyzed for this study. AJCC TNM staging used is as follows: IA correlates to patch or plaque covering less than 10 percent of skin; IB correlates to patch or plaque covering greater than 10 percent of skin; IIB correlates to tumor stage disease; IVA2 correlates to lymph node effacement by MF H Hispanic; NH Non-Hispanic; W White

A B C D Figure 1. A. Case 1 upon presentation with tumor stage MF/LCT. B. Close-up photo of the region biopsied. C. 6 months later, tumors on the abdomen. D. Close-up view of the area biopsied

A B Figure 2. A, B Low power, punch biopsies of case, 1 diffuse sheets of tumor.

A B C Figure 3. A. Case 2, biopsy of plaque stage disease. B, C. Low and high power photomicrographs of effaced lymph node and transformed disease.

Same Clone Present in Both Biopsies Table 2. Pathologic Characteristics 1st Biopsy 2nd Biopsy   Case # CD3 CD4 (%) CD8 (%) CD4:CD8 CD7 Clonal Beta TCR Clonal Gamma TCR TCR Beta TCR Gamma Same Clone Present in Both Biopsies 1 + 99 25:1 preserved ND 70 5 15:1 loss 2 80 10 8:1 Yes 3 No** 4 20 4:1 90 10:1 95 20:1 6 7 ND = No Data **Necrosis

Table 3. Summary of Data Patient Characteristics Male:Female 4:3   Male:Female 4:3 Median time from first sign to diagnosis (months; range) 18.5 (1.5 - 158) Median age at diagnosis (years; range) 60 (51 - 75) Median time to death from diagnosis (months; range) 43 (8 - 131) Median age at 1st biopsy (years; range) 62 (57 -75) Median age at 2nd biopsy (years; range) 62 (59 -75) Cases with large cell transformation 5 Tumor characteristics Number of skin biopsies (tumor/plaque) 11 (8/3) Skin lesions with large cell transformation (tumor/plaque) 7 (7/0) Number with tumor in lymph node 1* Patients with folliculotropism 5/6 Patients with follicular mucin 1/6 Tumor in other location 2* Median interval between biopsies (months, range) 6 (0 - 94) *A 2nd might have been node, but unclear, so placed in "other location"

Conclusions/Future Directions Evolution of CTCL in our patients was characterized in part by clinical, histologic, and immunophenotypic changes, however, these changes are not consistent or predictable We await the findings of our targeted mutational analysis to see if conservation or evolution of cancer-related mutations in CTCL better characterize progression of disease.

References Zackheim, H. S. (2005). Cutaneous T-cell lymphoma : Mycosis Fungoides and Sézary syndrome. Boca Raton, Fla., CRC Press. Quaglino P, Pimpinelli N, Berti E, et al. Time course, clinical pathways, and long-term hazards risk trends of disease progression in patients with classic mycosis fungoides: A multicenter, retrospective follow-up study from the Italian Group of Cutaneous Lymphomas. Cancer. Jun 6 2012.