1. 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

2. 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.

3. 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

4. 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.

5. 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.

6. 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

7. 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

8. 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

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

10. 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.

11. 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

12. 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 ( )
Median age at diagnosis (years; range)
60 ( )
Median time to death from diagnosis (months; range)
43 ( )
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"

14. 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.

15. 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