Eligibility criteria for intergroup trials E1694 and E1690 included patients with primary cutaneous melanoma thicker than 4 mm in the absence of regional node metastases (American Joint Committee on Cancer stage II B disease) or the presence of regional lymph node involvement (American Joint Committee on Cancer stage III disease) at diagnosis or at subsequent recurrence.23,24
All pathologic material received for each of the patients accrued into these Eastern Cooperative Oncology Group (ECOG)-led intergroup protocols was reviewed on entry, and the diagnosis of melanoma was confirmed along with the assessment of 15 characteristics of the primary tumor. Data were recorded on standardized ECOG pathology forms by the ECOG pathologist and study cochair (U.N.M.R.). This information included tumor size, ulceration, levels of invasion as defined by Clark et al,2
Breslow thickness, presence of partial regression, mitoses, and surgical treatment. For each case, the pathology slides were assigned ECOG protocol case sequence numbers at the pathology coordinating office of ECOG located at Northwestern University, Chicago, IL, such that patient information could not be linked to the slides by the reviewing pathologists. The information from the completed pathology forms was entered into the central ECOG database at the Coordinating Center in Boston, MA.
All slides were reexamined by 2 pathologists (U.N.M.R. and M.C.M.) following approval of the study protocol (IRB No. 020746) by the institutional review board at the University of Pittsburgh, Pittsburgh, PA, and the ECOG scientific review committees. Pathology slides of the primary tumor were available from 14 patients in the E1690 trial and 342 patients in the E1694 trial. This study of advanced-stage melanoma included cases at high risk by virtue of primary tumor thickness (≥4 mm) or because they were associated with regional lymph node metastases at diagnosis. The thickness of the primary tumor in this latter group ranged from 0.76 to 1.5 mm (49 cases). Of these 49 cases, 34 patients had tumors that were 1 mm or less in thickness, for which there was evidence of partial regression. In 94 cases, tumors ranged from 1.5 to 4 mm in thickness, and the rest were greater than 4 mm in thickness (188 cases).
The patterns of TILs were reevaluated according to consensus guidelines that were adopted for wider corroboration of the importance of TILs in melanoma. TILs were defined as those that percolated between tumor cells and surrounded individual tumor cells. The infiltrate was assessed quantitatively and reached an approximate tumor/lymphocyte ratio of 1:5.
The intensity of lymphocytic infiltrates and their locations were evaluated. Focal refers to a single collection of few lymphocytes; multifocal refers to multiple collections of few lymphocytes; and segmental is a large confluent collection of lymphocytes in the center or at the periphery of the tumor occupying at least a third but not the entire portion of the vertical growth phase of the tumor or at its circumference.
The intensity of lymphocytic infiltrates, albeit partly subjective, was evaluated. Grade 1 refers to few lymphocytes, no less than 10 per high-power field (×40) (nonbrisk); grade 2 to approximately 10 to 20 lymphocytes per high-power field; and grade 3 to a density of lymphocytes greater than 20 per high-power field throughout the lesion (brisk).
These data with respect to the tumor were assigned specific numbers for statistical computation. In each of the cases, the findings were entered on a standardized form.
Partial regression was defined as the presence of fibrosis of the papillary dermis with ectatic vessels, associated with flattening of overlying epidermal rete and a scant to absent melanoma in situ component in this area. In addition, variable amounts of lymphocytes and melanophages were associated with lesions in which the diagnosis of partial regression was made. The cases with sectioning artifacts, cases of suboptimal quality, or cases in which a small and inadequate portion of the primary tumor was submitted were excluded from the study. Any unusual findings were recorded in the comment section.
Five-year truncated OS and RFS data were used for this analysis because it was deemed most reasonable to use the 5-year OS and RFS data in assessing the TIL data. Patients who died or in whom disease progressed after 5 years were censored at 5 years for OS and RFS, respectively. For simplicity in terminology, OS and RFS truncated at 5 years are referred to as OS and RFS henceforth. The method of Kaplan and Meier was used to evaluate OS and RFS in univariate analysis. The distribution of OS and RFS by patterns of lymphocytic infiltration and their location were compared using the log-rank test. Other prognostic factors such as ECOG performance status, ulceration, Breslow thickness, Clark level of the primary tumor, presence of angiolymphatic and perineural invasion, stage and nodal involvement, and interferon treatment were also evaluated. The stratified log-rank test was also used. Cox proportional hazards regression models were used to evaluate the patterns of infiltration and locations, adjusting for other prognostic factors. Associations of categorical data were assessed by using the Fisher exact test. All P values reported in this article are based on 2-sided testing.