Tumor-Infiltrating CD8+ Lymphocytes Effect on Clinical Outcome of Muco-Cutaneous Melanoma.

BACKGROUND: Recent data have changed our views of prognostic factors in cutaneous melanoma, while some newer methods have yielded better prognostic information. Tumor-infiltrating lymphocytes are believed to represent the immune reaction/response to melanoma cells which is often found in melanocytic cancer. AIM AND
OBJECTIVE: We carried out an analysis, aiming to establish pooled estimates for clinical outcomes based on the presence of CD8+ T cell in melanocytic cancer.
MATERIALS AND METHODS: We have included 42 patients with primary cutaneous melanocytic cancer without preoperative treatments in our study. We next analyzed the proliferative activity of CD8+ T cells that infiltrated in tumor cell nests. The intratumoral and adjacent to invasive margin of tumor CD+ T-cell infiltration were analyzed which could also reflect antitumor immunity.
RESULTS: The total number of CD8+ cells especially adjacent to invasive margin of tumor was positively correlated with anatomical tumor thickness (P < .001) and not correlated with patient's age and sex. The stage of tumor which is related to vascular-neural invasion, regional lymph nodes involvement and tumor thickness shows positive correlation with CD8+ infiltration in tumor (P < .004, P < .005, P < .001), respectively. Acral melanoma shows more CD8 lymphocytes infiltration and also recurrence rate of tumor (P < .005).
CONCLUSION: We believe that CD8+ T-cell infiltration in primary cutaneous melanocytic cancer represents the immune reaction/response to melanoma which could be an important new therapy for melanoma although more research is needed on this treatment modality.

What was known?

Cytotoxic T cell have clinically significant antitumoral activity against human melanocytic cancer.

Introduction

The biological malignancy of human malignant neoplasm is determined by effects of biological malignancy of tumor cells and host reactions which is represented by immune and vascular host answers. In human cancers, however, such immune reactions are generally insufficient because of weak immunogenicity of cancer cells or impaired immunity in cancer-bearing patients.[1] The presumptive mechanisms of these included the absence of tumor-specific antigens, down-regulation of MHC class I molecule expression,[2] or secretion of immunosuppressive cytokines.[3] Cytotoxic CD8+ T lymphocytes are crucial components of tumor-specific cellular adaptive immunity that attack tumor cells presenting tumor-associated antigen peptide with major histocompatibility complex class I on their surface. CD8+ T cells produce interferon gamma following interaction with their tumor targets. The interferon gamma-dependent mechanisms of tumor cell cytostasis and killing consequently occur by cell cycle inhibition, apoptosis, angiostasis, and induction of macrophage tumoricidal activity.[45] Recently, functional inactivation of potentially tumor-reactive T cells was analyzed as an important mechanism of immune evasion.[4] Human melanocytic cancer is one of the targets of immunotherapy. Melanocytic cancer is a heterogeneous disease embracing a range of clinical patterns, biologic behavior, prognostic characteristics, and response to different types of treatment. The present study was designed to analyze CD8+ T cells in human melanocytic cancer, which showed that CD8+ T cells infiltrated into cancer cell nests could reflect antitumor immunity. However, it is still unclear whether the presence of CD8+ cytotoxic lymphocytes provides any prognostic information in human melanocytic cancer. Therefore, our aim was to analyze the influence of density and distribution of CD8+ cytotoxic lymphocytes on patient prognosis in well-characterized series of patients with primary cutaneous melanocytic cancer during about 3 year follow-up.

Materials and Methods

We retrospectively analyzed 42 patients with human melanocytic cancer with about 3-year follow-up data. We have excluded the patients with immunosuppressive problems or who suffered of chronic background disease. None of these patients received preoperative immunotherapy. The age of patients ranged from 25 to 81 years of age (mean, 57.9 years). They were 27 males (64.3%) and 15 females (35.7%) and male: female ratio was 1.8. The patients received complete resection of tumor with regional lymph node dissection who have been followed up for 3 years during 2011 and 2013 at Kermanshah University Hospitals. Resected specimens were fixed in formalin and embedded in paraffin for the routine histopathological diagnosis. After surgery, 31 patients received immunotherapy with interferon-alpha and interleukin-2 then 11 patients received Ipilimumab (Yervoy) which is a monoclonal antibody. The follow-up data showed that 26 patients survived with cancer also with recurrence and 16 patients died of this cancer because of metastasis. The tumor stage was classified into four according to TNM classification. Immunohistochemistry for CD8+ TILs was performed by using the antibody against human CD8 (clone C8/144B, dilution 1:100) in accordance with the protocol of the manufacturer (DakoCytomation, Glostrup, Denmark). The chromogen was 3,3-diaminobenzidine tetra hydrochloride (brown). We classified CD8+ T cells into three locations in two groups and three locations: (a) those distributed along the invasive margin of cancer location (peritumoral group); (b) those infiltrated in cancer stroma location and (c) intratumoral-nests location (intratumoral group). We used normal human tonsil tissue as a positive control of immunohistochemistry staining of CD8+ lymphocytes which are distributed mainly in the paracortical lymphoid tissue of tonsil.

We semi quantitatively scored the degrees of infiltration into four groups: 0, nil; I, mild; II, moderate; and III, severe. We counted numbers of CD8+ T lymphocytes each tumor core using a Nikon Eclipse 80i microscope (Nikon, Tokyo, Japan). CD8+ T cells were counted in two compartments in each tumor: intratumoral compartment (within the tumor cell nests and stroma) and within peritumoral compartment. The total number of CD8+ T cells was determined by combining the counts for the two compartments. The average numbers of 0, 1-19, 20-49, and over 50 were scored as 0, I, II, and III, respectively.[6] Scores were also rechecked randomly by a second observer. Interobserver agreement was found (κ =0.75).

Tumor-host interface was clearly confirmed in 42 cases. Two independent observers judged. When the results were not consistent, the two observers discussed to reach a consensus on the semi quantification scoring.

Statistical analysis

We quantified or semi quantified each variable as described above and then made correlation with the patients’ clinical outcome with Pearson Chi-square method for each variable and using computer.

Software Spss16. We judged correlation significant with Fisher's exact test, crosstab, one-way ANOVA and then reportedreported P value for correlation effects of each variable.

Results

We retrospectively analyzed 42 patients with human melanocytic cancer with about 3-year follow-up data. None of these patients received preoperative immunotherapy. The age of patients ranged from 25 to 81 years of age (mean, 57.9 years). They were 27 males (64.3%) and 15 females (35.7%) and the male: female ratio was 1.8. The most frequency of tumor was in acral areas with face and the least was in genitalia and upper extremity. Primary cutaneous melanocytic cancer of acral area shows the most total CD8+ T-cell infiltration but was not significant which is possibly related to the numbers of our specimen [Table 1].

Table 1

Frequency of melanocytic tumor location in our patients and CD8+ T lymphocytic infiltration in the different locations in human primary cutaneous melanocytic cancer (n=42)

Tumor infiltration lymphocytes recognized by conventional H and E stain were mainly detected along the tumor-host interface. We designated infiltration CD8+ T cell in contact to cancer cells [Figure 1].

Figure 1

Intratumoral area of melanocytic cancer tissue section infiltrated by CD8+ cells. (a) Peritumoral area of tumor infiltrated by CD8+ cells (original magnification ×200)

We first analyzed the proliferative activity of CD8+ T cells that infiltrated in tumor cell nests (intratumoral and adjacent to invasive margin of tumor), which could also reflect antitumor immunity.

Semi quantitative scoring of intratumoral (34%) and peritumoral (76%) CD8+ T-cell infiltration represented positive correlation with both tumor thickness (P < .001) and stage of tumor (P < .003). The total number of CD8+ cells was positively correlated with anatomical tumor thickness (P < .001) and not correlated with patient's age and sex. The stage of tumor, vascular - neural invasion and recurrence rate of tumor show positively correlation with CD8+ T-cell infiltration of tumor (P < .001, P < .004, P < .005) respectively [Figure 1]. Anatomical growth of tumor (superficial or nodular pattern) was affected from CD8+ T-cell infiltration which shows positive correlation between them (P < .001).

Recurrence rate of tumor represents positive correlation with peritumoral CD8+ T-cell infiltration (P < .005). Acral melanoma shows more CD8 lymphocytes infiltration (P < .21) [Table 2].

Table 2

Correlation between total numbers of CD8+ lymphocytic infiltration and clinicopathologic features in human primary cutaneous melanocytic cancer

We analyzed CD8+ T cells among different localization patterns to demonstrate, for the first time, that CD8+ T cells that infiltrated within melanocytic cancer cell nests can effect on clinical outcome of patient [Figure 2].

Figure 2

Correlation between stage of tumor, recurrence of tumor and total CD8+ T-cell infiltration in human primary cutaneous melanocytic cancer

Discussion

We analyzed CD8+ T cells among different localization patterns to demonstrate, for the first time, that the CD8+ T-cell infiltration within melanocytic cancer cell nests can affect on clinical outcome of patient. The impact of this factor is parallel with staging of melanocytic cancer. The effects by CD8+ T cells within cancer cell nests could be theoretically related to the effect or function of activated killer T cells. CD8+ T cells may migrate into cancer cell nests, exhibiting a higher proliferation MHC class I molecule required to be expressed by cancer cells for the recognition of cancer cells by T cells which provides evidence in colorectal cancer cells.[7] In cervical neoplasia, no clear correlation was reported between the expression of MHC class I molecules by neoplastic cells and infiltration of CD8+ T cells into the neoplastic tissue.[8]

In previous studies, it was believed that tumor-infiltrating lymphocytes can represent the immune reaction/response to melanoma cells which is measured by the level of lymphocytic infiltrate present at the base of the vertical growth phase of the tumor and is sometimes categorized as brisk, no brisk, or absent.[9] The most brisk tumor-infiltrating lymphocytes response is found in thin tumors.[10] The 5- and 10-year survival rates for melanoma with a vertical growth phase and a brisk infiltrate were 77% and 55%, respectively. For tumors with a no brisk infiltrate, the 5- and 10-year survival rates were 53% and 45%, respectively, and for tumors with absent tumor-infiltrating lymphocytes, the 5- and 10-year survival rates were 37% and 27%, respectively.[10] There is a need for a uniform definition of host response in terms of type and location of infiltrate before the role of tumor-infiltrating lymphocytes can be clarified. Tumor-infiltrating lymphocytes could be an important new therapy for melanoma.[1112]

Our results suggest that the cell-mediated immune reaction has an important role in the outcome of melanocytic cancer, as described in other tumor types such as colorectal carcinoma.[7]

As previously described, CD8+ T-cell count was correlated with higher histologic grade in colon cancer and this mechanism results that tumor escape from immune-mediated destruction.[7] Our results, however, suggest that the adaptive immune response plays a role in preventing tumor recurrence in humans. When the tumor is clinically apparent, the proposed tumor-associated factors (possibly the antigenic determinants or danger signals) and inflammatory elements may provide an efficient adaptive immune reaction that averts tumor progression.

The tumor might not stimulate an immune response in the early stages, perhaps because of a lack of danger signals. It is generally believed that antitumor immune responses are focused in the draining lymph nodes.[7] Our observations, however, suggest that this could also occur in the tumor site.

Despite the presence of a lymphocytic infiltrate in breast cancer, complete regression of breast cancer is extremely rare. Moreover, the fact that a tumor is present at all and its continued growth despite the presence of cytotoxic CD8 lymphocytes imply that the host immune response is not completely successful, presumably because of various tumor mechanisms to evade immunologic surveillance. Lymphocytes have been isolated from primary breast carcinomas and their functions have been tested in vitro (A review is available in Stewart and Heppner).[13]

Evidence suggests that tumors produce an array of immune inhibitory factors that exert either local or systemic effects on the host immunity.[14] However, patients with a measurable lymphocyte-mediated immune response against tumor-associated antigen had an excellent prognosis. In conclusion, our results provide evidence of clinical outcome importance of the cytotoxic T-cell population in primary cutaneous melanocytic cancer. This suggests that cytotoxic T cells have clinically significant antitumoral activity against human melanocytic cancer. The results of this study support further investigation of this line of potential therapeutic intervention.

What is new?

This study support further potential therapeutic intervention.