Clinical course
A 52-year-old Asian female patient was admitted to our clinic, with complaints of right cheek pain and an ulcerative protruding mass lesion in the right buccal area. We conducted punch biopsy for the buccal mass and this revealed well-differentiated squamous cell carcinoma. Computed tomography (CT), magnetic resonance imaging (MRI) and positron emission tomography/computed tomography (PET/CT) were conducted and these delineated a 2.9 × 2 × 1.4 cm buccal mass in the right lower cheek with right lymph node metastasis (level Ib, IIa and III), and there was no evidence of distant metastasis. We made a diagnosis of buccal cancer cT2N2bM0 (Figure 1A-B). She underwent wide surgical resection of the malignant tumor in the right buccal area, right comprehensive neck dissection (level I to V) and left selective neck dissection (level I to III). The defect that occurred after the tumor resection was reconstructed with an omental free flap. According to the pathologic TNM (tumor, node, metastasis) staging system, the final diagnosis was pT2N1 (right lymph node metastasis). Post-operative adjuvant radiotherapy was administered, covering the primary sites and regional nodes (right whole neck and left upper neck) (Figure 1C-D).
After the treatment mentioned above, we regularly checked the CT, MRI and PET/CT as scheduled in the outpatient clinic. During the follow-up period, intermittent swelling of the neck lymph nodes was observed. Therefore, we performed ultrasonography guided fine needle aspiration cytology to exclude recurrence, and there was no evidence of malignant tumor cells. She has remained in a disease-free state for 4 years. At post-treatment 4 years 6 months, a markedly enlarged lymph node in the contralateral lower neck (left level V) was found. CT and ultrasonography guided fine needle aspiration cytology showed regional lymph node recurrence, which was suggestive of metastatic squamous cell carcinoma, based on the necrotic features of the enlarged lymph nodes on the CT and the keratin debris on cytology (Additional file 1: Figure S1).
Salvage neck dissection was performed for these enlarged lymph nodes; however, surgical pathology revealed total regression of metastatic squamous cell carcinoma cells in the left level V lymph node and there was no evidence of malignancy in the other dissected lymph nodes (Figure 1E-F). Interestingly, the left level V lymph nodes were not involved in the previous surgery or radiation fields. In addition, the patient denied any self-remedy or other treatments.
Immunohistochemistry findings
To further characterize this rare event, we performed immunohistochemistry on the various immune cells. The patient submitted written informed consent for each procedure and our Institutional Review Board approved the use of the archived tissues (Approval No. 2013-08-088-001). We compared the immune cell profiles among the metastatic node, non-tumor bearing node (from the first surgery) and the tumor-regressed nodes (from the second surgery). The antibodies for staining were cytokeratin, Ki-67, CD4, CD8, CD20, CD68, Mannose receptor, Foxp3, CD56, CD86, CD11c, and CD123 (Additional file 2: Table S1).
Complete tumor-regression in the lymph node was confirmed by cytokeratin and Ki-67 staining (Figure 2). High expression of cytokeratin and Ki-67 levels were observed in the tumor cells in metastatic nodes (Figure 2A, D), meanwhile cytokeratin expression was noted in the keratin debris and no Ki-67(+) tumor cells were found in the tumor-regressed lymph node (Figure 2C,F). In the tumor-free lymph node, the cytokeratin expression was not detected (Figure 2B). And most of the cells were CD86(+) cells with a clump of CD68(+) or CD11c(+) cells (Additional file 3: Figure S2), suggesting high infiltration of dendritic cells and macrophages in the tumor-regressed lymph nodes. However, the distribution of CD4(+), CD8(+) and CD20(+) cells were similar or relatively homogenous in the area around the metastatic and regressed nodes (data not shown).
Marked differences between the metastatic node and tumor-regressed node were found in the incidence of Foxp3(+) regulatory T (Treg) cells and CD56(+) natural killer (NK) cells (Figure 3). There was nearly a two-fold increase in the density of Foxp3(+) Treg cells in the metastatic node (Figure 3A), compared with the non-tumor bearing lymph node (Figure 3B). Meanwhile, very few Foxp3(+) cells were noted in the tumor-regressed lymph node (Figure 3C). These findings suggested that the immune-suppressive microenvironment of Treg cells could be reversed in the tumor-regressed lymph node. In addition, a noticeable infiltration of CD56(+) NK cells was found around the keratin debris in the tumor-regressed lymph nodes (Figure 3F); however, in the metastatic and control lymph nodes, the CD56(+) cell population was negligible (Figure 3D-E). Tumor-free lymph nodes were negative for cytokeratin, and had lower expression of Ki-67, Foxp3 and CD56 than other lymph nodes.
