EP920 - A Progressive Anal Squamous Cell Carcinoma: Case of Clinical and Immunologic Failure of Treatment Response

Purpose/Background: The treatment of metastatic anal squamous cell cancer relies on systemic chemotherapy and immunotherapy as a second line option. This is a case of a 61-year-old female who was diagnosed with squamous cell carcinoma of the anus and was treated with the modified Nigro protocol. She subsequently had progression of her locally advanced tumor requiring systemic chemotherapy (Carboplatin and Paclitaxel) and immunotherapy (Nivolumab, an anti-PD1 therapy). The question arises as to why some cancers are non-responders to different cancer therapies. During her treatment she was enrolled in a translational research study evaluating the tumor microenvironment of her anal cancer.

Methods/Interventions: Under IRB approval (GICOS IRB #22198), peripheral blood and tissue specimens of her anal cancer were obtained from an exam under anesthesia at two time points: 1. 8 months after completion of the Nigro protocol, 2. 7 months after initiation of systemic chemotherapy and immunotherapy. The blood and tissue samples were processed into single cell suspensions and analyzed via spectral flow cytometry. Immune markers of activation, proliferation, exhaustion, integrins and gut homing receptors were assessed by flow cytometry.

Results/Outcomes: The immune composition of the patient's blood and anal cancer specimen were compared prior to initiation of systemic therapy and after. Her disease became more advanced despite initiation of chemotherapy and immunotherapy with progression of a large mass with an expanding anovaginal fistula with extension into the urethra (Figure 1).

The anal tumor characteristics prior to systemic therapy included a high percentage (50%) of regulatory T cells (Tregs) and expression of ICOS and PD-1 within CD4 conventional T cells (Figure 2A). After initiation of systemic therapy, her tumor immune microenvironment displayed similarly elevated Treg levels (43%), but 50 days after her last nivolumab dose no PD-1 expression was identified within the tumor and blood sample (Figure 2B). To understand whether PD-1 was still masked by nivolumab (an IgG4 isotype antibody) on the cell surface, we utilized an anti-IgG4 antibody to indirectly identify PD-1 expression. We found that all PD-1 remained completely bound by nivolumab despite the typical half-life of 25 days (Figure 2B).

Data from a prior study demonstrated that intracellular expression of Ki-67, a cell-cycle marker of activation, increases in patients with a positive response to Nivolumab treatment. This patient's tumor sample showed a decrease in Ki-67 expression, which points to her lack of response (Figure 2C).

Conclusion/Discussion: Despite nivolumab binding to create an inhibitory effect on PD-1 within the tumor and peripheral blood, the patient did not respond to treatment. The best monitoring technique to assess response to nivolumab is still under investigation, but could potentially include monitoring of activation markers, such as Ki-67, changes in Tregs and CD8 cytotoxic cells, which may tailor further therapy. Deeper investigation into the appropriate monitoring of both patients' clinical and immunologic response can be a critical component of anal cancer care given the increased application of immunotherapy and personalized treatment plans.

T2 MRI images demonstrating the patient's anal mass
A. Sagittal image demonstrating the mass extending into the vaginal wall and levator ani muscles prior to the initiation of systemic therapy. B. Sagittal image obtained after 6 months of systemic therapy demonstrating enlargement of the mass with an anovaginal fistula and increased involvement of the pelvic floor muscles.
Flow cytometry data for pre- and post-systemic chemotherapy samples
A. Flow cytomtery data of CD4+ T cells. Foxp3 and low IL-7R expression delineate Treg cells (left). PD-1 and ICOS expression is shown for CD4+ (Foxp3neg) T conventional cells (right). B. Tissue and blood samples obtained post systemic therapy demonstrate lack of PD-1 expression in the CD4 T conventional cells (highlighted in red). Addition of IgG4 antibody reveals a significant amount of nivolumab bound to PD-1 on the cell surface in both the tissue and blood sample (highlighted in green). C. Ki67 expression in CD4+ T cells prior to systemic therapy (left) and Ki-67 expression after initiation of nivolumab treatment (right). Levels demonstrate a decrease in cell activation.