Increased lymphocyte infiltration in patients with head and neck cancer treated with the IRX-2 immunotherapy regimen

Abstract

Twenty-seven subjects with squamous cell cancer of the head and neck received the neoadjuvant IRX-2 immunotherapy regimen prior to surgery in a Phase 2 trial. Pretreatment tumor biopsies were compared with the primary tumor surgical specimens for lymphocyte infiltration, necrosis and fibrosis, using hematoxylin and eosin stain and immunohistochemistry in 25 subjects. Sections were examined by three pathologists. Relative to pretreatment biopsies, increases in lymphocyte infiltration (LI) were seen using H and E or immunohistochemistry. CD3+ CD4+ T cells and CD20+ B cells were primarily found in the peritumoral stroma and CD3+ CD8+ T cells and CD68+ macrophages were mainly intratumoral. LI in the surgical specimens were associated with reductions in the primary tumor size. Improved survival at 5 years was correlated with high overall LI in the tumor specimens. Neoadjuvant IRX-2 immunotherapy regimen may restore immune responsiveness presumably by mobilizing tumor infiltrating effector lymphocytes and macrophages into the tumor.

Figures

Fig. 1

Relationship of VAS scoring for H&E lymphocyte infiltration and CD3± cells by immunohistochemistry. Photographs of three different surgical specimens stained with H&E or with IHC for CD3+ cells are shown with their corresponding VAS scores. The H&E sections (a, b, c) have their VAS scores listed below the picture and placed on the position of the VAS line that the pathologist would have made on the VAS scoring line. The VAS scores presented are the average of either three pathologists (H&E) or two pathologists (IHC). The VAS scores for the IHC staining of CD3+ lymphocytes are presented above the three matched sections (d, e, f). In the H&E sections, the small dark blue nuclei of the lymphocytes should be distinguished from the small dark nuclei with surrounding pink cytoplasm of the tumor cells. The intense brown/orange staining for CD3+ cells in e and f is readily contrasted to the few such positive cells in d. Enhanced fibrosis and necrosis can also be seen in sample c as compared to sample a. Sample b is intermediate in scoring

Fig. 2

Relationship of VAS scores and CD3± IHC in comparing initial pretreatment biopsies with surgical specimens. Biopsy and surgical specimens from subjects treated with IRX-2 immunotherapy regimen were stained for CD3+ positive cells. The photographs illustrate a patient that has minimal increase in CD3+ cells in the surgical specimen compared to the biopsy and a subject with a more significant increase in CD3+ cells. Note that although tumor histology cannot be readily assessed in IHC, both subjects had CD3+ cells located predominantly on “margins” in the biopsy sample while the subject with the increase in CD3+ cells in the surgical specimen had an enhanced intratumoral infiltration

Fig. 3

Assessment of histological features in biopsy and surgical tumor specimens. Specimens were stained by H&E or IHC. The VAS scores presented are the average of either three pathologists (H&E) or two pathologists (IHC). All subjects were treated with the IRX-2 immunotherapy regimen prior to surgical removal of the tumor. Lymphocyte Infiltration was assessed using a VAS of 0–100 mm where 100 represented the maximum infiltration seen in the specimens. The changes in each assessment were analyzed using one-sample t-tests. The graphs represent the change (Surgery [S] — Biopsy [B]) for the LIavg for individual subjects, sorted based on the magnitude of the change, a Lymphocyte infiltration for H&E-stained specimens by VAS scores are shown by box-and-whisker plots for each of the three pathologists’ reads as well as the average calculated using the value of the three pathologists for each subject, b Lymphocyte infiltration, fibrosis and necrosis as read by three pathologists on H&E specimens, c Lymphocyte infiltration assessed by two pathologists using both H&E stained and IHC stained specimens. LI for the IHC specimens was calculated by adding the VAS score for CD3+ and CD20+ cell infiltration. Also shown on the graph is the Lymphocyte Infiltration Average (LIavg) that was defined as the average VAS Score for all 5 reads (3 H&E and 2 IHC). d Change in LIavg for individual patients

Fig. 4

Lymphocyte infiltration subsets defined as the average VAS score by two pathologists. a The graph displays the VAS scores for biopsy versus surgery for subjects with IHC stained slides (n = 21). a The extent of leukocyte infiltration in the surgical samples. Biopsy samples were not stained for CD4+ and CD8+. The change from biopsy to surgery was analyzed using a one-sample t-test. b Location of infiltrating lymphocytes in primary tumor (surgical sample). Location in the tumor of the lymphocyte infiltration was defined using a 0–100 VAS scale that was from peripheral (peritumoral (0) or far left) or intratumoral (100 for far right). The graph displays the average VAS scores for biopsy versus surgery in subjects with IHC stained slides. (n = 21)

Fig. 5

Centrally reviewed radiologic change in primary tumor following the TRX-2 immunotherapy regimen versus lymphocyte infiltration, a The change in tumor size following treatment with IRX-2 immunotherapy regimen was defined by radiologic assessment and is plotted against the lymphocyte infiltration in the surgical specimen defined by the LIavg. The LIavg in the tumor specimen correlated with percent change in tumor size (P < 0.05, Pearson correlation). Also plotted b, c, d are the results for LI by IHC (CD3+ and CD20+) average and for CD3+ average and CD20+ average. The x-axis displays percent tumor change and the y-axis displays the average value for tumor specimen of subjects with IHC-stained slides. All 3 IHC assessments were correlated with change in tumor size (P < 0.05, Pearson correlation, see Table 2)

Fig. 6

Kaplan-Meier estimates of overall survival by intensity of lymphocyte infiltration assessed using H&E or IHC of the resected primary tumor after IRX-2 immunotherapy regimen. Kaplan-Meier estimates of overall survival for subjects in the Phase 2 trial are presented on the graph as a function of high- and low lymphocyte infiltration in the surgical specimens after the IRX-2 immunotherapy regimen. The upper graph is for LIavg and the lower graph is for CD3+ T-cell average. The cut-offs selected for identifying the 2 subsets of subjects are indicated in the legends for the graphs. The number of subjects in each are as follows: LIavg n = 18 best survival versus n = 7 worst survival; CD3+ n = 18 best survival and n = 6 worst survival, a LIavg versus Survival (P < 0.05). b LI CD3 versus Survival (P < 0.01)