CD4/CD8 ratio is a prognostic factor in IgG nonresponders among peptide vaccine‐treated ovarian cancer patients

Abstract The identification of useful biomarkers is an urgent issue in cancer treatment, particularly for immunotherapy, as only some patients experience benefits from this treatment. The early induction of the IgG response has been reported as a useful biomarker of favorable prognosis for cancer patients treated with a personalized peptide vaccination, but a portion of these patients (IgG nonresponders) fail to achieve an early induction of IgG response yet experience long‐term survival. It is thus necessary to identify other biomarkers of favorable prognosis among these patients. Here we report the usefulness of classical T‐cell markers (ie, the CD8 content and the CD4/CD8 ratio in peripheral blood) in IgG nonresponders among advanced or recurrent ovarian cancer patients treated with a personalized peptide vaccination. Among IgG nonresponders (n = 25), the overall survival (OS) of the increased‐CD8 group (n = 7) was significantly longer than that of the decreased‐CD8 group (n = 18; P = .018), and the OS of the patients with a decreased CD4/CD8 ratio (n = 10) was significantly longer than that of the patients with an increased ratio (n = 15; P = .0055). Thus, an increased content of CD8 and a decreased CD4/CD8 ratio are each favorable prognosis markers in IgG nonresponders treated with a personalized peptide vaccination.

The early induction of IgG response has been described as one of the useful biomarkers of favorable prognosis for cancer patients treated with a personalized peptide vaccination. [10][11][12][13][14][15][16] The portion of these patients who did not show an early induction of IgG response, ie, the IgG nonresponders, has also included patients who nevertheless achieved long-term survival. It is thus necessary to identify other biomarkers of favorable prognosis that will contribute to further treatment planning for IgG nonresponders treated with a personalized peptide vaccination, eg, whether to continue the vaccination or switch to other methods.
Here we report the usefulness of 2 classical T-cell markers, the CD8 content and the CD4/CD8 ratio in the peripheral blood of IgG nonresponders among advanced or recurrent ovarian cancer patients treated with a personalized peptide vaccination.

| Clinical samples
Peripheral blood samples were obtained from patients with a histological diagnosis of ovarian cancer who were enrolled in a personalized peptide vaccination during the period from January 2009 to December 2015. The study protocols were approved by the Kurume University Ethics Committee and were registered with the UMIN Clinical Trial Registry (UMIN3083 and UMIN1482). All of the patients were enrolled in these studies after providing informed consent. The entry criteria and precise vaccination protocols have been reported. 10 Briefly, a maximum of 4 peptides selected (based on the individual patient's HLA type and preexisting immunity) from a vaccine peptide panel consisting of 31 CTL-epitope peptides derived from a variety of tumorassociated antigens were s.c. injected together with the oil adjuvant Montanide ISA 51 VG (Seppic). One vaccination cycle consisted of a 1 vaccination per week for 6 or 8 weeks. The patients' characteristics are summarized in Table 1. The characteristics of the 26 patients enrolled in the study during the period from January 2009 to July 2012 who were subjected to the initial analysis (named the "discovery cohort") were reported by Kawano et al. 10 The patients of the "validation cohort" were enrolled in the more recent study conducted during the period from August 2012 to December 2015. All the patients described in the present study completed at least 1 cycle of vaccine treatment.

| Flow cytometry
Peripheral blood mononuclear cells (1 × 10 5 ) were suspended in PBS supplemented with 20% human AB serum and then incubated for 30 minutes on ice with the appropriate dilution of the Ab anti-CD4-FITC (clone RPA-T4), anti-CD8-PerCP-Cy5.5 (clone RPA-T8), or anti-CD279 (PD-1)-APC (clone MIH4); all of these Abs including isotype-matched negative controls were purchased from BD Biosciences. The stained cells were analyzed on a BD FACSCanto II flow cytometry system with FACS Diva software (BD Biosciences).

| Measurement of peptide-reactive IgG and CTLs
The IgG levels of the serum samples subjected to the vaccine candidate panel consisting of 31 CTL-epitope peptides were measured using the Luminex analyzer (Luminex) as described. 10 The patients' IgG levels are expressed as fluorescence intensity units, and if the values were increased to 2-fold or more of the patient's prevaccination level, the response was considered augmented.
The CTL responses were measured by an interferon-gamma ELISPOT assay as described. 10 The vaccination-induced CTL responses were evaluated by the difference between the number of spots produced in response to each corresponding peptide and that produced in response to the control HIV peptide, and if the value was increased to 2-fold or more of the prevaccination number, the response was considered augmented regardless of whether or not the peptides were used for the vaccination.

| Statistical analyses
The survival curves were plotted by the Kaplan-Meier method. We compared the increased and not increased (or decreased) groups for each variable by using a Cox hazard model. We used Pearson's χ 2 test to analyze the T cell responses specific to the vaccinated peptides, Fisher's exact test to analyze the IgG responses to the vaccine TA B L E 1 Characteristics of ovarian cancer patients treated with peptide vaccine who were analyzed in this study 2 or more 0 0 a Patients' data of the discovery cohort have been reported previously. 10 peptides, the pairwise Student's t test to analyze the T cell phenotypes, and Fisher's exact test to analyze the relationships among the CD4/CD8 ratio, the CTL response, and PD-1 expression. JMP version 13 software (SAS) was used for the analyses, and P values less than .05 were considered significant.  Table 2).

| Relationship between vaccination-induced alterations of T-cell subsets and prognosis in discovery cohort
We divided the 26 patients of the discovery cohort based on high and low CD4 or CD8 contents and based on the pre-and postvaccination CD4/CD8 ratios. We analyzed the relationships of each of these 3 parameters to the patients' OS by determining the Kaplan-Meier plot. The median of each T-cell subset percentage or ratio was used for the definition of "high" and "low" contents or rates. The results showed that the CD4 T cell content and the CD8 T cell content at both pre-and postvaccination were not correlated with the OS (data not shown).
We next divided the patients into groups based on decreased vs increased (ie, altered) contents of CD4 and CD8 and the CD4/ CD8 ratio at pre-and postvaccination, and the relationship between these parameters with the patients' OS was analyzed by Kaplan-Meier plots ( Figure 1A). The OS of the increased-CD8 group of patients (n = 9) was significantly longer than that of the de- T-cell groups in terms of OS.

| Relationship between vaccination-induced alterations of T-cell subsets and prognosis in validation cohort
To further test our results obtained from the discovery cohort, we evaluated an additional 26 patients enrolled more recently as the validation cohort. Surprisingly, opposite tendencies were observed in the validation cohort: the OS of the decreased-CD8 group (n = 20) was significantly longer than that of the increased-CD8 group (n = 6) (HR 0.34; 95% CI, 0.13-0.97; P = .044), and the OS of the increased-CD4/CD8 ratio group (n = 13) tended to be longer than that of the   Table 1, the characteristics of the patients in the validation cohort were not significantly different from those of the patients in the discovery cohort.
We further analyzed the IgG and CTL responses of the discovery and validation cohorts, and we observed a significant difference in the IgG response between the 2 groups. The IgG response rate after the first vaccination cycle in the discovery cohort was 34.6%, which is significantly lower than that of the validation cohort (69.2%; P = .0254). These results indicated that the majority of patients in the discovery cohort were IgG nonresponders, whereas the majority of the validation cohort patients were IgG responders.
To test these results, we divided both the validation and discovery cohorts into IgG responder and nonresponder groups and undertook a survival analysis; the results are illustrated in Figure 2.
In the IgG nonresponder group (the major group) of the discovery cohort (n = 17), our initial findings were confirmed: the OS of the increased-CD8 group (n = 5) was significantly longer than that of the decreased-CD8 group (HR 0.14; 95% CI, 0.02-0.54; P = .0029), and the OS of the decreased-CD4/CD8 ratio group (n = 6) was significantly longer than that of the increased-CD4/CD8 ratio group (HR 0.23; 95% CI, 0.05-0.77; P = .016) (Figure 2A). Similar tendencies were also observed in the IgG nonresponders of validation cohort (n = 8), but without significance as this number of nonresponder patients was too small ( Figure 2B).
We next merged the IgG subgroups of the discovery and validation cohorts and undertook a survival analysis (Figure 3). In the IgG nonresponder group (n = 25) of the merged cohort, our initial findings were confirmed, ie, the OS of the increased-CD8 group (n = 7) was significantly longer than that of the decreased-CD8 group (P = .018), and the OS of the decreased-CD4/CD8 ratio group (n = 10) was significantly longer than that of the increased-CD4/CD8 ratio group (HR 0.26; 95% CI, 0.09-0.68; P = .0055; Figure 3A). These results suggest that an increase in the CD8 content and a decrease in the CD4/CD8 ratio are both favorable prognosis markers following treatment of IgG nonresponders with personalized peptide vaccinations. However, no drastic increase in the percentage of CD8 T cells or decrease in the CD4/CD8 ratio was observed in the patients with good prognoses. It is also notable that in the IgG responder group of our present merged cohort, the OS of the patients with an increased CD4/CD8 ratio was also significantly longer than that of the patients whose CD4/CD8 ratio was not increased (HR 0.40; 95% CI, 0.17-0.95; P = .037; Figure 3B).

| Relationships among CD4/CD8 ratio, the CTL response, and PD-1 expression
We reported that induction of CTL responses and the increase of circulating PD-1 + CD8 T cells were favorable prognosis markers Features of delayed hypersensitivity (such as a skin reaction) have also been reported as biomarkers. [20][21][22] In addition to immune response-related biomarkers, we have reported inflammation-related markers such as C-reactive protein, interleukin-6, serum amyloid A, and high-mobility group box 1. [23][24][25][26][27][28] More recently, we reported that the level of circulating PD-1 + CD8 T cells was a biomarker for vaccine therapy in patients with non-small cell lung cancer or cervical cancer. 11,17 Our present findings indicated that the circulating CD8 T cell content and the CD4/CD8 ratio were each a possible vaccine therapy biomarker in patients with ovarian cancer. We observed that in the IgG nonresponders, an increase in the CD8 content and a decrease in the CD4/CD8 ratio were both favorable prognosis markers following peptide vaccination. The decrease in the CD4/CD8 ratio was reflected mainly by the increase in the CD8 T cell content. We also analyzed circulating PD-1 + CD8 T cells in ovarian cancer patients, and no correlation was observed between the PD-1 + CD8 T cell content and prognosis. It is unclear why a decreased CD4/CD8 ratio or an increased CD8 content was a favorable prognostic marker in the IgG nonresponders but not in the IgG responders. One of the possible mechanisms is as follows. All of the patients enrolled in this study had a history of chemotherapy, and the effects of the chemotherapy on the patients' lymphoid cells might be carried over during the vaccination period. In the IgG nonresponders, the reactivity of B cells and CD4 T cells against vaccine peptides as well as tumor antigens might be suppressed. If the antigen reactivity of CD8 T cells (ie, CTLs) has remained in the IgG nonresponders, the numbers of CD8 T cells against the vaccinated peptides as well as tumor antigens are expanded and contribute to a favorable prognosis. In this case, the CD8 contents are increased and the CD4/CD8 ratio is decreased.
In the IgG responders, the reactivity of B cells and CD4 T cells has remained and expanded in response to the vaccine peptides as well as tumor antigens. In this case, the expansion of CD8 cells has less effect on the CD4/CD8 ratio. In conclusion, the usefulness of the 2 classical T cell markers (the CD8 content and the CD4/CD8 ratio in peripheral blood) as biomarkers in IgG nonresponders among advanced or recurrent ovarian cancer patients treated with personalized peptide vaccinations was revealed by the results of our present analyses.