Tryptophan metabolism enzymes are potential targets in ovarian clear cell carcinoma

Abstract Aim As the second most prevalent subtype of epithelial ovarian cancers, ovarian clear cell carcinoma (OCCC) is known for its chemoresistance to conventional platinum‐based therapy. In this work, we examined the tryptophan (Trp) metabolism enzymes' differential expression in patients with OCCC to assess the potential for personalised treatment. Methods A total of 127 OCCC tissues were used to construct tissue microarrays, and immunohistochemistry (IHC) staining of the Trp enzymes IDO1, IDO2, TDO2 and IL4I1 was performed. The correlations between Trp enzyme expression and clinical characteristics were analysed. Results Positive IDO1, IDO2, TDO2 and IL4I1 staining was identified in 26.8%, 94.5%, 75.6% and 82.7% of OCCC respectively. IDO1‐positive samples were more common in the chemoresistant group than in the platinum‐sensitive group (46.7% vs. 19.8%). Moreover, positive expression of IDO1, TDO2 and IL4I1 was related to advanced stage, metastasis, bilateral tumours, endometriosis and tumour rupture (p < 0.05) respectively. Univariate analysis revealed a significant association between bilateral tumours, lymph node metastasis, advanced stage, distant metastasis and aberrant cytology with a poor prognosis for OCCC, while the absence of residual tumour was correlated with a favourable outcome (p < 0.05). However, only bilateral tumours and lymph node metastases were related to a poor prognosis after multivariate analysis. Conclusion This is the first study to investigate the expression of the Trp enzymes IDO1, IDO2, TDO2 and IL4I1 in OCCC tissues. IDO2, TDO2 and IL4I1 were detected in the majority of OCCC. Clinical traits were correlated with IDO1, IDO2, TDO2 and IL4I1 expression. IDO1 may be used as a therapeutic target given the large percentage of chemoresistant cases with IDO1 expression. These results will aid the development of personalised therapies for OCCC.


| INTRODUCTION
Ovarian cancer is the most common gynaecological cancer, with a total of 198,412 deaths in 2019. 1 Epithelial ovarian cancer can be classified into high-grade serous ovarian carcinoma (HGSOC), low-grade serous ovarian carcinoma (LGSOC), mucinous carcinoma (MC), ovarian clear cell carcinoma (OCCC), endometrioid carcinoma (EC) and other types based on histopathology. 2Compared with HGSOC (70%), OCCC accounts for only 10% of epithelial ovary cancers 3 and has a poor prognosis. 4][7][8] From a molecular standpoint, the increased antioxidant capacity of the cancer cells and the surrounding microenvironment was the primary cause of chemoresistance in ovarian cancer cells. 9,10NQO1 is an antioxidant enzyme that takes part in ovarian cancer onset and progression. 10ts modulator, Nrf2 mediates antioxidant pathway and serves as a resistance mechanism in proteasome inhibitorresistant cells. 11Moreover, suppression of the NF-κB signalling pathway, a key factor involved in innate immune response and inflammation, led to apoptosis and reduced cell proliferation in drug-resistant ovarian cancer cells. 9For chemoresistant OCCCs, immunotherapy directed at the tumour microenvironment may therefore be the best option.
The enzyme IDO1 has been reported to be a therapeutic target in cancers, with evidence from pharmacological and genetic studies. 29Interestingly, the function of IDO1 in HGSOC is controversial.One study indicated that high IDO1 predicted longer survival and less sensitivity to carboplatin in HGSOC patients. 30Another study reported that HGSOC tumours with a high level of IDO1 might be resistant to therapy and were associated with significantly lower overall survival and progression-free survival. 31DO1 can catabolise Trp and suppress the proliferation of CD8+ effector T cells, natural killer cells and myeloidderived suppressor cells (MDSCs).In line with this, high IDO expression in ovarian cancer cells was found to be correlated with lower levels of tumour-infiltrating lymphocytes, advanced surgical stage and decreased survival. 13Similarly, TDO2 catabolises the degradation of Trp to Kyn and is upregulated in ovarian cancer tissues compared with normal ovarian tissues, promoting the proliferation, migration and invasion of ovarian cancer cells. 32ecently, overexpression of IL4I1 was shown to activate AHR and promote tumour progression. 22Furthermore, elevated metabolism of l-phenylalanine and l-tyrosine by IL4I1 in ascites was associated with advanced disease stage, suggesting a role of IL4I1 in HGSOC progression. 33o date, the function of Trp metabolism in OCCC remains unclear.Therefore, in the present study, we investigated the in situ expression of enzymes of Trp metabolism in OCCC, including IDO1, IDO2, TDO2 and IL4I1, using immunohistochemistry (IHC) analysis of tissue microarrays (TMAs).This study aimed to explore the roles of Trp metabolism in OCCC.
only bilateral tumours and lymph node metastases were related to a poor prognosis after multivariate analysis.

Conclusion:
This is the first study to investigate the expression of the Trp enzymes IDO1, IDO2, TDO2 and IL4I1 in OCCC tissues.IDO2, TDO2 and IL4I1 were detected in the majority of OCCC.Clinical traits were correlated with IDO1, IDO2, TDO2 and IL4I1 expression.IDO1 may be used as a therapeutic target given the large percentage of chemoresistant cases with IDO1 expression.These results will aid the development of personalised therapies for OCCC.

K E Y W O R D S
immunohistochemistry, ovarian clear cell carcinoma, tryptophan metabolism

| Patients and tumour specimens
This study recruited 127 patients diagnosed with OCCC.All patients received surgical treatment at our hospital between 2019 and 2022.The patients' diagnoses were confirmed by two experienced pathologists blinded to the first diagnosis based on the WHO guidelines introduced in 2020.Clinical and demographic data were collected and analysed.Tumour size was measured based on the longest axis of the primary tumour, and the larger tumour was considered if bilateral tumours existed.Abnormal cytology referred to malignant cells in ascites or peritoneal washings.Chemotherapy mainly included conventional platinum-based therapy after surgery.Chemoresistance or platinum resistance was identified as recurrence within 6 months after completion of platinum-based therapy.
Formalin-fixed, paraffin-embedded tissues were used to generate the TMA for IHC staining as previously described. 34The TMA was constructed by an expert pathologist from our hospital.Briefly, representative 1.8 mm tissue cores from the tumour area were selected for the purpose of constructing TMA blocks.Routine 4 μm sections were cut from the TMA blocks and stained with haematoxylin & eosin (H&E) to confirm the tumour purity and pathologic parameters.
This study was approved by the Institutional Review Board of Our Hospital (I-22PJ112).Informed consent was obtained from all patients.
Composite scores for IDO1, IDO2, TDO2 and IL4I1 were determined by two experienced pathologists based on both the intensity of staining and proportion of positive cells.The intensity score was determined on a scale of 0-3 (0-negative, 1-weak, 2-moderate, 3-intense staining), and the representative images for different intensity were shown in Figure S1.In addition, the percentage of positive cells was scored on a scale of 1-4 (1: 0%-24%, 2: 25%-49%, 3: 50%-74%, 4: 75%-100%).Sections with composite scores (the product of the intensity score and the percentage score) greater than or equal to 4 points were considered positive.

| Statistical analysis
Statistical analyses of IDO1, IDO2, TDO2 and IL4I1 expression determined by IHC and clinical characteristics were carried out using nonparametric statistics.Pearson's coefficient was calculated to evaluate the relations between the expression of IDO1, IDO2, TDO2 and IL4I1 and clinical characteristics.Univariate and multivariate statistical analyses were applied to identify factors related to patient prognosis.The data were analysed with SPSS version 24.0 for Windows (SPSS Inc., Chicago, IL, USA).A value of p < 0.05 was considered statistically significant.

| Demographic and clinical data of patients with OCCC
A total of 127 tissue samples from patients with OCCC were analysed in the present study.The clinicopathological characteristics of the OCCC patients are summarised in Table 1.The average age of the OCCC patients was 51 years, and 95 (74.8%) patients had early-stage disease (Stage I & II).Additionally, bilateral tumours were detected in 17 (13.4%)patients.The mean value of the longest axis of the primary tumour was 12.1 cm.Thirteen patients were diagnosed with lymph node metastasis, and 11 were diagnosed with distant metastasis.Although 46 (36.2%) tumours had ruptured, the majority of patients did not have residual tumours.A history of endometriosis was reported in 86 (67.7%) women, and 123 (96.9%) patients received chemotherapy after surgery.The total number of patients who experienced chemotherapy resistance and recurrence was 15 (15.6%) and 23 (18.1%) respectively.During the follow-up period, two patients died, and the progression-free survival and overall survival were 16 and 18 months respectively.According to the IDO1, IDO2, TDO2 and IL4I1 IHC results, 34 (26.8%), 120 (94.5%), 96 (75.6%) and 105 (82.7%) patients had positive IHC staining respectively.

| Representative images of morphology and IHC results
H&E sections showed typical morphology of OCCC with clear cells displaying tubulocystic or papillary architecture, as seen in Figure 1A,B.OCCC samples did not express ER (Figure 1C), PR (Figure 1D), WT-1 (Figure 1E) based on IHC staining.More importantly, the positive HNF1B (Figure 1G) and Napsin A (Figure 1H) staining distinguished OCCC from other types of ovarian cancers. 40Representative examples of the expression of four Trp metabolism enzymes (IDO1, IDO2, TDO2 and IL4I1) in OCCC were shown in Figure 2.

| Correlation between IDO1, IDO2, TDO2 and IL4I1 expression and clinical information
Interestingly, IDO1 expression was much lower than that of the other three genes in OCCC tissues (Table 1).We investigated the interrelationships of IDO1, IDO2, TDO2 and IL4I1 expression and the relationship of IDO1, IDO2, TDO2 and IL4I1 expression with clinical features; the results are shown in Table 2 and Table 3 respectively.As shown in Table 2, IDO1-positive cases were more likely to express IL4I1 (p = 0.039), while TDO2 was often co-expressed with F I G U R E 2 Positive or negative staining results of four tryptophan metabolic enzymes.The scale bar is 250 μm.IL4I1 (p = 0.011).Double-positive staining of IDO1 and IL4I1 was detected in 32 samples (94.1% of IDO1-positive samples).Among 96 TDO2-positive samples, 84 (87.5%) exhibited simultaneous IL4I1 expression, while 21 tissues with TDO2-negative staining were IL4I1 positive.Furthermore, we analysed the relationship between IDO1, IDO2, TDO2 and IL4I1 expression and some clinical features, such as advanced stage, bilateral ovarian carcinoma, lymph node metastasis, distant organ metastasis, abnormal cytology, tumour rupture, complications (endometriosis or thrombosis), cancer recurrence, chemotherapy drug resistance and death.As shown in Table 3, IDO1 expression was related to advanced stage (p = 0.041) as well as lymph node metastasis (p = 0.033).Negative TDO2 expression was significantly correlated with bilateral tumours (29% vs. 8.3%, p < 0.01) and endometriosis (51.6% vs. 72.9%,p < 0.05).Similarly, tumour rupture was observed to be related to negative IL4I1 expression (54.5% vs. 32.4%,p < 0.05).

| Factors related to OCCC prognosis
We analysed the influence of IDO1, IDO2, TDO2 and IL4I1 expression and clinical characteristics on OCCC prognosis, and the results are shown in Table 4.In the univariate statistical analysis, there was no significant correlation of progression-free survival with IDO1, IDO2, TDO2 or IL4I1 expression in OCCC patients, tumour rupture or chemoresistance.In contrast, bilateral tumours, lymph node metastasis, advanced stage, distant metastasis and abnormal cytology were significant risk factors negatively correlated with OCCC patient outcome (p < 0.05), with odds ratios (ORs) of 7.6, 5.5, 4.6, 4.3 and 2.7 respectively.Furthermore, lack of residual tumour after the first surgery was a protective factor for prognosis.However, only bilateral tumours and lymph node metastasis were shown to be significant risk factors related to OCCC prognosis in the multivariate statistical analysis (p < 0.01), with ORs of 4.4 and 3.7 respectively.
In addition, 96 OCCC patients were grouped into resistant and sensitive groups based on sensitivity to different platinum agents to assess the distribution of Trp catabolising enzyme expression.As shown in Table S2, we examined the relationship between chemosensitivity and the expression of the four proteins.We found that more than half of the OCCC samples in both the platinum-resistant and platinum-sensitive group expressed IDO2, TDO2 and IL4I1.Although the overall IDO1 positive expression rate was low in platinumresistant samples, they showed a trend for being more likely to be IDO1 positive (p = 0.056).Our present study was the first to evaluate the expression of the Trp enzymes IDO1, IDO2, TDO2 and IL4I1 in OCCC, and the positive expression rates were considerably high, indicating a possible benefit of Trp enzymetargeted therapy in OCCC.More than 75% of samples stained positively for IDO2, TDO2 and IL4I1 on IHC, while approximately 26.8% of OCCC samples were IDO1 positive.Chemoresistant cases were more likely to be IDO1-positive than platinum-sensitive cases (46.7% vs. 19.8%).Furthermore, higher IDO1 expression was significantly correlated with advanced tumour stage and metastasis (Table 3).Consistently, positive IDO1 expression was associated with advanced stage in both ovarian serous carcinoma 32,41 and OCCC.Considering that the failure of IDO1 inhibitors in clinical trials might be a result of compensation by IDO2 or TDO, 26 IDO1/IDO2 or IDO1/TDO dual-target inhibitors might be better choices for the treatment of OCCC.Correlation analysis showed that the OCCC cells that were IL4I1 positive were mainly IDO1 positive, while the majority of OCCC cells were positive for both TDO2 and IL4I1 (Table 2).To our knowledge, this is the first report of such relationships in OCCC tissues.Previously, it was shown that l-tryptophan is a major substrate of both IDO1 and IL4I1, and high levels of its metabolites were associated with IL4I1 activity in ascites from HGSOC patients. 33Compared with that in normal tissue, TDO2 expression was upregulated in ovarian cancer tissues. 32The same was found to be true for IL4I1, a high level of which promoted ovarian progression. 42n a study of Merkel cell carcinoma (MCC), MCC cells with lower IDO1 expression but a tumour microenvironment with lower TDO2 and AhR expression had a longer overall survival rate. 43To target the IDO1/TDO2-AhR pathway, IDO1, TDO2 and AhR expression should be investigated in cancer patients who plan to receive immunotherapy.

T A B L E 3 Relations between expression of Trp catabolising enzymes and clinical information.
We also analysed independent risk factors related to OCCC outcomes, including IDO1, IDO2, TDO2 and IL4I1 expression and clinical features.5][46][47] In conclusion, positive IDO1, TDO2 and IL4I1 expression was considered as unfavourable factor of OCCC.Unfortunately, in our prognostic analysis, IDO1, IDO2, TDO2 and IL4I1 expression was not significantly associated with the OCCC survival rate, as shown in Table 4.Among the four clinical characteristics related to OCCC outcome, only bilateral tumours and lymph node metastasis of OCCC remained independent risk factors related to prognosis (Table 4).Previous studies showed that higher IDO1 expression in high-grade ovarian adenocarcinoma is associated with better prognosis, including better overall and progression-free survival rates. 48,49In contrast, one study of HGSOC patients found that IDO1 expression was higher in platinum-resistant patients than in platinum-sensitive patients, and higher IDO1 expression was correlated with unfavourable prognosis. 41According to an analysis of gene expression data from The Cancer Genome Atlas and Genotype-Tissue Expression project, the mRNA level of TDO2 is upregulated in ovarian serous cystadenocarcinoma in comparison to normal tissue, and higher TDO2 expression resulted in cancer progression. 32In a single-cell study of IL4I1 in serous ovarian cancer, Kaplan-Meier curve analysis showed that higher IL4I1 expression was associated with poor OS. 42 Certainly, our observational study has some limitations.For instance, our work lacks data from functional experiments or clinical trials to verify Trp metabolism enzyme expression in OCCC.The IDO1 expression pattern in our OCCC cohort was different from that in ovarian serous carcinoma, previously reported to range from 59% to 71%. 32,41Efforts should be made to validate our findings.

| CONCLUSION
In conclusion, our study might be the first to demonstrate the expression of four Trp metabolism enzymes (i.e.IDO1, IDO2, TDO2 and IL4I1) in OCCC tissues and their relationships with each other and clinical features.Most OCCC samples expressed IDO2, TDO2 and IL4I1.The expression of IDO1, IDO2, TDO2 and IL4I1 was related to different clinical characteristics.In terms of clinical characteristics, bilateral tumours were a significant risk factor predicting poor prognosis.These findings have the potential to optimise personalised therapies for OCCC patients.

F I G U R E 1
Typical histopathological and immunohistochemical images of OCCC.(A) Haematoxylin and eosin (H&E) figure of OCCC at low power (100×).(B) H&E figure of OCCC at high power (400×).(C-H).Immunohistochemical results of OCCC (400×).OCCC does not express ER (C), PR (D) or WT-1 (E).It shows a wild-type immunohistochemical staining pattern of P53 (F).Staining results of HNF1B (G) and Napsin A (H) are positive in OCCC.The scale bar is 200 μm.
stage (III/IV) Clinicopathological data of patients with ovarian clear cell carcinoma (n = 127).
T A B L E 1a Tumour size was the measurement of the longest axis of the primary tumour.b Abnormal cytology meant malignant cells in ascites or peritoneal washings.c Data deficiency due to lack of medical records.

IDO1 p value Pos IDO2 p value Pos TDO2
Prognostic value of clinicopathological parameters in OCCC (PFS, n = 96).a survival (PFS) was used in univariate and multivariate prognostic analysis.Some cases were not involved in prognostic analysis because of incomplete medical records.
T A B L E 4 *p < 0.05 as significance.a Progression-free