BHLHE40 is a basic helix-loop-helix transcription factor that is involved in multiple cell activities including differentiation, cell cycle, and epithelial-to-mesenchymal transition. While there is growing evidence to support the functions of BHLHE40 in energy metabolism, little is known about the mechanism. In this study, we found that BHLHE40 expression was downregulated in cases of endometrial cancer of higher grade and advanced disease. Knockdown of BHLHE40 in endometrial cancer cells resulted in suppressed oxygen consumption and enhanced extracellular acidification. Suppressed pyruvate dehydrogenase (PDH) activity and enhanced lactated dehydrogenase (LDH) activity were observed in the knockdown cells. Knockdown of BHLHE40 also led to dephosphorylation of AMPKα Thr172 and enhanced phosphorylation of pyruvate dehydrogenase E1 subunit alpha 1 (PDHA1) Ser293 and lactate dehydrogenase A (LDHA) Tyr10. These results suggested that BHLHE40 modulates PDH and LDH activity by regulating the phosphorylation status of PDHA1 and LDHA. We found that BHLHE40 enhanced AMPKα phosphorylation by directly suppressing the transcription of an AMPKα-specific phosphatase, PPM1F. Our immunohistochemical study showed that the expression of BHLHE40, PPM1F, and phosphorylated AMPKα correlated with the prognosis of endometrial cancer patients. Because AMPK is a central regulator of energy metabolism in cancer cells, targeting the BHLHE40‒PPM1F‒AMPK axis may represent a strategy to control cancer development.

Immune checkpoint inhibitors (ICIs) enhance antitumoral immune mechanisms and are used to treat various types of solid tumors including those that are microsatellite instability (MSI)-high. Uterine endometrial cancer is one of the most frequent tumor types that shows MSI-high, and, consequently, opportunities to use ICIs for endometrial cancer treatment are increasing. While using ICIs, it is important to monitor and manage various immune-related adverse events (irAEs). Here, we report two cases of secondary adrenal insufficiency during treatment of endometrial cancer with pembrolizumab. Both cases showed appetite loss and general fatigue after the 6th or 12th cycle of pembrolizumab. They were admitted to our hospital because of remarkable hyponatremia. Both cases showed no adrenocorticotropic hormone (ACTH) or cortisol response to CRH loading tests. Other pituitary hormone levels were normal, and MRI revealed no sign of hypophysitis in either patient. They were diagnosed with secondary adrenal insufficiency due to isolated ACTH deficiency and recovered soon after the administration of hydrocortisone and hydration. Thus, we should be aware of irAEs with the use of ICIs. In particular, adrenocortical insufficiency is sometimes lethal without appropriate treatment. Because the clinical symptoms are fatigue, appetite loss, and nausea, patients might be misjudged to have symptoms related to cancer. Checking serum morning cortisol before ICIs use and monitoring serum sodium levels could provide clues to diagnose secondary adrenal insufficiency.

Clear cell carcinoma of the uterine cervix is a rare subtype. We report the case of a 14-year-old girl who presented with abnormal genital bleeding, a 7 cm mass arising from the uterine cervix, and swollen right inguinal lymph nodes. Histological and immunohistochemical examinations showed clear cell carcinoma. We performed a modified-radical hysterectomy, bilateral salpingo-oophorectomy, right inguinal lymph node biopsy, bilateral pelvic lymph node biopsy, and para-aortic lymph node biopsy, and diagnosed uterine cervical cancer stage IVB (clear cell carcinoma, pT2bN2M1). We administered paclitaxel-carboplatin+bevacizumab+pembrolizumab as adjuvant chemotherapy. Pembrolizumab is an immune checkpoint inhibitor associated with characteristic adverse events called immune-related adverse events (irAEs). In this case, arthritis and autoimmune pancreatitis were observed as irAEs during the treatment, but the patient was able to continue treatment. There is limited clinical experience with immune checkpoint inhibitors for pediatric patients. When remission is achieved, a better long-term prognosis is expected than in adults, so it is necessary to follow up with attention to late-onset irAEs.

Background: A molecular classification of endometrial cancer was developed based on an analysis of The Cancer Genome Atlas. In this classification, the group characterized by abnormal p53 immunohistochemical expression showed the poorest prognosis. However, there may be no need to apply a molecular classification in low-grade endometrial cancer. In this study, we investigated the clinical significance of abnormal p53 immunohistochemical expression in low-grade endometrial cancer. Methods: We obtained nine frozen samples of endometrial cancer [low-grade endometrial cancer with wild-type p53 expression (EC^lop53^wt group): n = 3, low-grade endometrial cancer with abnormal p53 expression (EC^lop53^ab group): n = 3, and high-grade endometrial cancer (EC^hi group): n = 3]. RNA sequencing was performed for each sample. All the samples passed RNA quality control. In addition, an immunohistochemical analysis was performed for 44 formalin-fixed paraffin-embedded samples. Results: Differentially expressed genes were identified in the RNA sequencing results (1811 genes between the EC^lop53^ab group and the EC^hi group, and 1088 genes between the EC^lop53^ab group and the EC^lop53^wt group). In a principal component analysis, the EC^lop53^ab group was more similar to the EC^lop53^wt group than to the EC^hi group. In the immunohistochemical analysis, L1CAM expression was significantly less frequently observed in the EC^lop53^ab group than in the EC^hi group. Moreover, p21 expression tended to be more frequently observed in the EC^lop53^ab group than in the EC^hi group. Conclusions: In this study, the RNA sequencing and immunohistochemical results revealed that the EC^lop53^ab group is a separate entity from the EC^hi group. While the abnormal p53 group is considered the most prognostically unfavorable in molecular classification, these findings suggest that routine molecular profiling is not necessary for patients with low-grade endometrial cancer. However, there is insufficient evidence to modify adjuvant treatment in low-grade endometrial cancer patients. Further investigation is needed on the clinical application of molecular classification to low-grade endometrial cancer.

Low-grade, low-stage endometrial carcinomas are generally associated with an excellent prognosis. However, some of these tumors recur, although the prognostic factors are largely unknown. Recently, reports have described associations among tumor immune microenvironment (TIME), epithelial-mesenchymal transition (EMT), and circulating tumor cells (CTCs), factors that have also been reported to be associated with recurrence in endometrial carcinoma. Therefore, we conducted the present study to clarify the association between recurrence and TIME- and EMT-related factors in low-grade, low-stage endometrial carcinomas. This study included 80 Japanese patients with low-grade, stage IA endometrial endometrioid carcinoma (20 in the recurrence group and 60 in the no-recurrence group). The institutional review board of Kyushu University approved this study (project number: 24101-00). The median follow-up was 94.5 months (range, 10-298). Immunohistochemistry was performed to evaluate regulatory T cells, marginal and intratumoral tumor-associated macrophages, and tumor cell expression of C-X-C motif chemokine ligand 12 (CXCL12) and Secreted Protein Acidic and Rich in Cysteine (SPARC). In 13 of the 20 recurrent cases, recurrence occurred distant from the primary site, at lung or lymph nodes. Immunohistochemistry revealed that the numbers of FoxP3-positive cells, and marginal and intratumoral CD68-positive cells in the recurrence group were significantly higher than in the no-recurrence group. In addition, tumor cell expression of CXCL12, a chemokine involved in macrophage recruitment, was significantly higher in the recurrence group. Similarly, tumor cell expression of SPARC, an EMT-associated protein, was also significantly higher in the recurrence group. These findings suggest that a tumor microenvironment permissive of tumor cell dissemination may be associated with recurrence. Recurrence in low-grade, stage IA endometrial carcinoma was associated with increased FoxP3-positive cells, increased marginal and intratumoral CD68-positive cells, and higher tumor cell expression of CXCL12 and SPARC. However, these findings suggesting a tumor microenvironment permissive of tumor cell dissemination should be interpreted as exploratory and hypothesis-generating, given that the involvement of CTCs was not directly evaluated in this study.

The administration of immune checkpoint inhibitors (ICIs) is increasing in endometrial cancer, especially in the mismatch repair (MMR)-deficient group. To prevent unnecessary immune-related adverse events, ICIs need to be administered to more appropriate patients. The tumor immune microenvironment has been reported to be a predictive marker of the efficacy of ICI therapies. This study evaluated CD8, FoxP3, CD68, PD-L1, and β-catenin expression in endometrial endometrioid carcinoma, grade 1 (G1) with DNA mismatch repair protein loss (MMR loss), and their association with clinicopathological features. We retrospectively analyzed tumor samples from 107 patients with endometrial endometrioid carcinoma, G1 (MMR-deficient group: n=67; MMR-proficient group: n=40). Overall, 47 cases of MLH1/PMS2 loss and 20 cases of MSH2/MSH6 loss were observed. The patients with low intraepithelial CD8 expression significantly more frequently exhibited deep myometrial invasion, and the elderly group (≥60 y) significantly more frequently showed low stromal CD8 expression. In addition, FoxP3-positive cell count and FoxP3/CD8+ ratio were significantly correlated with the International Federation of Obstetrics and Gynecology 2023 stage and lymph node metastasis. In the Kaplan-Meier analysis, the patients with low intraepithelial or stromal CD8 expression had shorter progression-free survival (PFS) than those with high intraepithelial or stromal CD8 expression, albeit not significantly. We clarified that the tumor immune microenvironment had an impact on clinicopathological features within the group with MMR loss, which is the main target for ICIs, limited to endometrioid carcinoma, G1. Further studies are needed, including on patients administered ICIs.