數位空間分析研究原發性子宮癌肉瘤腫瘤間及腫瘤內免疫表現的異質性

Abstract

論述重點 子宮癌肉瘤是比較少見的婦科癌症，和一般子宮內膜癌相比，他有比較特殊的組織型態，一般子宮內膜癌為單一上皮癌類型的組織，然而子宮癌肉瘤是同時表現有上皮癌及肉癌兩種類型的組織型態。它的治療方式為手術並術後輔助化學治療 +/-放射線治療，然而其預後相比常見型態的子宮內膜癌差。 近幾年，對免疫及分子醫學的研究逐漸進步，免疫治療也隨之蓬勃發展。以前研究表明子宮體癌腫瘤內浸潤的淋巴細胞在疾病的進展扮演重要角色，而腫瘤細胞也會藉由免疫調節能力去改變那些淋巴細胞的表現，進而影響癌症進程。然而目前對子宮癌肉瘤這種兩種組織型態組成的特殊癌症的腫瘤免疫表現的了解尚不多。 現在治療方向走向精準醫療，了解腫瘤的異質性在精準醫療中扮演很重要的角色，腫瘤間的不同性質造成同樣一種治療卻有不同療效。而腫瘤內的多樣性表現，包含腫瘤細胞不同表現以及腫瘤微環境的多樣表現，也會造成治療效果表現受影響，進而影響疾病結果。 在此篇研究中，我們將利用數位空間分析技術，研究擁有特殊雙組織型態的子宮癌肉瘤的腫瘤間以及腫瘤內的免疫微環境表現的異質性。

方法或程序 本研究回溯追蹤2010-2020年間在台大醫院初次診斷為子宮癌肉瘤並接受手術的病患，記錄其各種臨床治療及表現，並取得其子宮內膜癌組織的蠟塊切片。對切片脫蠟後以免疫螢光染色，接著在數位空間分析儀的幫助下，量化不同組織型態、不同腫瘤位置內的各種免疫蛋白的表現。最後對取得的資料進行統計分析。

結果 有23位診斷為子宮癌肉瘤的病人被收錄進此次研究。在不同的組織型態以及不同的腫瘤中的免疫表現都有所不同。在上皮癌型態組織中的免疫蛋白的表現和肉癌型態組織中的免疫蛋白表現作比較，會發現多數免疫蛋白聚集在肉癌型態的組織中，尤其fibronectin和SMA這兩者的表現量在肉癌型態組織中表現量尤其顯著高出在上皮癌型態組織 (p=0.009, p=0.01). 另外，在特別針對CD45+的免疫蛋白分析中，發現CD163在肉癌型態組織中的表現量也顯著高於在上皮癌型態組織中的表現量 (p<0.0001)。 在23位被診斷患有子宮癌肉瘤的病人中，有7位病人的腫瘤並沒有復發，相反的有16位病人的腫瘤有復發。分析這兩類病人的腫瘤免疫表現，發現這兩類病人在上皮癌型態組織中的免疫表現不一樣，雖然免疫表現量都不像肉癌型態組織中的高，但會發現相較於復發腫瘤，未復發腫瘤的上皮癌型態組織中表現相對高量的CD66b (p<0.0001)。相反的，復發腫瘤的上皮癌型態組織中表現相對高量的CD56 (p<0.0001)。兩類腫瘤在肉癌型態組織中的免疫表現也有些許不同，在造血幹細胞分化而來的免疫細胞表現分析中，未復發腫瘤的肉癌型態組織中有表現較高比例表現旺盛免疫細胞浸潤，相較之下復發腫瘤的肉癌型態組織中表現旺盛免疫細胞浸潤的比例較低 (p=0.03)。

討論及結論 子宮癌肉瘤是一有特殊組織型態的癌症。研究其腫瘤免疫微環境發現，肉癌型態的組織中免疫蛋白表現豐富，尤其是fibronectin和SMA的表現，這兩個標記都和上皮間質轉化(epithelial-mesenchymal transition, EMT)有關，這呼應到目前子宮癌肉瘤形成的假說，目前普遍認為子宮癌肉瘤中的肉癌型態組織是由上皮癌型態組織轉化而來。另外，肉癌型態組織，在針對骨髓造血幹細胞分化而來的免疫細胞做特別分析時，發現明顯較多的CD163表現。CD163是抑制型巨噬細胞 (M2 macrophage)的一個標記，這種腫瘤相關的巨噬細胞和腫瘤的惡化有關，以前研究以也發現肉癌型態組織傾向局部擴散。 分析有無惡化的兩種腫瘤的免疫微環境，發現沒有惡化的腫瘤其上皮癌型態組織的免疫表現傾向表現CD66b標記，而有惡化的腫瘤其上皮癌型態組織免疫表現傾向表現CD56標記。有兩種免疫細胞被發現以CD66b做標記，腫瘤相關中性球細胞和癌症預後有關，但目前對預後影響好壞皆有報告。多核型骨髓來源抑制細胞 (PMN-MDSC)也以CD66b為標記，它和免疫抑制以及腫瘤細胞複製有關。針對CD66b在不同癌症有不同預後的報告。CD56是自然殺手細胞的標記，自然殺手細胞能力有兩面向，傾向胞殺能力的多數伴隨有其他免疫細胞表現，而單獨出現的多傾向免疫抑制能力。此處看到的自然殺手細胞並無大量伴隨其他免疫細胞。另外，在沒有惡化的腫瘤，其肉癌型態組織中的免疫表現，相較於有惡化的細胞，骨髓造血幹細胞來源的免疫細胞有較高比例的表現，細看其內免疫細胞，有和免疫監控 (immune surveillance)相關的，也有和免疫編輯(immunoediting)有關的，相信在沒有惡化的腫瘤中，其肉癌型態組織尚處於免疫攻擊和腫瘤細胞抵抗的平衡拉鋸狀態。 子宮癌肉瘤在傳統治療下的預後不佳，免疫治療是一新的治療武器，在現今免疫療法興起以及精準醫療興起的時代，了解其腫瘤免疫微環境表現的異質性相信能提供我們針對此癌症未來治療的方向。

Key Points Uterine carcinosarcoma is an uncommon gynecological cancer, and the histology pattern is different from typical endometrial cancer. Uterine carcinosarcoma simultaneously expresses of both carcinoma and sarcomatous components. The standard treatment involves surgery with adjuvant chemotherapy +/- radiation therapy. However, its prognosis is poor. In recent years, research in immunology and molecular medicine has made significant progress, leading to the flourishing development of immunotherapy. Previous studies have indicated the significant role of infiltrating lymphocytes within endometrial cancer progression. Tumor cells also modulate the expression lymphocytes by immunoediting, thereby influencing the cancer progression. However, our current understanding of the tumor immune expression in uterine carcinosarcoma, who is composed of two distinct histological components, is limited. Current trends in treatment focus on precision medicine, emphasizing the importance of understanding tumor heterogeneity in achieving optimal outcomes. Variability in tumor properties can result in different treatment responses despite using the same therapeutic approach. The diversity in tumor expression, including varied expressions of tumor cells and the tumor microenvironment, can impact treatment efficacy and subsequently influence disease outcomes. In this study, we aim to utilize the digital spatial profiling to explore the heterogeneity in the tumor-to-tumor and intra-tumor immune microenvironment expression of uterine carcinosarcoma with its distinctive dual histological pattern. Methods or Procedures This study retrospectively traces patients diagnosed with uterine carcinosarcoma and received surgery at National Taiwan University Hospital between 2010 and 2020. Various clinical treatments and expressions were recorded, and paraffin-embedded tissue sections from primary tumors of uterine carcinosarcoma were obtained. Following deparaffinization of the sections, immunofluorescent staining was applied, and with the assistance of a digital spatial profiling instrument, the expression of various immune-related cell markers in different histological patterns and tumor locations was quantified. Finally, statistical analysis was performed on the acquired data. Results A total of 23 patients diagnosed with uterine carcinosarcoma were included in this study. Variations in immune expression were observed among different histological patterns. Most immune-related cell markers were found to be more concentrated in the sarcoma components than in the carcinoma components. Particularly, the expression levels of fibronectin and SMA were significantly higher in sarcoma components than in carcinoma components (p=0.009, p=0.01). Additionally, in a specific analysis focusing on CD45+, the expression level of CD163 in sarcoma components was significantly higher than in carcinoma components (p<0.0001). Seven patients experienced no tumor recurrence, while 16 had tumor recurrence. Analyzing the immune expression in these two groups, the immune expressions in carcinoma components were different. Compared to recurrent tumors, carcinoma components of non-recurrent tumors exhibited a relatively higher expression of CD66b (p<0.0001). Conversely, carcinoma components of recurrent tumors showed a relatively higher expression of CD56 (p<0.0001). There were also slight differences in immune expression in sarcoma components between the two tumor groups. In the analysis of immune cell infiltration derived from hematopoietic stem cell differentiation, sarcoma components of non-recurrent tumors exhibited a higher proportion of vigorous immune cell infiltration, compared to the recurrent tumors (p=0.03). Discussion and Conclusion Uterine carcinosarcoma is a cancer with a distinctive histological pattern. Studying the tumor immune microenvironment revealed abundant expression of immune-related cell markers in the sarcoma components, particularly fibronectin and SMA. The markers are associated with epithelial-mesenchymal transition (EMT), supporting the hypothesis that sarcomatous-type tissues in uterine carcinosarcoma originate from a transformation of carcinoma-type tissues. Additionally, in a specific analysis of immune cells derived from hematopoietic stem cell differentiation showed significantly higher CD163 expression in sarcoma components. CD163 is a marker of M2 macrophages, which are related to tumor progression. Previous studies have suggested the sarcoma tended to local spread. Analyzing the immune microenvironment of tumors with and without progression, it was found that the carcinoma components of non-recurrent tumors tended to express the CD66b marker, while the carcinoma components of recurrent tumors tended to express CD56. CD66b marks tumor-associated neutrophils with biphasic effect on cancer outcomes. CD66b also marks polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), and it associated with immune suppression and tumor cell replication. CD56 is a marker for natural killer (NK) cells with dual role in immune surveillance and immunosuppression. NK cells coexisting with other immune cells tended to show cell-killing ability, while those without coexisting with other immune cells were similar to helper cells. Besides, the sarcoma component of tumors without recurrence exhibited a higher proportion of immune cell expression derived from bone marrow hematopoietic stem cells compared to recurrent tumors. The cells were associations with immune surveillance and immunoediting, indicating a delicate balance between immune attack and resistance in the sarcoma component of nonrecurrent tumors. Uterine carcinosarcoma has a poor prognosis under traditional treatment, and immunotherapy emerges as a new therapeutic approach. In the era of rising immunotherapy and precision medicine, understanding the heterogeneity in tumor immune microenvironment expression is believed to guide future treatment directions for this cancer.