優化停經前荷爾蒙受體陽性乳癌荷爾蒙治療：臨床前期實驗與臨床試驗

Abstract

乳癌近三十年在台灣與東亞地區快速增加，然而其好發年齡、病理型態、分子分類及病人癒後都與西方國家不同，主要為停經前婦女，並且以荷爾蒙受體陽性的類型為主，其發生原因至今仍然不清楚。荷爾蒙受體陽性的乳癌病人，其荷爾蒙用藥，近四十年主要有tamoxifen及aromatase inhibitors；針對停經前婦女在這四十年間，主要的用藥僅有tamoxifen，並無太多進展。東亞地區主要是停經前荷爾蒙受體陽性的乳癌為最多，這方面的研究在歐美非常鮮少。尋找優化現有荷爾蒙治療用藥，改善荷爾蒙受體陽性乳癌病人治療成績，尤其是停經前婦女的治療，是我們亟待解決的問題。 本研究主軸是以優化停經前荷爾蒙受體陽性乳癌的荷爾蒙治療為出發，由臨床前期實驗，銜接到臨床試驗。第一部份是mTOR抑制劑與荷爾蒙治療併用相關的biomarker，以了解是否亞洲病人適用此治療方式，第二部分是PI3K抑制劑與荷爾蒙治療併用的臨床前期實驗與臨床試驗，第三部分是以荷爾蒙治療與免疫抑制劑併用的臨床試驗設計，實際針對停經前荷爾蒙受體陽性乳癌的新治療注入活水。 (1)以荷爾蒙治療併用mTOR抑制劑： PIK3CA乃荷爾蒙受體陽性乳癌最常見的突變之一，且BOLERO-2的成功亦暗示了，荷爾蒙治療藥物併用PI3K/AKT/mTOR路徑上的抑制劑，是一個可行的治療方向。同一時期TAMRAD試驗的結果，在biomarker的分析中，發現當tamoxifen併用everolimus時，LKB1可能可以預測療效。在BOLERO-2與TAMRAD兩個臨床試驗案，僅納入停經後婦女；而對於在東亞我們面對的，主要是停經前荷爾蒙受體陽性的乳癌病人，tamoxifen是主要的治療藥物選擇，是故，若在停經後婦女有效果，我們認為也許在停經前婦女的荷爾蒙受體陽性乳癌，tamoxifen併用Everolimus可能也會有角色，因此我們一方面展開了LKB1在亞洲乳癌預後角色的translational study，一方面想尋求是否有機會領導tamoxifen併用everolimus在亞洲停經前荷爾蒙受體陽性乳癌的臨床試驗。 LKB1本身是AMPK的上游蛋白質，在腫瘤發生過程中，是一個腫瘤抑制基因。在TAMRAD試驗的分析中，低的LKB1表現量，可以預測everolimus的藥效。我分析了台大、馬偕檢體的LKB1免疫染色，與英國兩個METABRIC datasets基因表現量的資料，主要想探討LKB1在停經前乳癌病人的角色。LKB1在我的研究中發現，與荷爾蒙受體的表現有高度相關，然而對於整體存活率來說，只有在HER2陽性的病人族群， LKB1的表現量才與OS有關。原先進行這個translational study的初衷，是希望對於everolimus併用Tamoxifen是否可能在亞洲的停經前荷爾蒙受體陽性乳癌有效，做一些評估。雖然後續因為everolimus在藥廠並沒有進一步進行臨床試驗的意願，同時我們的細胞實驗發現，eveolimus/tamoxifen僅有additive效果，不如PI3K抑制劑和tamoxifen併用時，是synergistic效果，我們沒有繼續以everolimus/ tamoxifen的組合往下做研究延伸，轉而以PI3K抑制劑與tamoxifen的組合進行臨床前與臨床試驗發展。 (2) 以荷爾蒙治療併用PI3K抑制劑： PIK3CA是荷爾蒙受體陽性乳癌最常見的突變，而且由everolimus的成功推測，若以PI3K抑制劑併用荷爾蒙治療，可能亦有機會強化荷爾蒙治療在荷爾蒙受體陽性乳癌的效果。本研究的第二個研究子題，乃環繞PI3K抑制劑併用tamoxifen的臨床試驗設計與臨床前期研究。身為腫瘤內科醫師，對於藥物發展除了臨床前期研究的支持外，另一個重要的環節，在於臨床試驗的設計與執行，本人參與設計了B-YOND 臨床試驗。此Phase Ib的試驗案，我們主要收納停經前荷爾蒙受體陽性乳癌病人，測試在第一線荷爾蒙治療使用tamoxifen時，併用buparlisib或是alpelisib的治療效果，並且尋找出後續在Phase II時的，最大耐受劑量。 在此臨床試驗的設計過程中，除了對於亞洲停經前荷爾蒙陽性乳癌病人更加了解外，同時對於估計藥物效果與副作用，以有意義的統計假設進行設計，對於臨床試驗執行面的了解，包括與Sponsor的溝通、經費的取得、整體預算的編列，追蹤病人給藥各方面，有更全面的學習。目前初步的分析仍在進行中，預計在2019年年底會有正式的研究成果發表。而根據SOLAR-1臨床試驗在停經後婦女的結果，PI3K抑制劑併用tamoxifen/goserelin在停經前婦女預期也會有不錯的效果。 另一方面，本研究主要以細胞實驗與動物實驗，來確認PI3K廣泛型抑制劑buparlisib與PI3K-alpha專一型抑制劑alpelisib，和tamoxifen併用的效果與機轉，以對B-YOND臨床試驗提供更多可能會有效的證據。 首先以具有不同PI3K/AKT途徑上突變的荷爾蒙受體陽性乳癌細胞株進行實驗，以MTT活性存活測試，並計算藥物合併指數(combination index)，用來比較併用藥物時的效果。buparlisib或alpelisib與tamoxifen併用，其藥物合併指數皆小於1，表示皆有加乘效果。此加乘不只侷限在PI3K/AKT路徑有突變的細胞株。進一步發現MCF-7和ZR75-1細胞株接受Buparlisib或Alpelisib與Tamoxifen併用後，細胞停留在SubG1的比例會高於單用tamoxifen、buparlisib或是alpelisib。以Annexin-V與PI進行染色時可發現，tamoxifen併用buparlisib或alpelisib的細胞，會增加Annexin-V+/PI+的細胞比例，表示這些藥物治療，主要是透過apoptosis死亡；西方墨點法分析接受上述藥物治療的細胞蛋白質時，可發現同時會透過降低磷酸化的4EBP1，磷酸化的GSK3β，磷酸化的p70S6K，來影響下游的訊息傳導。 為了證明上述tamoxifen併用buparlisib或alpelisib，主要透過哪個路徑影響藥物效果，我們以MCF-7細胞株當作對照組，一組MCF-7細胞株轉殖了mock，一組MCF-7細胞株轉殖了AKT，此乃為了強迫AKT的表現。AKT會一直表現的MCF-7細胞，在接受了tamoxifen併用buparlisib或alpelisib時，藥物合併指數會大於一，表示原本的加乘效果消失。且其下游的磷酸化的4EBP1，磷酸化的GSK3β，磷酸化的p70S6K，亦不受到抑制。另一方面，我們想比較是否荷爾蒙受體在tamoxifen併用buparlisib或alpelisib時，具有角色。是故我們將MCF-7細胞株與ZR-75-1細胞株轉殖了具有Y537S突變的荷爾蒙受體，並且分別以tamoxifen併用buparlisib或alpelisib進行比較。當細胞具有Y537S突變的荷爾蒙受體時，則藥物合併指數亦大於一，表示原先的加乘效果也消失了。上述兩組實驗證實了，tamoxifen併用buparlisib或alpelisib的加乘效果，需仰賴正常的AKT與無突變的荷爾蒙受體。 基於細胞實驗的結果，本研究進一步進行裸鼠動物實驗。tamoxifen併用buparlisib或alpelisib用以治療裸鼠的MCF-7腫瘤實驗中，buparlisib或alpelisib併用tamoxifen，相較於對照組或是單用tamoxifen、buparlisib或alpelisib的組別，皆讓MCF-7的腫瘤生長變慢，小鼠有較長的存活。我們取下裸鼠的MCF-7腫瘤，進行免疫染色分析，發現與前述的MCF-7細胞株在西方墨點法分析的結果相近，buparlisib或alpelisib併用tamoxifen會降低磷酸化的Akt，磷酸化的S6，磷酸化的p70S6K。 (3)荷爾蒙治療併用免疫治療的臨床試驗設計 有鑑於本研究中亞洲的乳癌病人有不少具有慢性發炎的特性，同時發現亞洲女性荷爾蒙受體陽性乳癌中，管腔B型為多，而且具有較高的免疫反應性，是故本研究的假說是，以免疫治療並用荷爾蒙治療，會有不錯的療效。本人參與了PEER臨床試驗的設計與計畫書的撰寫，並且負責病人檢體translation study，以找出療效預測因子。PEER納入停經前荷爾蒙陽性乳癌，未曾接受過化學治療者，給予荷爾蒙治療exemestane/ leuprolide，併用免疫治療pembrolizumab。本人亦同時在發展免疫健全小鼠的乳癌模式，預備進一步探討荷爾蒙治療併用免疫治療的機轉。目前臨床試驗還在收案中，但是可評估的十一位病人當中，有四位達到部分緩解，並且持續有效中。不僅這是在第二或第三線已經對荷爾蒙治療產生抗藥性的狀態下，很突出的治療效果。同時，也是少數在荷爾蒙陽性停經前乳癌病人，接受免疫治療，能夠有這麼高的反應率的組合。 整體而言，本研究內容，針對如何優化停經前荷爾蒙受體陽性乳癌的荷爾蒙治療，由PI3K/mTOR路徑的抑制劑與荷爾蒙治療併用，或是近期仍持續進行中的免疫治療與荷爾蒙治療併用。不管是以臨床前期的細胞或動物模式，來證實荷爾蒙治療併用PI3K抑制劑的效果與機轉，同時支持PI3K抑制劑併用荷爾蒙治療；或是仍在進行中的荷爾蒙陽性乳癌在免疫健全小鼠的實驗模式，進一步了解免疫治療與荷爾蒙治療併用的有效機轉，並且同時進行臨床試驗與病人檢體的研究。針對東亞主要的停經前荷爾蒙受體陽性的乳癌病人，我們的研究方向逐漸可以讓這群病人的治療，再往前邁進一步。

Breast cancer has emerged rapidly in Taiwan and East Asia in recent 3 decades. The age distribution, pathologic features, molecular subtypes, and patient prognosis of breast cancer patients in East Asia are all very different from those in western countries. The majority of breast cancer patients in East Asia is premenopausal and estrogen receptor positive. The etiology of this emerging breast cancer is still unknown. For estrogen receptor positive breast cancer patients, hormonal therapy developed in the past 40 years are tamoxifen, aromatase inhibitors, and fulvestrant. As for hormonal therapy specific for premenopausal patients, tamoxifen is the only drug in the past 40 years. Premenopausal breast cancer patients are endemic in East Asia, but not in western countries. Therefore, the research focusing on premenopausal breast cancer patients is not popular in western countries. Optimizing current hormonal therapy to improve treatment results in premenopausal hormonal receptor positive breast cancer patients is an unmet medical need. The main focus of this research is to optimize the hormonal therapy for premenopausal estrogen receptor positive breast cancer patients. We started from preclinical experiments to clinical trials. The first part of the study, I investigated the biomarker from mTOR inhibitor and tamoxifen combination. I would like to understand if this combination is feasible for Asian patients or not. The second part of the study is the combination of PI3K inhibitor and hormonal therapy in preclinical experiments and clinical trials. The last part is hormonal therapy in combination with immunotherapy in a clinical trial setting. (A)Hormonal therapy and mTOR inhibitor PIK3CA mutation is one of the most common mutations in estrogen receptor positive breast cancer. The success from BOLERO-2 trial also suggests that combining hormonal therapy and PI3K/AKT/mTOR inhibitors is a feasible treatment strategy. At the same time, biomarker study from TAMRAD trial discovered LKB1 as a potential efficacy predictor for everolimus in combination with tamoxifen. However, in BOLERO-2 and TAMRAD study, they only enrolled postmenopausal women. We wondered if tamoxifen and everolimus would also be effective for premenopausal patients in East Asia. Therefore, we started from understanding the role of LKB1 in Asian breast cancer patients and hoping to have a chance leading tamoxifen/ everolimus clinical trials in premenopausal patients in Asia. I have analyzed the tumor slides from National Taiwan University Hospital and Mackay Memorial Hospital by LKB1 immunohistochemical staining and gene expression levels from the two METABRIC datasets. The aim is to understand the role of LKB1 in premenopausal estrogen receptor positive breast cancer. However, LKB1 is associated with estrogen receptor positivity, but not prognosis. It was only in the HER-2 positive group that LKB1 is prognostic for overall survival. Despite our hypothesis was LKB1 might play an important role in estrogen receptor positive breast cancer, the result did not support this hypothesis. On the other hand, the pharmaceutical companies also do not plan to further invest on everolimus. We then moved to another target, PI3K, on the same thread. (B)Hormonal therapy and PI3K inhibitor We aim to investigate if PI3K inhibitor is a good partner to enhance hormonal therapy efficacy in estrogen receptor positive breast cancer. We utilized human breast cancer cell lines harboring different PI3K pathway alterations to demonstrate a synergistic effect between tamoxifen and PI3K inhibitor. I used siRNA to silence PIK3CA, myristoylated AKT to constantly activate AKT, and mutant estrogen receptor transfection to mitigate estrogen receptor function. These strategies demonstrate that the synergistic effect of tamoxifen and PI3K inhibitor rely on AKT, PIK3CA, and functioning estrogen receptor. Furthermore, MCF-7 xenograft treated with tamoxifen and PI3K inhibitor also demonstrated tumor shrinkage. This suggests the rationale to support the design of clinical trial, B-YOND. We have enrolled patients into either tamoxifen/ goserelin/ alpelisib or buparlisib. (C)Hormonal therapy and immunotherapy Chronic inflammation is a common characteristic in Asian breast cancer patients. Luminal B subtype, which is more immunoreactive, is enriched in Asian breast cancer patients. We hypothesize that combining hormonal therapy and immunotherapy would be efficacious in Asian breast cancer patients. I have designed the PEER clinical trial and translational studies with Professor Lu. In this study, we enrolled premenopausal estrogen receptor positive breast cancer patients, who had not received chemotherapy for metastatic disease. Hormonal therapy (exemestane and leuprolide), and immunotherapy (pembrolizumab) were given together in this study. Among the 11 evaluable patients, 4 patients achieved a partial response. This extraordinary response is superior to usual response rates in the 2nd line and 3rd line of hormonal therapy in estrogen receptor positive breast cancer patients. I am also working on establishing an immunocompetent estrogen receptor positive murine model to study the mechanism underlying this combination therapy. In conclusion, my study helps to understand the mechanism of hormonal therapy in combination with targeted therapy or immunotherapy. We have provided evidence from preclinical investigations to consolidate the ideas before moving into clinical trials. This is a very good training for me as a medical oncologist working from bench to bedside and backwards.