Neoadjuvan kemoterapi alan meme kanserli hastalarda androjen reseptör ekspresyonunun tedavi yanıtla ilişkisi ve prognostik önemi
The relationship between androgen receptor expression and treatment response, as well as the prognostic significance, in breast cancer patients receiving neoadjuvant chemotherapy
Yazar: ABDULLAH ÜMİT
Danışman: PROF. DR. ALİ MURAT TATLI
Akdeniz Üniversitesi / Tıp Fakültesi / İç Hastalıkları Ana Bilim Dalı
Derece: Tıpta Uzmanlık, 2023
Konu: Onkoloji = Oncology ; İç Hastalıkları = Internal diseases

Özet:
GİRİŞ: Meme kanseri; klinik, patolojik ve moleküler açıdan çeşitli spektrumları içeren, farklı biyolojik alt tiplerden oluşan heterojen bir hastalık grubudur. Küresel olarak her yıl iki milyondan fazla vakaya neden olan ve kadınlarda en sık teşhis edilen malignitedir. Aynı zamanda kadınlarda kanser ölümlerinin önde gelen nedenlerinden birisidir. Meme kanserinde bilinen tüm prognostik / prediktif belirteçler ve sınıflamalar göz önüne alındığında; aynı tedavi yöntemleri uygulandığında bile hastalar arasında tedaviye cevap, rekürrensler, tümör davranışı ve genel prognoz açısından önemli farklılıklar olduğu gözlenmektedir. Bu durumlar prognozu etkileyen başka faktörlerin de 
bulunduğu düşüncesini ortaya çıkarmaktadır. Meme tümörlerindeki bu heterojenitenin yol açmış olduğu hedefe yönelik tedavilerdeki zorluklar aşılmaya çalışılmaktadır. Androjen reseptörü, tüm meme kanseri tiplerinde en sık eksprese edilen (%47-%90) seks steroid reseptörü olmasına rağmen 
androjenlerin ve androjen reseptörü ekspresyonunun; meme kanserindeki prognostik ve prediktif değerleri ile klinik olarak hastalık seyri ve tedavi yanıttaki önemi hakkında yeterli klinik çalışma mevcut değildir. Literatürde, özellikle neoadjuvan kemoterapi alan hastalarda AR durumunun önemini açığa çıkarmak için yapılan çalışma sayısı azdır.
 
AMAÇ: Bu çalışma androjen reseptör ekspresyonu durumunun, meme kanserindeki diğer biyolojik belirteçler ile olan ilişkisini, klinik sonuçlar üzerine etkili prediktif ve prognostik önemini, neoadjuvan kemoterapi sonrası patolojik yanıtı tahmin etme gücü ve sağkalım üzerine etkilerinin gösterilmesi amaçlanmıştır. Özellikle tedavi seçeneği sınırlı ve mevcut tedavilere karşı dirençli olan meme tümörlerinde geliştirilebilecek olan hedefe yönelik yeni tedavi yöntemleri için androjen reseptörünün de seçenekler arasında bulunabileceğinin, mevcut diğer tedavilere verilen yanıtlarla prediktif ilişkisinin ve neoadjuvan tedavilere daha iyi yanıt verecek hastaların belirlenmesinde rutin kullanılabilecek ek bir prognostik belirteç olarak öneminin gösterilmesi amaçlanmıştır.

GEREÇ ve YÖNTEM: Bu çalışma retrospektif olarak neoadjuvan kemoterapi alan evre 2 ve evre 3 meme kanserli hastalarda tüm moleküler alt tipleri kapsayacak şekilde planlanmıştır. Akdeniz Üniversitesi Tıp Fakültesi Hastanesi Tıbbi Onkoloji kliniğinde meme kanseri tanısıyla takip edilip Neoadjuvan 
kemoterapi almış ve sonrasında cerrahi olmuş toplam 176 hasta çalışmaya dahil edilmiştir. Androjen reseptör antikorları ile IHK olarak meme tümör dokularında AR ekspresyonu durumu incelenerek; Androjen reseptörünün, çeşitli klinopatolojik parametreler, neoadjuvan kemoterapi sonrası patolojik 
yanıt durumu ve sağkalım durumları ile ilişkisi analiz edilmiştir. Androjen reseptörü antikoru ile IHK olarak tümör hücrelerindeki %1 ve üzeri nükleer boyanmayı AR ekspresyonu pozitif durum olarak kabul edilmiştir. Neoadjuvan kemoterapi sonrası patolojik yanıt durumları RCB (Residual Cancer Burden) skorlama sistemi kullanarak değerlendirilmiştir.

BULGULAR: Bulgular: Çalışmaya katılan hastaların tamamı kadındır ve yaş ortalaması 49,8±10,5 yıldır. Hastaların %87,5'i IDC, NST; %4,5'i ILC tipindeydi. Hastaların moleküler subtiplerine göre sırasıyla %32,4'ü Luminal B HER2 negatif, %27,8'i Luminal B HER2 pozitif, %18,8'i TNMK, %14,2'si HER2 pozitif (non-luminal) ve %6,8'i ise Luminal A subtipinde saptandı. Çalışmaya katılan hastaların %66,5'i HR pozitif iken %42'si HER2 pozitif izlendi. Hastaların tanı anı doku biyopsilerinde ortalama TİL yüzdesi %27±28,7 saptandı. Hastaların neoadjuvan kemoterapi öncesi %59,1'inde AR ekspresyonu pozitif saptandı. Androjen reseptörü pozitifliği; daha küçük tümör boyutu (p=0,024), daha düşük histolojik grade (p=0,022), daha düşük Ki-67 skoru (p=0,004), hormon reseptör pozitifliğinde artış, triple negatif meme kanseri sıklığında azalma gibi olumlu prognostik faktörler ile istatistiksel anlamlı ilişkili gösterdi. Hastaların %42'sinde neoadjuvan kemoterapi sonrası patolojik tam yanıt saptandı. Androjen reseptörü pozitif grupta patolojik tam yanıt oranı %44,2 iken androjen reseptörü negatif olan grupta %38,9'du. Tüm hasta grubunda androjen reseptör pozitifliği ile patolojik tam yanıt arasında istatiksel anlamlı ilişki saptanmadı. Luminal B HER2 + hastalarda, androjen reseptör pozitif boyanma yüzdesi arttıkça tümör boyutunda küçülme izlendi (p=0,031). Triple negatif meme kanserli hastalarda ise androjen reseptörpozitiflik yüzdesinin artması; yaşın ileri olması, daha büyük rezidü invaziv tümör boyutu ve daha yüksek RCB skoru ile ilişki gözlendi (sırasıyla p=0,020; 
p=0,007; p=0,017). Luminal B HER2 + grupta androjen reseptör pozitif olan hastalarda neoadjuvan kemoterapi sonrası rezidü invaziv tümör boyutu ve RCB skoru androjen reseptör negatif olan hastalara göre daha düşük saptandı (sırasıyla; p=0,037 ve p=0,037). Triple negatif meme kanserli grupta androjen reseptör pozitif olan hastalarda, neoadjuvan kemoterapi sonrası RCB skorunda 
ve tümör boyutunda androjen reseptör negatif olan hastalara göre istatistiksel anlamlı yükseklik saptandı (sırasıyla; p=0,019 ve p=0,005). Patolojik tam yanıt olan hastalarda neoadjuvan kemoterapi öncesi ortanca TİL yüzdesi patolojik tam yanıt olmayan hastalara göre daha yüksek saptandı (p=0,006). Yapılan sağkalım analizi çalışmalarında tüm hastalarda androjen reseptörü ekspresyonu 
pozitifliği ile genel sağkalım ve hastalıksız sağkalım arasında anlamlı ilişki saptanmadı. Hastalar androjen reseptörü boyanma yüzdesine göre sınıflandırıp sağkalım açısından karşılaştırıldığında; androjen reseptörü boyanma yüzdesi >%50 olan hastalarda genel sağkalım hızının, androjen reseptörü boyanma yüzdesi %0-50 olan hastalara göre daha düşük olduğu saptandı (p=0,041). 
Ancak genel sağkalım üzerine etkili risk faktörlerinin multivariate COX Regresyon Analizi ile değerlendirilmesi sonuçlarında androjen reseptörü boyanma yüzdesinin sağkalıma etkisi gösterilemedi. Multivariate COX Regresyon analizi sonucuna göre nüks olan hastaların 8 kat, 50 yaş üzerindeki hastaların ise 5 kat fazla mortal seyrettiği gösterildi. Yapılan sağkalım analizlerinde TİL boyanma yüzdesinin genel sağkalım üzerine anlamlı bir etkisi saptanmadı. Ancak TIL skoru >%10 olan hastalarda 3 yıllık ve 5 yıllık hastalıksız sağkalım hızlarında TIL skoru <%10 olan hastalara göre istatistiksel anlamlı artış izlendi (p=0,042).

SONUÇ: Çalışmamızın temel amaçlarından birisi olan androjen reseptörekspresyon durumunun patolojik tam yanıt üzerine etkisi tüm hasta grubunda değerlendirildiğinde; androjen reseptörü ekspresyon durumu ile patolojik tam yanıt arasında anlamlı ilişki saptanmadı. Ancak androjen reseptörü durumuna göre neoadjuvan kemoterapiye verilen tedavi yanıtının moleküler alt tiplerde farklılık göstermesi; androjen reseptörünün, meme kanseri moleküler alt tiplerinde farklı biyolojik davranışlar sergilediğini ve neoadjuvan kemoterapiye verilen patolojik yanıtı predikte etme ile prognostik öneminin moleküler alt tiplerde farklı olduğunu göstermektedir. Androjen reseptör ekspresyonu pozitifliğinin bazı olumlu prognostik faktörler ile istatistiksel anlamlı ilişkili tespit edilmiş olması; androjen reseptörü ekspresyonunun neoadjuvan kemoterapi alan alan meme kanserli hastalarda olumlu bir prognostik ve prediktif belirteç olarak değerlendirilebileceğini göstermektedir.
Çalışmamızda yüksek TIL yüzdelerinin, tüm moleküler alt tiplerde androjen reseptöründen bağımsız olarak neoadjuvan kemoterapiye verilen patolojik yanıtı artırdığını ve özellikle %10 ve üzerindeki TIL yüzdelerinin hastalıksız sağkalım üzerine olumlu prognostik özellikler taşıdığını gösterdik. Ayrıca bu sonuçlar tümör mikro çevresindeki azalmış TIL varlığının, azalmış tümör bağışıklığı ve prognozda kötüleşme ile ilişkili olduğunu da desteklemektedir.

ANAHTAR KELİMELER: Meme Kanseri, Neoadjuvan Kemoterapi, Androjen 
Reseptörü, Patolojik Tam Yanıt, Prognoz, Sağkalım

Abstract:
Introduction: Breast cancer is a disease group that encompasses various spectra clinically, pathologically, and molecularly, consisting of different biological subtypes. Globally, it is the most commonly diagnosed malignancy, causing over two million new cases each year. Breast cancer, along with lung, stomach, liver, and colon cancer, plays a significant role in cancer-related mortality. Neoadjuvant therapy allows for reducing the extent of surgery, preserving axillary lymph nodes, and achieving lower complications and better cosmetic outcomes. Additionally, neoadjuvant therapy carries predictive characteristics, enabling the anticipation of various information about the treatment regimen that is expected to provide a better response than the available treatment modalities in case of recurrence during postsurgical follow-ups. It also allows for early assessment of treatment response, thus holding predictive value. Breast cancer has been divided into different molecular subtypes based on gene expression analyses and molecular studies, which have implications for prognosis, treatment management, and clinical perspectives. The therapeutic, predictive, and prognostic importance of molecular markers such as ER, PR, and HER2 has been 
well-established for predicting clinical outcomes and managing the disease. 
However, despite considering all these parameters and classifications, and even with the application of the same treatment methods, significant differences in treatment response, recurrences, tumor behavior, and overall prognosis are observed among patients. This suggests the presence of other factors that influence prognosis. Efforts are being made to overcome the challenges in targeted therapies caused by the heterogeneity of breast tumors.


Objective: Despite androgen receptor (AR) being the most commonly expressed sex steroid receptor (47%-90%) in all types of breast cancer, there is a lack of sufficient clinical studies regarding the prognostic and predictive values of androgens and AR expression in the diagnosis of the disease, as well as their clinical significance in disease progression and treatment response in breast cancer. In the literature, there are few studies specifically focusing on the importance of AR status in patients receiving neoadjuvant chemotherapy (NACT). This study aims to demonstrate the relationship between AR expression and other biological markers in breast cancer, their predictive and prognostic significance on clinical outcomes, their ability to predict pathological response after NACT, and their impact on survival.


Materials and Methods: This study was planned retrospectively to include all molecular subtypes of stage 2 and stage 3 breast cancer patients who received neoadjuvant chemotherapy (NAKT). A total of 176 patients who were diagnosed with breast cancer and followed up at the Medical Oncology Clinic of Akdeniz University Faculty of Medicine Hospital, received neoadjuvant chemotherapy, and underwent subsequent surgery were included in the study. The data regarding the demographic, clinical, and pathological characteristics of the included patients were retrospectively collected through file scanning method from the hospital's medical record systems and archives.The AR expression status in breast tumor tissues was examined using androgen receptor antibodies through immunohistochemical analysis. The relationship between androgen receptor and various clinical and pathological parameters, pathological response after NACT, and survival outcomes was analyzed. Nuclear 
staining of 1% or higher in tumor cells with AR antibody was considered as a positive AR expression status. The pathological response after neoadjuvant chemotherapy was evaluated using the Residual Cancer Burden (RCB) scoring system. The overall survival duration is defined as the time between the diagnosis date and the date of death or the time until the end of the study/last visit if the patient is still alive. Disease-free survival duration is defined as the time between the date of 
surgery after NACT and the date of the first recurrence, or if no recurrence occurs, the time between the date of surgery and the date of death, or if the patient is in remission, the time between the date of surgery and the end of the study/last visit.


Results: All patients included in the study were female, with a mean age of 49.8±10.5 years. The average tumor size before neoadjuvant chemotherapy was 39.1±19.3 mm. Among the patients, 87.5% had invasive ductal carcinoma (IDC),not otherwise specified (NST), and 4.5% had invasive lobular carcinoma (ILC). When analyzing the distribution of patients based on molecular subtypes, 32.4% 
were identified as Luminal B HER2-negative, 27.8% as Luminal B HER2-positive, 18.8% as triple negative, 14.2% as HER2-positive (non-luminal), and 6.8% as Luminal A subtype. Among the participants, 66.5% were hormone receptor (HR) positive, while 42% were HER2 positive. The mean percentage of tumorinfiltrating lymphocytes (TIL) in the diagnostic tissue biopsies of the patients was found to be 27%±28.7. When the pre-NACT tissue biopsy samples of the patients were examined using immunohistochemical analysis methods, positive AR expression was detected in 59.1% of the patients. In our study, positive AR expression was found to be significantly associated with favorable prognostic factors such as smaller tumor size (p=0.024), lower histological grade (p=0.022), lower Ki-67 score (p=0.004), and an increase in hormone receptor positivity and a decrease in triple-negative phenotype. Among the 176 patients included in the study, pathological complete response (pCR) was observed in 74 (42%) patients. The statistical significance of pCR on 
disease-free survival (DFS) and overall survival (OS) could not be demonstrated. The pCR rate was 44.2% in the AR-positive group and 38.9% in the AR-negative group. There was no statistically significant relationship between AR positivity and pCR in the entire patient group. In Luminal B HER2+ patients, as the percentage of AR-positive staining increased, tumor size reduction was observed (p=0.031). In triple-negative breast cancer patients, an increase in AR positivity percentage was associated with older age at diagnosis, larger residual invasive tumor size after NACT, and higher RCB score after NACT (p=0.020, p=0.007, p=0.017, respectively). In the Luminal B HER2+ group, patients with positive AR expression had significantly smaller residual invasive tumor size and lower RCB score after NACT compared to patients with negative AR expression (p=0.037 and p=0.037, respectively). In the triple-negative breast cancer group, patients with positive AR 
expression had statistically higher RCB score and tumor size after NACT compared to patients with negative AR expression (p=0.019 and p=0.005, respectively).When the TIL scores before NACT were compared between the groups with and without pCR, it was found that the median TIL percentage before NACT was significantly higher in patients with pCR compared to those without pCR (p=0.006).In our study, the survival analysis showed a 1-year disease-free survival (DFS) rate of 100%, a 3-year DFS rate of 95.2%, and a 5-year DFS rate of 91.7% for all patients. However, no significant relationship was found between AR expression positivity and DFS or overall survival (OS). When patients were classified based on AR staining percentage and compared using the Log-Rank analysis, it was 
observed that patients with AR staining percentage >50% had a lower DFS rate compared to those with AR staining percentage of 0-50% (p=0.041). However, the impact of AR staining percentage on DFS could not be demonstrated as an influential risk factor. This indicates a significantly increased mortality risk in patients who experienced recurrence and in those who were above 50 years old.
In the survival analysis conducted on the patients included in the study, no significant effect of TIL (Tumor-Infiltrating Lymphocyte) staining percentage on overall survival (GSK) was observed. However, when comparing the DFS(Disease-Free Survival) rates based on TIL staining percentage, a statistically significant increase in the 3-year and 5-year DFS rates was observed in patients 
with TIL score >10% compared to those with TIL score <10% (p=0.042). This suggests that a higher TIL score (>10%) is associated with improved disease-free survival at the 3-year and 5-year follow-up periods.


Conclusion: When evaluating the impact of AR expression status on pCR in the entire patient group, no significant association was found between AR expression status and pCR. However, when evaluating treatment response in different molecular subtypes, AR positivity was found to be associated with a better pathological response to NACT in Luminal B HER2-positive patients and a worse pathological response to NACT in TNBC patients. These findings suggest that the role of AR expression in treatment response may vary depending on the molecular subtypes.
We have demonstrated that increasing positive staining percentage of AR is associated with better pathological response to NACT and smaller residual invasive tumor size in Luminal B HER2+ patients, indicating favorable prognostic outcomes. Our study has shown that AR expression in HER2-positive patients is associated with positive clinicopathological characteristics, and based on these clinicopathological data, we have shown that Luminal B HER2+/AR+ patients have a better prognosis compared to Non-Luminal HER2+/AR+ patients. This finding constitutes one of the most significant contributions of our study to the literature.On the contrary, in TNBC, we found that increasing positive staining percentage of AR is associated with a decrease in pathological response to NACT and worse prognostic characteristics. These findings suggest that AR exhibits different biological behaviors in molecular subtypes of breast cancer and that the predictive and prognostic significance of AR expression in predicting pathological response to NACT varies among molecular subtypes.
In our study, positive AR expression was found to be significantly associated with favorable prognostic factors such as smaller tumor size (p=0.024), lower histological grade (p=0.022), lower Ki-67 score (p=0.004), increased HR positivity, and decreased TNBC subtype. Additionally, AR-positive patients showed an increased frequency of HER2 positivity (p=0.016) and HR positivity (p=0.001), as well as a lower frequency of TNBC subtype and a higher frequency of Luminal A 
and Luminal B HER2+ subtypes (p=0.001). These findings indicate the predictive significance of AR positivity in patients eligible for targeted therapies. These results suggest that AR expression can be considered as a favorable prognostic and predictive marker in patients with breast cancer receiving NACT.
In our survival analysis studies, the 1-year DFS rate was 100%, the 3-year DFS rate was 95.2%, and the 5-year DFS rate was 91.7% for all patients. However, no significant association was found between AR expression positivity and DFS or overall survival (OS). Due to the limited number of patients and total events encountered in specific molecular subtypes, survival analyses comparing AR positive and AR-negative groups could not be performed. Additionally, when patients were classified based on AR staining percentage and compared using the Log Rank test, it was found that patients with AR staining percentage >50% had lower DFS compared to those with AR staining percentage of 0-50% (p=0.041). However, when evaluating the impact of influential risk factors on DFS using 
multivariate Cox regression analysis, the effect of AR staining percentage on survival could not be demonstrated.
In our study, we demonstrated that high tumor-infiltrating lymphocyte (TIL) percentages increased the pathological response to neoadjuvant chemotherapy (NACT) independently of AR expression in all molecular subtypes. Specifically, TIL percentages of 10% or higher were associated with positive prognostic features on disease-free survival (DFS). These findings suggest that increased TIL presence in the tumor microenvironment is associated with enhanced tumor immunity and 
improved treatment response. In conclusion, we believe that increasing the number of patients in our study, extending the clinical follow-up period, and encountering more events would provide a clearer understanding of the impact of AR positivity and AR staining percentage on the pathological response to NACT and their prognostic effects on survival, both for the entire study population and within specific molecular subtypes.

 
Key Words: Breast Cancer, Neoadjuvant Chemotherapy, Androgen Receptor, 
Pathological Complete Response, Prognosis, Survival.