الفهرس 
IntroductionOnly 14 pages are availabe for public view
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Abstract
Despite the progress of tailored therapeutic strategies in patient with breast cancer, there is an unmet medical need for additional biomarkers that would guide therapy making it more personalized. In adjuvant setting many prognostic and predictive factors are available but this is not the scenario in the metastatic settings rendering it very difficult to give a tailored therapy for each patient. KI 67 proliferation index had limited value in predicting the behavior of disease along with contradictory data about the reliability of Ki-67 to assess tumor proliferation due to its relatively low inter-laboratory reproducibility. PD L1 testing, is one of the most important predictive markers in the last few years and considered the backbone in prescribing immunotherapy in many solid tumors, however this test is expensive and at sometimes required a licensed or accredited lab which are few in our country. Based on all the above mentioned factors; there is a need for a reliable and reproducible quantification of the immune infiltrate within the heterogeneous microenvironment of tumors in order to support therapy selection in oncology. One of these reliable and reproducible characteristics can be achieved in tumor infiltrating lymphocytes (TILs) which is more practical, affordable than others. Several studies stated that TILs have been shown to provide prognostic and potentially predictive values in primary breast cancer in adjuvant status and also in the neoadjuvant setting, however, limited data characterizing the immunobiology of metastatic breast cancer are available (213) (214). Because of most trials used TILs in the neoadjuvant and adjuvant settings and the data are scanty in the metastatic phase; accordingly we focused on advanced phase of this disease. Our study showed that, TILs has a positive correlation with progression free survival which translated into a good clinical outcome with significant p value 0.043. These data are in concordance with that published by Stephen J. et al. whom stated a relationship between TILs and survival in the setting of advanced breast cancer(138).
Also the positive correlation finding between the Stromal infiltrating lymphocytes (SILs) and survival was evident in our study with a significant p value <0.001. This also matched with SJ et al who examined the associations between the stromal TILs and progression free survival in patients with advanced HER 2 –positive breast cancer who were treated with trastuzumab added to pertuzumab and docetaxel in the first –line setting as a part of CLEOPATERA trial . Also in our study, TILs IHC staining had a significant correlation with Disease free survival ; with a significant p value 0.035. This finding is matched with several studies in literature (127) (121) (129) (130) (131) (132), which concluded that; a high TILs level is an independent indicator of good prognosis and has been validated in large cohorts of patients in the adjuvant setting. Also a high level of TILs is associated with improved disease free survival and overall prognosis. Kolberg et al. found that; although the presence of high TILs is associated with negative prognostic parameters such as high KI67 and HR-negative status among patients with HR-positive HER2-negative EBC, patients with high TILs show a favorable 5-year DFS in both HR-positive/HER2-negative and triple-negative breast cancer.(215) Contradictoey data mentioned by Dankert et al. stated that; patients with luminal tumors with a low level of TILs were associated with improved OS. This could be a chance of finding a sign of different biology of immunologic infiltrate in this subtype the study showed that tumor infiltrating lymphocytes may be linked to a reduced endocrine response (140). Also, Loi et al. investigated the relationship between TILs and clinical outcomes using 2009 samples from the BIG 02-98 adjuvant study. TILs were associated with improved DFS and OS, but only in patients with TNBC (127). In our study we tested another two variables which are CD8 and CD4 positive lymphocytes and we found that: Positive correlations between CD8 & CD4 with progression free survival this presented with a significant p values 0.010 and 0.025 respectively. Based on our study results, CD8 positive lymphocytes was positively correlated with overall survival with a highly statically significant p value <0.001 which is concordant with Hunborg et al. data (216). Our results matched with Song et al. who suggested that elevated levels of CD8 suppressor T lymphocytes represent a novel independent predictor of PFS (217). Mohamed et al. found that; tumor-infiltrating CD8 T lymphocytes have antitumor activity as judged by their favorable effect on patients’ survival and could potentially be exploited in the treatment of breast cancer (218). Hunborg et al. reveled that; Numbers of tumor-infiltrating CD4+ T cells in breast cancer were positively correlated with tumor stages and lymph nodal status but negatively correlated with RFS and OS. However, CD8+ T cell numbers were negatively correlated with high tumor stages and positive lymph node status and positively correlated with clinical outcomes of RFS and OS (216). Although chemotherapy’s main mechanism of action is believed to be induction of apoptosis, because of interference with DNA replication and evidence of DNA damage, studies suggest that part of the antitumor effect occurs through modulation of the immune system .There is increasing evidence of an interaction between TILs and sensitivity to targeted therapy and chemotherapy (153) (154). The most commonly used regimens in our study; taxane based/ taxane-platinum based (for those previously treated with anthracycline) and anthracycline based chemotherapy (for those who were anthracycline naïve). TILs in our study had positive correlation with chemotherapy (particularly first line of treatment) with significant p value 0.001. Moreover CD8 & CD4 were positively with significant p value 0.011 & 0.004 respectively. TH et al. found that ;accumulation of enhanced CD8+ activation and CD11c+ dendritic cells in breast tumors were demonstrated in mice treated with cisplatin (155). Demaria et al. elevation of TIL percentage has been demonstrated after taxane neoadjuvant therapy, and this reaction correlated with clinical response (156). SR et al. stated that; anthracyclines can stimulate CD8+ lymphocyte proliferation in tumors.(157) Conversely, studies on CD8+ depleted mice demonstrated an increased resistance to anthracyclines, suggesting that immunity is indispensable for an optimal antitumor effect (157). D. C et al. demonstrated a correlation between TIL level and benefit from carboplatin (136). In our study; CD8 lymphocytes had correlations with the nodal status, tumor size positive LN, PR status , HR status and molecular subtype Mahmoud et al. found that; the total number of CD8 cells was positively correlated with tumor grade (P .001) and inversely correlated with patient’s age at diagnosis, estrogen receptor–alpha (ER- ), and progesterone receptor (PgR) expression (Mann-Whitney U test, P .001) (218) According to our data; CD4 lymphocytes had positive correlations with age group, grade, PR status SILs, and TILs. MATKOWSKI et al. found that; high correlations between CD4 expression and lymph node status and intensity of lymphatic infiltrate (219). In our study a highly significant correlation between the stromal infiltrating lymphocytes (SILs) and age group, lymphovascular invasion, tumor infiltrating lymphocytes, significant value with N status, ER status and PR status. Also we found a significant correlation between TILs IHC as a variable and some clinicopathalogical parameters, namely M status (0.030), CD4 IHC staining (p value 0.001 ) and SILs IHC staining (p value <0.001). Miyoshi et al. published a study with conclusion that; the TIL distributions were significantly associated with nodal metastasis (P=0.004), ER status (P=0.045), progesterone receptor (PgR) status (P=0.002), tumor grade (P=0.021), and the Ki67 labeling index (LI) (220). TILs IHC staining has a significant correlation with first sites of metastases, which is lung metastases , this is matching with Takada et al. who found that; Lung metastasis, was the most common site of distant metastasis and associated with higher TILs density in a study included ER positive/HER 2 NEU negative patients (221). Coast of doing TILs, SILs, CD8 and CD4 are relatively low in Comparison with the high coast and highly complex testing like PDL-1. To the best of our knowledge, our study is one of the first studies in Egypt assessing the prognostic value of TILs in metastatic breast cancer patients. However there are few limitations facing our study: Small number of patients, this may attributed to the difficulty in obtaining the paraffin blocks either from patients or from cancer center. Difficulty in taking biopsy from distant metastases like few, small pulmonary nodules to assess the level of TILs in the metastatic site. Lack of standardization in methods of evaluation of TILs and SILs. To the best of our knowledge, our study is one of the first studies in Egypt assessing the prognostic value of TILs in metastatic breast cancer patients. With a median follow up 43 months (36-60 months) and after analyzing the data we found the following: Strong correlations had been demonstrated between the percentage of tumor infiltrating lymphocytes and disease free survival. TILs, SILs, CD8 positive lymphocytes and CD4 positive lymphocytes were positively associated with progression free survival. CD8 positive lymphocytes were associated with improved overall survival. In the present era of tailored therapy and immunotherapy, finding a predictive marker will allow better selection of targeted agents in addition to chemotherapy regimens aiming to provide a better service to our patients in spite of the limited resources of our country. Our recommendations: Evaluation of the TILs & SILs in histopathalogical examination of biopsy in neoadjuvant/adjuvant/ metastatic cases and including them in the pathology report. Making a large national study comparing the value of TILs toward PDL 1 testing in patients subjected to immunotherapy. Facilitating a multicenter study between different faculties of medicine and recruitment a large number of patients with specific breast cancer subtyping with standard therapy. Facilitating researches in cancer centers and cooperation with different faculties of medicine with elimination of the difficulties and obstacles facing the postgraduate candidates.