POSSIBILITIES TO ASSESS THE EFFICACY OF THE NEOADJUVANT TREATMENT FOR LOW RECTAL CANCER, IN ORDER TO APPLY A SPHINCTER SAVING PROCEDURE

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1 UNIVERSITY OF MEDICINE AND PHARMACY GR. T. POPA IAȘI POSSIBILITIES TO ASSESS THE EFFICACY OF THE NEOADJUVANT TREATMENT FOR LOW RECTAL CANCER, IN ORDER TO APPLY A SPHINCTER SAVING PROCEDURE PhD THESIS (ABSTRACT) SCIENTIFIC COORDINATOR, Viorel SCRIPCARIU, MD, PhD Professor of Surgery PhD candidate, Maria-Gabriela ANIŢEI IAŞI 2013

2 Invest in people! Project co-financed from the European Social Fund by the Sectoral Operational Program Human Resources Development Education and professional training supporting the economic growth and the society development based on knowledge primary axis Main intervention field: 1.5 Doctoral and post-doctoral program supporting the research Project title: Doctoral fellowships for the increase of competitiveness in the medical and pharmaceutical field Contract Identification Number: POSDRU/88/1.5/S/58965 Beneficiary: Gr. T. Popa University of Medicine and Pharmacy of Iasi Partner : Iuliu Hatieganu University of Medicine and Pharmacy of Cluj Napoca Keywords: - low rectal cancer; - radio (chemo) therapy, - anal sphincter preservation, - immune status, - immune score, - quality of life "You must try to climb very high, if you want to be able to see very far away... " - Constantin Brâncuși "People can see in different things, what they have in their mind " - Unknown author - 1

3 CONTENT OF THE PhD THESIS INTRODUCTION 4 A. CURRENT STAGE OF KNOWLEDGE ABOUT RECTAL CANCER 7 Chapter 1 Surgical anatomy of the rectum and mesorectum 9 Chapter Epidemiology of the rectal cancer Particular elements of the rectal cancer 14 Chapter 3 Diagnosis of the low rectal cancer Definition of the low rectal cancer Stages of the low rectal cancer Imagistic explorations in staging of the low rectal cancer 21 Chapter 4 Treatment for low rectal cancer The role of the multidisciplinary team in the low rectal cancer Radio/chemotherapy in the low rectal cancer Principles of radiotherapy in locally advanced low rectal cancer Side effects of the neoadjuvant treatment for the locally advanced rectal cancer Predictive factors in response to the neoadjuvant treatment for low rectal cancer Surgical treatment for low rectal cancer Criteria of operability and resectability in low rectal cancer Quality criteria of surgical resection Operative management in low rectal cancer ESMO recommendations regarding the treatment for locally advanced, low rectal cancer NCCN recommendations regarding the treatment for locally advanced, low rectal cancer 45 Chapter 5 Prognostic factors in the low rectal cancer 46 2

4 Capitolul 6 Quality of life in patients with low rectal cancer, with radiotherapy 50 Chapter The connection between the colorectal cancer and the immune system Immune infiltrate in tumor cells - the tumor microenvironment Role of cytotoxic T lymphocytes in cancer The importance of immune score in colorectal cancer 55 B. PERSONAL CONTRIBUTIONS TO THE STUDY OF THE RECTAL CANCER 57 Motivation and objectives of the PhD study 59 Chapter 8. STUDY I Optimising the treatment for locally advanced, low rectal cancer the experience of St. Spiridon Hospital, III Surgery Unit, Iasi Aim and objectives Material and methods Selection of patients Pre-therapeutic evaluation of patients Neoadjuvant treatment Imagistic evaluation of the neoadjuvant treatment s tumor response Surgical treatment - low anterior resection (very low) of the rectum with total mesorectum excision - technique Pathological evaluation of the surgical specimen Results Imagistic appreciation of the neoadjuvant treatment s eficacity in order to descent the limit of surgical resection Possibilities for anal sphincter preservation Pathological evaluation of the tumor response to the neoadjuvant treatment Discussion 88 Chapter 9. STUDY II - Prognostic value of tumor immune infiltrate for the patients with rectal cancer Aim and objectives Material and methods 95 3

5 Selection of patients Evaluation of the immune score Statistical analysis Statistical analysis Evaluation of the immune score, as a prognostic factor in rectal cancer Evaluation of the immune infiltrate s impact on the evolution of the patients with rectal cancer, eligible for the first-line surgical treatment Evaluation of the immune score s impact on the evolution of the patients with rectal cancer, eligible for first-line surgical treatment Multivariate analysis of prognostic factors in rectal cancer for patients eligible for first-line surgical treatment Evaluation of the immune infiltrate s impact on the evolution of the patients with rectal cancer, eligible for first-line neoadjuvant treatment Discussion 109 Chapter 10. STUDY III - The analysis of tumor immune infiltrate on the rectal tumor biopsies, and the correlation with the response to the neoadjuvant treatment Aim and objectives Material and methods Selection of patients Immunohistochemistry technique of tumor tissue biopsies Acquisition of immunohistochemistry slides in electronic format Automatic analysis of immunohistochemistry staining Pathological examinations of the tumor response to the neoadjuvant radiochemotherapy Statistical analysis Results Evaluation of T lymphocytes CD3 + and CD8 + in biopsies of rectal tumors collected before treatment Tumor regression score (Dworak) evaluated after surgery in rectal cancer after radio(chemo)therapy Correlations between intratumoral immune infiltrate 4

6 (T lymphocyte CD3 + and CD8 +) and tumor regression after neoadjuvant treatment for locally advanced rectal cancer Discussion 138 Chapter 11. STUDY IV - Quality of life in patients with low rectal cancer, radio(chemo)treated, that underwent a operation with curative visa Aim and objectives Material and methods Results Quality of life assessment in patients undergoing the neoadjuvant treatment, and the comparison between the results and the EORTC reference values Comparison between the quality of life of patients with stoma and anal sphincter preservation Comparison of quality of life between male and female patients Discussion 150 Chapter 12 Originality, limits and the innovative contributions of the PhD thesis 152 Chapter 13 Conclusions and perspectives 154 References 156 Used abbreviations 170 Annexes 171 Papers published and presented during the PhD studies 5

7 INTRODUCTION In the last 30 years, the management of the treatment for rectal cancer has known several important moments, represented by: - the introduction of the neoadjuvant treatment (long and shortterm radiotherapy, brachytherapy) [1,2]; - the introduction of the concept of total mesorectal excision (TME) [3]; - the introduction of the local excision for early rectal carcinoma (T0, T1) by transanal endoscopic microsurgery (TEMS); -"improvement" of new tumor staging classification (TNM 5,6,7) based on the results of clinical trials; - the introduction of microsatellite instability testing (MSI); - introduction of a "targeted therapy" through molecular testing (KRAS mutation). All these changes have made possible important progress in the treatment and evolution of the rectal tumors, causing increased operability of these tumors, decreased local recurrence under 10% [4,5], increased numbers of surgical interventions for anal sphincter preservation, thus improving the quality of life for those patients. At the moment, in numerous specialty centers, the surgical resection procedure tends to gradually replace the abdominoperineal excision of the rectum, with an increase in anal sphincter preservation from under 10% in 1970, to over 80% in 2000 [2]. The current standards of treatment in rectal cancer are the neoadjuvant radiochemotherapy for the tumors T3-T4 N0 or any T, N1-2 M0, followed by surgical interventions with a radical cancer visa[6]. One of the biggest challenges in the coming years is to identify the prognostic and predictive factors in neoplasia, and to find new approaches adapted to these criteria CURRENT STAGE OF KNOWLEDGE ABOUT RECTAL CANCER The current stage of knowledge, structured in 7 chapters, presents the current data on rectal cancer. The first two chapters address elements of surgical anatomy of the rectum and mesorectum, and epidemiology data. In chapter 2, the focus was on emphasizing the particular elements of rectal neoplasm, the significant differences between colon cancer and rectal cancer in terms of anatomically, therapeutically, as well as in what concerns the development of neoplasia. 6

8 Chapter 3 defines the locally advanced, low rectal cancer, presents the staging data of rectal tumors, both in terms of imagistics, and pathologically. It underlines the importance of accurate staging of the tumor, in the decision regarding therapy, and in the prognosis of patients. It presents the methods of diagnosis for rectal neoplasm, with a detailed presentation of the loco-regional evaluation methods, and the distant disseminations balance. It s emphasised the important role of the MRI and of the evaluation of the sphincter device s function in the therapeutic sequence decision. In chapter 4, it presented the multimodal treatment of low rectal cancer, the role of the multidisciplinary team, and the decision tree, which generally depends on: the location of the tumor in relation to the edge of the anal wall, the extension of the tumor in the rectal wall (T), and the ganglionic invasion (N). It outlines the principles and the side effects of the neoadjuvant treatment, and a review of the various factors for predicting the tumor response to the neoadjuvant therapy. The surgical treatment details the criteria for operability and resectability, the surgical options, the modalities for rectum exposure, the methods of rectal resections and of restoring the digestive continuity, emphasizing the individualized nature of the treatment. The end of this chapter contains the current ESMO (2010) and NCCN (2012) recommendations. Chapter 5 presents various factors with prognostic significance in low rectal cancer, as well as the 7th edition TNM pathological staging recommendations: the tumor deposits (also called, satellite nodules''), the circumferential resection margins, the degree of tumor regression after radiotherapy, the k-ras gene analysis, the perineural invasion, the microsatellite instability, the heterogeneity loss on chromosome 18q. Chapter 6 presented aspects of quality of life for patients with low rectal cancer, treated with radiotherapy, the risk factors for postoperative dysfunctions, as well as the previous resection of rectum syndrome. The general part ends with the description of the relationship between colorectal cancer and the immune system, the immune infiltrate in tumor cells and the importance of the immune score in colorectal cancer as a prognostic factor significantly associated with disease-free survival and overall survival. 7

9 PERSONAL CONTRIBUTIONS TO THE STUDY OF RECTAL CANCER Motivation and objectives of the PhD study Many of the patients with low rectal cancer go to the physician with stage II-III disease. Locally advanced rectal neoplasm is a common and well-known reality in our country, although the tumor becomes early symptomatic (through the presence of the rectoragy), and accessible for clinical examination (through digital rectal examination). After confirming the pre-therapeutic anatomical and pathological staging, the patient is subjected to the long-term neoadjuvant therapy (50.4 Gy administered in a dose of Gy for 28 days, associated with chemotherapy), followed by surgical treatment performed at least in 6 weeks time after the end of radiochemotherapy. Thus, the surgical treatment for locally advanced, low rectal cancer with curative visa is performed after at least 11 weeks from the time of the diagnosis. TNM clinical classification (ctnm), achieved through MRI examination or pelvic CT, is an important criteria of decision for the application of the neoadjuvant treatment and for the choice of surgical technique. The predictive factors of the tumor response to the neoadjuvant treatment are not used in the current practice. For some patients, preoperative clinical and imagistic reassessment shows a stationary status of the tumor, sometimes a further tumor development, both locally and distance (the presence of liver, lung and brain metastases). For these patients, the neoadjuvant radiotherapy delays the surgical act, and allows the occurrence of adverse reactions and side effects induced by radiotherapy. Tumor response to neoadjuvant therapy may mean tumor regression, tumor downstaging and downsize. Tumor regression (TRG) is assessed by the anatomopathological evaluation of the tumor-fibrosis ratio. There can be TRG without tumor downstage, as there also can be downstage tumors with reduced TRG. Complete tumor regression is downstage tumor ypt0, being an independent prognostic factor for the relapse-free survival. Worldwide, the research objectives in this field are aimed to assess the predictive factors of prognostic and response to the neoadjuvant therapy, useful in the therapeutic decision when choosing a targeted therapy. The recent literature shows the importance of the host immune system, of the tumor s 8

10 immunological profile, and of different immunological biomarkers in the tumor progression prognosis. Based on these findings observed in my current practice and recent specialized literature, and guided by the experts in the field, I decided to analyse the importance of the clinical factors, imagistic and immunological factors as predictors of the tumor response to the neoadjuvant treatment (radio / chemotherapy) for the locally advanced, low rectal cancer, in order to apply a surgical procedure to preserve the anal sphincter. The main objective of the study is to evaluate the tumor immune infiltrate as a predictive factor for the response to the neoadjuvant therapy in the locally advanced, low rectal cancer, and to determine the significant levels of tumor immune status before treatment, in order to establish the indication to perform the neoadjuvant treatment. The secondary objectives are: - assessing the possibilities of preserving the anal sphincter in the locally advanced, low rectal neoplasm; - drawing up a pre-therapeutic evaluation protocol of patients with low rectal cancer, required for the application of the neoadjuvant treatment for decreasing the limit of surgical resection; - the prognostic value of tumor immune infiltrate performed on tumor biopsies taken before treatment from patients with rectal cancer; - the microscopic evaluation of the tumor response to the neoadjuvant therapy through the evaluation of the tumor-fibrosis ratio; - the analysis of the quality of life in patients with locally advanced, low rectal cancer, undergoing neoadjuvant therapy. In order to achieve these objectives, I split the personal research in 4 studies: STUDY I - "Optimising the treatment for locally advanced, low rectal cancer" a retrospective-prospective study, descriptive, the experience of St. Spiridon Hospital, III Surgery Unit, Iasi, ; STUDY II - "The prognostic value of tumor immune infiltrate in patients with rectal cancer," retrospective study, Geoges Pompidou European Hospital, Paris, France, ; STUDY III - "Analysis of intratumoral immune infiltrate on the rectal tumor biopsies and its correlation with the response to the 9

11 neoadjuvant treatment" retrospective study, experience of St. Spiridon Hospital, III Surgery Unit, ; STUDY IV - "Quality of life in patients with low rectal cancer, radio(chemo)treated, that underwent a operation with curative visa ", retrospective study, III Surgical Unit, Iasi St. Spiridon Hospital, To perform these studies, I analyzed two groups of patients diagnosed with rectal cancer, who underwent radical surgery. The first group was represented by patients investigated and treated in St. Spiridon Hospital, III Surgery Unit for a period of six years, between , diagnosed with locally advanced rectal cancer, with the tumor located between 5 and 8 cm distance from the anal verge, undergoing the neoadjuvant treatment. The second group of patients comes from the database of the European Georges Pompidou Hospital Paris, France, from which were removed the patients diagnosed with rectal cancer between , who had not received the neoadjuvant treatment. My research topic tries to find explanations for the different situations that I faced in my surgical practice, as a resident doctor. Starting with the information from the bedside of the patients, I have continued the tumor evaluation in the laboratory, at the molecular level, and I looked then for the explanations for the patients evolution, thus individualizing the applied treatment. This study received a favorable ethical approval issued by the Research Ethics Committee of the University of Medicine and Pharmacy, Gr T. Popa'', Iasi. Chapter 8 - STUDY I - Optimisation of the treatment for the locally advanced, low rectal cancer - the experience of St. Spiridon Hospital, III Surgery Unit, Iasi Aim and objectives The aim of this observational, retrospective-prospective study is to evaluate the optimisation of the surgical treatment for the low rectal cancer, neoadjuvant radiotreated, in the III Surgery Unit, St.Spiridon Hospital Iasi, as surgical technique. The general objectives of the study are: - the imagistic appreciation of the neoadjuvant treatment s effectiveness, in order to descend the surgical resection limit; - the evaluation of the possibilities of preserving the anal sphincter; - the anatomopathological evaluation of the tumor response to the neoadjuvant treatment; 10

12 - the quality of the surgical procedure through the evaluation of the surgical specimen. Because the follow-up of the patients was for a short period of time, I did not want to evaluate the results of the multimodal treatment at distance. Material and methods Among the rectal neoplasm cases that undergo a surgery treatment with radical visa, from January 2006 to December 2011, from the III Surgery Unit St. Spiridon Hospital Iasi, were included in this study patients that had tumors located around the distance of 6 cm (5-8 cm) from the anal verge, or its lower limit was placed at least at 2 cm from the internal anal sphincter, and the patients underwent the long term neoadjuvant radiotherapy treatment. Among the patients diagnosed with rectal cancer, 115 cases met the inclusion criteria (table 8.I). Tabel 8.I. Demographic, clinical and pathological characteristics Patients characteristics number of patients % average age (range), years 61,66 + 9,98 (40-82) Sex M [n (%)] 72 (62,60) W [n (%)] 43 (37,40) Pretherapeutic classification (ctnm) T2 [n (%)] 19 (16,52) T3 [n (%)] 67 (58,26) T4 [n (%)] 29 (25,21) N0 [n (%)] 28 (24,34) N1 [n (%)] 54 (46,95) N2 [n (%)] 23 (20) Nx [n (%)] 10 (8,7) tumor location (cm)* 6,7 cm+ 0,9 (5-8) Histologic grade (biopsy) well 19 (16,2) moderately differentiated 76 (66,08) poorly diferentiated 6 (5,21) unknown 14 (12,17) *distance from the anal verge The patients received the neoadjuvant treatment, which consisted of external radiotherapy that varied between 30 Gy and 52.9 Gy, with a dose per fraction ranged from 1.8 to 2 Gy, 5 days per week (Monday-Friday), generally for 5 or 6 weeks, using the Linear Accelerator Clinac 2100, with 10 MV photons. The neoadjuvant chemotherapy was associated for 80 patients and consisted of: 11

13 - capecitabine (Xeloda) administered per os, the dose ranged from 1000 mg / day to 3000 mg / day, depending on the tolerance - 63 cases; - cisplatin combined with 5-FU - 8 cases; - irinotecan combination with oxaliplatin - 6 cases; - other chemotherapy regimens - 3 cases. Preoperative, the imagistic appreciation of the tumor response to the neoadjuvant treatment was not possible in all cases, due to the limitations related to the operation of these devices, and the patients addressability, or through the inability to compare the post-therapy evaluation with the pre-radiotherapy examination. Since the imagistic investigations (particularly the MRI) could not be made both pre-and post-irradiation, the indication for surgical treatment was chosen based on the initial imagistic staging. The surgical treatment of oncological radicality visa was applied to all patients (R0 resection). All patients had a complete excision of the mesorectum, carried out according to the standards [7]. For the patients whose oncological and anatomic conditions allowed, and depending on the surgeon's experience, the surgical resection procedure was performed by preserving the anal sphincter, with colo-rectal / colo-anal manual or mechanical anastomosis [8]. Low anterior resection of the rectum with total mesorectum excision (RAJR with TME) and colo-rectal anastomosis (mechanical or manual) was performed for the tumors located at 7-8 cm of the anal edge. For the lower tumors, but without the invasion of the anal sphincter, and with the distal edge of the resection of at least 1 cm, it was practiced a very low anterior resection of the rectum with complete excision of the mesorectum and mechanical colo-anal anastomosis. The surgical specimens were evaluated by the anatomopathologist, according to the 6th edition American Joint Committee on Cancer classification - TNM. It was rated the degree of tumor differentiation and the presence of venous, lymphatic and perineural invasion (VELIPI). The quality of the surgery was appreciated through macroscopic evaluation of the mesorectum, according to the criteria developed by Quirke [9], as well as through the distal and circumferential margins of surgical resection. According to the TNM classification, the response to the neoadjuvant treatment was considered as: - total (complete response - CR), when no tumor cells were found after surgery, N0; 12

14 - partial (partial response - PR), when there was a reduction in T or N stage; - lack of response (NOR), when the ptnm stage was stationary to ctnm. Results The preoperative staging, as well as the appreciation of the tumor response to the neoadjuvant treatment was made by abdominal-pelvic ultrasound, chest radiography and digital rectal examination for all patients. For 58 patients (50.43%) it was possible to assess the abdominal-pelvic CT scan and for 23 patients, pelvic MRI examination. Endorectal ultrasonography was performed for 14 patients. At surgery, 7 patients (6.08%) had liver metastases that were not seen at the initial assessment (staging M1). Pelvic MRI examination showed a reduction in tumor size (Fig. 8.20) for 17 of the 23 examined patients (73.91%), while 6 patients showed an unchanged TNM stage as against the pre-therapeutic examination. A B Fig Down-size of the tumor; pelvic MRI image, sagittal section-native T2; A- pre-radiotherapy image; B- post-radiotherapy image Surgery was performed after an interval that varied between 21 and 164 days after the completion of the neoadjuvant therapy, with an average of days. Surgical techniques were represented by: - low anterior / very low resection of the rectum (RAJR) with colorectal/colo-anal anastomosis - 38 cases (33.04%), of which 18 cases with mechanical anastomosis, and 20 cases with manual anastomosis, performed transabdominal; - extended Hartmann operation with TME - 13 cases (11.30%); 13

15 - abdomino-perineal excision of the rectum (EAP) - 64 cases (55.65%). The side protection ileostomy, Brooke type, was performed in 32 cases (84.21%) for the patients who underwent colo-anal anastomosis, or when the safety of anastomosis was doubtful (the uncertain air test). The protective ileostomy was closed at 6-8 weeks postoperatively, after assessing the integrity of the anastomosis by clinical examination (digital rectal examination) and barium enema. For 6 cases, the ileostomy closure was performed after weeks, due to the presence of a blind fistula at the level of the colo-anal anastomosis, showed by X-ray. The restoration of the digestive continuity after Hartmann operation was performed for 6 patients (46.15%), after an average period of 9 months, through mechanical anastomosis. The complete response to the neoadjuvant treatment (CR) was present in 6 cases (5.217%) for the patients who have had radiotherapy with chemotherapy (Fig. 8.28). A Fig Pathological complete response: no tumor cells A- mucus; B- fibrosis, chronic inflammation (Hematoxylin-eosin, objective x 4) The number of nodes examined ranged from 0 to 40 lymph nodes, with an average of nodes ( SD). Of these, the maximum number of positive nodes examined in a single surgical piece was of 30 nodes (30 positive nodes out of 31 nodes examined), and the average was of 4.94 positive nodes. For 45 patients, a total of more than 12 nodes were found and examined. The regression of the nodal status (cn versus ypn) was present for 16 patients from a total of 77 patients (20.77%). The small number of nodes found after surgery (0-4 nodes) in 13 patients (11.30%), the status ypn could not be assessed. T and N pretherapeutic assessment and post-radio(chemo)therapy is presented in table 8. V. B 14

16 Table 8.V. TN status before treatment (ctnm) as compared with the post-therapeutic (ptnm) before radiotherapy n (%) after radiotherapy n (%) T (5,21) T (2,60) T 2 19 (16,52) 36 (31,30) T 3 67 (58,26) 62 (53,91) T 4 29(25,21) 8 (6,95) N 0 28 (24,34) 44 (38,26) N+ 77 (66,95) 58 (50,42) N x 10 (8,69) 13 (11,30) The quality of the surgery technique was evaluated through the macroscopic appreciation of the mesorectum, and through the evaluation of the distal and circumferential resection margins. Fig Total mesorectal excision in the rectal fascia plane, posterior aspect (Prof. Dr. V. Scripcariu collection) The distal resection limit was evaluated for 42 cases (82.35% of patients for whom the anastomosis resectional procedure or the extended TME Hartmann surgery applied) (Fig. 8.33); two of them have experienced the lower edge of resection of 8 mm, 6 cases had the lower edge of resection of 10 mm, and in 36 cases it was over 10 mm. For the other 9 patients, it was noted that the circumferential resection edge is at a "distance" from the tumor invasion front. Fig Distal resection margin more than 1 cm (Prof. Dr. V. Scripcariu collection) 15

17 Discussion The management of the locally advanced, low rectal cancer was achieved through the participation of specialist surgeons in gastroenterology, pathologists, radiologists specialised in imagistic diagnosis, medical oncologists, radiotherapists. Due to the difficulties of access to imagistic methods, 10 patients (8.7%) could not be assessed the nodal status before the treatment, these patients being subjected to neoadjuvant therapy due to the following factors: the advanced ct status, the tumor location on the anterior rectal wall, the patient s age, the descent of the lower limit of resection with anal sphincter preservation. In terms of imagistics, I consider that the pre-therapeutic evaluation of patients was poor, mainly due to the difficult accessibility to these explorations (faulty device, long waiting period until the exploration moment...). Preoperative exploration for the evaluation of the tumor response to the neoadjuvant treatment was poor for the same reasons mentioned above. There was no case of complete clinical response, the re-biopsy of the tumor area after the neoadjuvant treatment has not been carried out, and nor was the tumor regression evaluated after radiochemotherapy, using the irigography. Location the tumor around the distance of 6 cm from the anal verge requires the evaluation of the anal sphincter, in order to rule out the tumor invasion, and not to turn an abdominal colostomy into a perineal colostomy, which is much more difficult to control. In the studied group, the anal sphincter function was assessed anamnestic and through digital rectal examination, as the evaluation by conventional manometry or 3D high-resolution manometry, useful in the separate evaluation of the internal and external anal sphincter, was not available. This assessment could be useful in the decision to preserve the anal sphincter, to create a reservoir, increasing the quality of life. The side effects of the neoadjuvant therapy could be managed through a symptomatic treatment or through the reduction of Xeloda dose, and in 4 cases the chemotherapy was discontinued due to nephrotoxicity (3 cases) and leucopenia grade III (1 case). In the group of the patients studied, anal sphincter preservation was achieved in 38 cases (33.04%) with a manual anastomosis / mechanical anastomosis ratio of 18/20. There is an increase in the 16

18 number of anal sphincter preservation surgeries since 2009, the main reasons being represented by: - the increasing availability of imagistic investigations of preoperative tumor staging; - the increasing accessibility to modern equipment, circular stapler and contour stapler needed for effect the mechanical anastomosis; - the learning curve for surgeons; - the increasing effectiveness of the neoadjuvant treatment on the rectal tumor, by increasing the total dose of radiation, in combination to chemotherapy to raise the tumor cells to the action of radiation, and by increasing the interval between the end of the neoadjuvant treatment and surgery. Assessment of tumor resectability and the choice of surgical technique to preserve the anal sphincter was a decision taken most often by the surgeon at the time of the operation. The evaluation of the T regression showed PR for 42 patients (36.52%). Anatomopathological complete response (pcr), present in 5.22% of patients, a lower rate than that of literature, ranging between 9% and 36% [10, 11]. This result could have the following causes: - the indication of the neoadjuvant therapy was based on the TNM pre-therapeutic staging, often poor due to the difficulties of access to the necessary investigations (especially MRI); - the type of neoadjuvant therapy that ranged in , as dictated by the medical oncologist and radiotherapist; - the non-medically causes - periods of time in which the machine did not work, making the patients to have their radiotherapy done in other centers; - the total dose of radiation applied to the first 2 years was lower, under 50 Gy; - the association of chemotherapy has been performed since 2008; - the interval between the end of radiotherapy and surgery ranged between 21 and 164 days (only one case), this interval increased in 2010 and 2011, to at least 6 weeks. A total of 7 patients (6.08%) had liver metastases at the time of the preoperative evaluation. These metastases were not highlighted during the pre-treatment evaluation. One reason could be the liver evaluation done only through ultrasounds, which may reveal metastases when they are large, which is an observerdependent investigation. For patients with tumors that require 17

19 neoadjuvant treatment, the liver should be evaluated through CT scan or MRI, thus achieving a better pre-therapeutic staging and the application of a proper treatment. In current practice, the TNM system is used to assess the response to the neoadjuvant treatment. But, the T3 or T4 stage assessed before treatment by imagistic methods can be modified after the radiotherapeutic treatment, meaning that the place of the tumor cells can be taken by post-radical fibrosis. This may explain the long-term prognosis differences of these patients. Chapter 9 - STUDY II - Prognostic value of tumor immune infiltrate for the patients with rectal cancer The aims and objectives of the study The aim of this study is to determine whether the tumor immune infiltrate, recently evaluated as the immune score, could be a useful prognostic marker for the patients with rectal cancer. The objectives are: - assessing the immune score of the surgical resection specimen, as a prognostic factor for the patients with rectal cancer; - assessing the impact on the evolution of the immune status of patients with rectal cancer, eligible for surgical treatment of first line; - assessing the impact of the immune score on the evolution of the patients with rectal cancer, eligible for surgical treatment of first line; - assessing the immune score of the surgical resection specimen as a prognostic factor for the patients with rectal cancer, eligible for neoadjuvant treatment of first line; - comparing the accuracy of the immune score with the TNM classification, as a prognostic factor for the patients with rectal cancer. Material and methods This study is a retrospective one, which includes a cohort of 144 patients diagnosed with rectal cancer in the period between , in the Laennec Hospital/ Hospital Européen GeorgesPompidou (HEGP) Paris, France, diagnosed with tumor adenocarcinoma) located at <15 cm from the anal verge, undergoing surgery with curative visa, with TME (Table 9.I). 18

20 Tabel 9.I. Clinical and pathological characteristics - HEGP cohort of 144 patients- 144 HEGP cohort characteristic No. of pts(%) Median age (range), years 69,63 (31,5-92,7) Sex M [n (%)] 82 (57) W [n (%)] 62 (43) Tumor location low rectum [n (%)] 22 (15) middle rectum [n (%)] 43 (30) upper rectum [n (%)] 79 (55) Pathological stage (pt, pn, pm) Tis [n (%)] 9 (7) T1 [n (%)] 15 (10) T2 [n (%)] 43 (30) T3 [n (%)] 62 (43) T4 [n (%)] 15 (10) N0 [n (%)] 111 (77) N1-N2 [n (%)] 33 (23) M0 [n (%)] 134 (93) M1 [n (%)] 10 (7) puicc-tnm stage 0 9 (7) I 51 (34) II 49 (34) III 26 (18) IV 10 (7) Histologic grade weell 111 (77) moderately differentiated 17 (12) poorly differentiated 3 (2) undifferentiated 1 (1) Unknown 11 (8) Relapse 41 (28,47) local 13 (9) distal 31 (21) local + distal 3 (2) Surgical resection R0 136 (94) R1 8 (5) The adjuvant therapy consisted of 5-FU for 18 patients and of radiotherapy for 3 patients (45Gy). The postoperative evaluation was performed according to the current standards, with an average follow-up period of 78.2 months. The range of progression / death varied between 0 and 247 months. From this cohort of 144 patients, I made 2 groups: - group A, represented by 111 patients - I included the patients with rectal cancer, eligible for first-line surgical treatment: TNM stage rectal tumors - I and IV, superior rectal tumors (clinico-pathological features presented in table 9.II ); 19

21 - group B, represented by 33 patients - I included the patients with rectal cancer, eligible for first-line neoadjuvant treatment: the middle and lower rectal, stage T3-T4, N0 / N +. The quantification of the immune infiltrate in the 2 tumor areas was performed using a workstation for image analysis called Spot Browser, ALPHELYS, according to the details submitted by Galon et al. [12]. The analysed images had a high resolution (740x540 pixel resolution 1.181μm/pixel), which allowed a good quality evaluation. The immune score was assessed by quantifing the infiltration of lymphocytes T CD3+ and CD8+ cells from the two areas of the tumor: the center of the tumor (CT) and the invasion front (IM - defined as the area that comprises, in equal proportions, the periphery of the tumor and the area immediately next of the tumor tissue) (fig. 9.3). Fig The establishment of immune score based on quantification of populations of CD3 + and CD8 + lymphocytes in the tumor center and the invasion front (Prof. Dr. F. Pagès collection) The cell density was measured as the number of positive cells on the surface of the tissue, separated for the two regions: CT and IM. Depending on the density, the patients were classified according to the immune score I0 - I4. The patients classified as I4 are those patients with high densities of CD3+ and CD8+ T cells, both in CT and in IM (meaning 4 high densities - 4 Hi). The patients classified as I0 are those patients with low densities of CD3+ and CD8+ T cells, both in CT and in IM (no high density-0 Hi). (Fig. 9.3) Statistical Analysis The parametric test Student's T-test and the non-parametric test Wilcoxon-Mann-Whitney were used to identify markers that show a significant expression different among the groups of patients. For 20

22 CD3 and CD8, the cut-off value of the cell density of the 2 areas (CT and IM) was the median. Kaplan-Meier curves were performed to evaluate the difference between DFS and OS. The significance of the groups of patients was calculated using the log-rank test. To determine the Hasard Ratios (HRs), I used the multivariate Cox model. HRs was corrected as suggested by Holländer et al. [13]. The test results are expressed as the value of p in determining the meaning of p <0.05. For uni-and multivariate analysis we used the statistical program R and StartView Results A.Evaluation of the immune score as the prognostic factor in rectal cancer For the 144 patients, I applied the univariate analysis for various clinical and anatomopathological parameters for DFS and OS. The results of the Kaplan Meier curves showed that the pathological stage (ptnm), the T stage, the nodal status, the LNR (lymphnode ratio, measured as the ratio between the number of positive nodes and total number of nodes examined) and VELIPI (venous, lymphatic, perineural) significantly influences the DFS and OS (p <0.05 for all of these factors). Age is a factor which influences the OS (p = ). I assessed the tumor immune infiltrate represented by the density of CD3+ and CD8+ T cells in both the CT and the IM. The patients were classified according to the density of the immune cells into Hi (high density of CD3+ T cells, or CD8+) and Lo (low density of CD3+ T cells, or CD8+). The cell density was related to the DFS and OS. A positive association was observed between CD8+ in CT, and between CD3+ in CT and the evolution of the patients (Table 9.IV). Table 9.IV. Immune infiltration for CD3+ and CD8+ cells in tumor regions and clinical outcome DFS and OS (cohort of 144 patients with rectal cancer) Nr.pts (%) Disease Free Survival Overall Survival CD3CT 5 yr % (95% CI) HR (95% CI) P value* 5 yr % (95% CI) HR (95% CI) 21 P value* Lo 47 (37.3) 48.7 ( ) 2.22 ( ) ( ) 2.43 (1.48-4) Hi 79 (62.7) 73.2 ( ) 1.0 (reference) 68.6 ( ) 1.0 (reference) CD3IM Lo 59 (45.7) 49.7 ( ) 2.47 ( ) ( ) 1.42 ( ) Hi 70 (54.3) 77.9 ( ) 1.0 (reference) 63 ( ) 1.0 (reference) CD8CT Lo 55 (42) 46.3 ( ) 2.86 ( ) ( ) 2.17 ( ) Hi 76 (58) 78.2 ( ) 1.0 (reference) 71.7 ( ) 1.0 (reference) CD8IM Lo 50 (42) 50.6 ( ) 1.69 ( ) ( ) 1.16 ( ) Hi 69 (58) 72.5 ( ) 1.0 (reference) 61 ( ) 1.0 (reference)

23 Disease-Free Survival (%) Disease-Free Survival (%) Overall Survival (%) Disease-Free Survival (%) Overall Survival (%) I associated the two analysed areas (CT and IM), and I classified the patients into 3 categories: - HiHi - characterized by high density in the cells marked in both the CT and the IM; - HiLo - characterized by increased density in the labeled cells in CT (or IM) and low density in the labeled cells in IM (or CT); - LoLo - characterized by low density in the labeled cells in both the CT and the IM. Analyzing the Kaplan-Meier curves for DFS and OS according to the density of the immune infiltrate (the association of CT and IM regions), I observed a good prognosis for the patients with increased cell density in both analyzed areas (HiHi), compared with patients showing low cell density (LoLo ), both for CD3+ and CD8+ (fig. 9.4, fig. 9.5) CD3 CT/IM LoLo (28pts) HiHi (47pts) Het (40pts) CD8 CT/IM HiHi (47 pts) Het (30 pts) 60 HiHi (47pts) LoLo (32 pts) LoLo (28pts) Het (40pts) 20 HiHi vs. LoLo: HR 3.43 ( ), pv 0.02 HiHi vs. LoLo: HR 3.07 ( ), pv HiHi vs. LoLo: HR 2.74 ( ), pv Fig Kaplan-Meier curves for the duration in months of DFS and OS according to T cell (CD3+) density evaluated in combined tumor regions (CT and IM) HiHi vs. LoLo: HR 2.06 ( ), CD3 CT/IM 100 CD8 CT/IM HiHi (47pts) Het (40pts) HiHi (47 pts) Het (30 pts) LoLo (28pts) 40 LoLo (32 pts) 3.43 ( ), pv HiHi vs. LoLo: HR 3.07 ( ), pv Fig Kaplan-Meier curves for the duration in months of DFS and OS according to T cell (CD8+) density evaluated in combined tumor regions (CT and IM) Thus, the evaluation of intratumoral immune infiltrate for the CD3+ and CD8+ T lymphocytes in CT and IM classify the patients into subgroups with distinct clinical outcomes (DFS and OS). B. Evaluation of the immune infiltrate impact on the development of the patients with rectal cancer, eligible for surgical treatment of first line 22

24 For the patients from group A, I evaluated the clinical and anatomopathological prognostic factors on DFS and OS. The univariate analysis showed the same results as for the complete cohort of 144 patients also in what concerns the DFS. In addition to this, the overall survival (OS) is influenced by the age of the patients, the pn stage and the presence of the tumor emboli (VELIPI). I obtained results with the same statistical significance for the separate assesment of the impact of the infiltrate in the CD3+ or CD8+ T cells in the 2 areas (CT and IM) on the DFS and OS, even a stronger influence than for the cohort of 144 patients (CD3 and CD8 for DFS and OS, HR> 4.5 and p <0.004). Analyzing together the immune status in the 2 regions (CT and IM) for CD3 and CD8, I obtained more important differences between the groups of patients in terms of DFS and OS, compared to each region (example: for CD8+ and combined DFS-analysis of the two regions (CT and IM) obtained HR = 5.88 between the group of patients HiHi and LoLo, unlike HR = 3.66 and HR = 1.87 between group Hi and Lo, from the separate analysis of CT, respectively, IM) (Table 9.VI). Tabel 9.VI. immune infiltration for CD3+ and CD8+ cells in tumor regions and clinical outcome - DFS and OS (cohort of 111 pts eligible for primary surgery) Disease Free Survival Overall Survival Characteristic No. of pts (%) 5 yr % (95% CI) HR (95% CI) P value* 5 yr % (95% CI) HR (95% CI) P value* CD3CT 0.53 ( ) ( ) 0,0024 Lo 34 (35.1) 59 ( ) 1, ( ) 2,37 Hi 63 (64.9) 74.1 ( ) ( ) 1.0 CD3IM 0.27 ( ) ( ) 0,0025 Lo 7 (6.9) 21.4 ( ) 3, ( ) 3,55 Hi 95 (93.1) 70.5 ( ) ( ) 1.0 CD8CT 0.27 ( ) 0, ( ) 0,0001 Lo 60 (58.8) 57.3 ( ) 3, ( ) 3,46 Hi 42 (41.2) 86.5 ( ) ( ) 1.0 CD8IM 0.54 ( ) 0, ( ) 0,0734 Lo 16 (17.2) 45.1 ( ) ( ) 1,85 Hi 77 (82.8) 70.1 ( ) ( ) 1.0 CD3CTIM 0.5 ( ) 0.49 ( ) LoLo 7 (7.7) 21.4 ( ) 4.57 ( ) 0, ( ) 5.18 ( ) 0,0002 Het 25 (27.5) 65.5 ( ) 1.15 ( ) 0, ( ) 1.88 ( ) 0,0502 HiHi 59 (64.8) 71.9 ( ) ( ) 1.0 CD8CTIM 0.44 ( ) 0.44 ( ) LoLo 13 (15.3) 38.9 ( ) 5.88 ( ) 0, ( ) 6 ( ) 0,0000 Het 40 (47.1) 56.3 ( ) 2.98 ( ) 0, ( ) 3.43 ( ) 0,0011 HiHi 32 (37.6) 85.7 ( ) ( ) 1.0 *Hazard Ratio corrected (Hollaender et al. 2004). **logrank P value corrected. # patients with immunological data available, NA-non aplicable C. Assessment of the immune score s impact on the evolution of the patients with rectal cancer, eligible for first-line surgical treatment 23

25 According to the classification of the immune score (I), the allocation of patients was as follows: I4-35%; I3-28%; I2-25%; I1-7% and I0-5%. The risk of recurrence increases from I4 to I0, with HR for DFS of 1, 1.69, 2.69, 3.1 and (the Log rank test corrected p = ) and for OS, HR 1, 2.63, 4.45, 4 and (the Log rank test corrected p = ) (Table 9.VII). Tabel 9.VII. immune score and clinical outcome DFS and OS (cohort of 111 pts eligible for primary surgery) Nr. of pts (%) Disease Free Survival 5 yr % (95% CI) HR (95% CI) P value* Overall Survival 5 yr % (95% CI) HR (95% CI) P value* Caracteristici Immune score 1.81 ( ) 0, ( ) 0,0003 I0 4 (4.8) 0 (NA-NA) NA (NA-NA) Na 0 (NA-NA) NA (NA-NA) NA I1 6 (7.2) 62.5 (32-100) 3.1 ( ) 0, ( ) 4 ( ) 0,0192 I2 21 (25.3) 53.9 ( ) 2.69 ( ) 0, ( ) 4.45 ( ) 0,0006 I3 23 (27.7) 64.3 (46-90) 1.69 ( ) NA 48.7 ( ) 2.63 ( ) 0, (70.7- I4 29 (34.9) 99.7) 1.0 (reference) 81.7 ( ) 1.0 (reference) *Hazard Ratio corectat (Hollaender et al. 2004). **logrank P value corectat Kaplan-Meier curves for DFS and OS, according to the immune score for group A of patients, are shown in Figure Fig Kaplan-Meier curves for DFS and OS according to the immune score (I0, I1, I2, I3, I4) In addition to this, where the patients are grouped as I0 and I1, significant differences are obtained between the groups of patients, depending on the immune score, for DFS (Figure 9.7.). 24

26 . Fig Kaplan-Meier curves for DFS according to the immune score (I0-1, I2, I3, I4) D. Multivariate analysis of prognostic factors for the patients with rectal neoplasm, eligible for first-line surgical treatment I applied the Cox regression for significant prognostic factors in the univariate analysis (age, T stage, N stage, RLN, VELIPI+, immune score) for DFS and OS. I combined the immune score with the clinical and anatomopathological parameters, and the immune score with LRN remained significant for DFS and OS (Table 9.VIII). Table 9.VIII. Multivariate Cox Proportional Hazard Analysis for DFS and OS among patients with rectal cancer eligible for primary surgery Disease-Free Survival Overall Survival HR (95% CI) P value HR (95% CI) P value Model before stepwise (stepaic) selection Age 1.14 ( ) ( ) Tumor (T) stage 1.63 ( ) ( ) N stage 0.42 ( ) ( ) LNR (ratio positive lymph nodes) 4.03 ( ) ( ) VELIPI+ (tumor emboli) 0.79 ( ) ( ) Immunoscore* (I0 to I4) 0.62 ( ) ( ) Model after stepwise (stepaic) selection Age 1.66 ( ) Tumor (T) stage 1.59 ( ) LNR 1.88 ( ) ( ) Immunoscore* (I0 to I4) 0.62 ( ) ( ) UICC TNM Staging 1.43 ( ) ( ) Immunoscore* (I0 to I4) 0.55 ( ) ( ) NOTE: All categorical covariates were transformed into numeric codes before they entered into the Cox model. Note: all categorical covariates were transformed into numeric codes before they entered into Cox model; HR-hazard ratio; AIC-Akaike information criterion *factor corrected with Holländer et al (2004) E. Evaluation of the immune infiltrate s impact on the evolution of the patients with rectal cancer, eligible for first-line neoadjuvant treatment (chemoradiotherapy) 25

27 I evaluated the density of CD3+ and CD8+ T cells in CT and IM in the patients from group B. I divided the patients according to the average cell density in Hi Lo and appreciated the local and distal recurrence in these patients, depending on cell density. I applied the T- Student test, respectively, the Wilcoxon-Mann-Whitney test. For the patients with increased density of CD3+ and CD8+ in the two examined regions, I observed a low rate of local recurrence (Fig. 9.8), while for the patients with low densities there is a high relapse rate. The density of the immune infiltrate in situ can be correlated with the presence of relapse after surgical treatment with curative visa. Fig Comparison of the mean densities of CD3+ and of CD8+ cells in the CT and IM regions of tumor from patients eligible for neoadjuvant treatment with (black bars) or without (white bars) tumor recurrence Discussion With this retrospective study, applied to a cohort of patients with rectal cancer who have not received neoadjuvant treatment, I 26

28 confirmed the immune infiltrate s impact (represented by CD3+ and CD8+ T cells) on the prognosis of these patients. A recent study, of meta-analysis type, summarises the importance of cytotoxic T cells and Th1 lymphocytes in the development of patients with cancer [180], including melanoma, breast neoplasm, urothelial, renal, prostate, lung and colorectal cancer. The applicability of this research has been translated into clinical evaluation by introducing the concept of immune score (from I0 to I4), defined on the basis of the density of the two lymphocytic populations (CD3+ and CD8+), located both in the center of the tumor, as well as in the tumor invasion front. The SI has not been yet validated in rectal cancer, where the therapeutic management also includes the neoadjuvant treatment that can change the patient s prognosis. The 144 studied patients are part of a historical cohort of patients, operated when the total mesorectum excision was routinely used, but the neoadjuvant treatment was not included in the treatment guidelines. I divided the cohort into 2 groups, and have evaluated the immune infiltrate s importance as a prognostic factor for DFS and OS for the 2 groups. The univariate analysis of clinical, anatomopathological factors and of the immune infiltrate (CD3+, CD8+) showed a favorable effect of the immune factor in the evolution of patients for both DFS and OS. This study revealed SI s prognostic power for the patients who are eligible for first-line surgical treatment. Thus, for the patients with low SI (12% of patients, I0 and I1), the prognosis is very poor, and for the patients with high SI (35% of patients, I4), the prognosis is very good. To emphasize the importance of SI regarding the observed statistical association, I grouped the patients with SI 0 and 1 (both groups have a very poor outcome after surgery), leading to an increased number of analysed patients and of relapse events. The multivariate analysis in which group I0 and group I1 were merged (Fig. 9.7), showed again the prognostic power of SI. In the multivariate analysis, I evaluated the SI with the TNM classification. Applying the multivariate Cox regression for the UICC-TNM stage, the immune score remains a prognostic factor with an important significance in rectal cancer, strongly significantly associated with DFS, superior to the TNM classification, in the case of the patients who, according to current standards, are eligible for first-line surgical treatment. On overall 27

29 survival, the immune score is an important prognostic factor in this group of patients. Along SI, in the multivariate analysis the LNR, which is a prognostic factor with a superior conventional nodal assessment, also has an important role, even after the neoadjuvant treatment. The LNR, like the SI, reflects aspects of antitumor immune function. Lymph nodes should be evaluated by their anti-tumor function, by activating and enhancing the lymphocytes, and should not be considered merely a tumor mechanical filter. For group B of patients who, according to the current treatment guidelines, should receive neoadjuvant radio(chemo)therapy, I confirmed that the in situ immune status influences the progression of the disease. Thus, the patients without local or distal recurrence showed a significantly higher density of CD3+ and CD8+ lymphocytes in both the examined regions The confirmation of the results requires evaluating of a multicentric cohort of patients, using the same strategy as the one validated for colon cancer [14]. Chapter 10 - STUDY III - The analysis of tumor immune infiltrate on the rectal tumor biopsies, and the correlation with the response to the neoadjuvant treatment The aim and objectives of the study The involvement of the immune system in the tumor response to radiotherapy is suggested since 1979 [15], showing that the tumor s decrease is not only the result of the direct effect of radiation on tumor cells, but that it is also influenced by the host s immune response. The aim of this study is to evaluate the tumor immune infiltration as predictive factor for the response to the neoadjuvant radio(chemo)therapy in patients with locally advanced, low rectal cancer. The objectives are: - correlations between the tumor immune infiltrate of the biopsies performed at the moment of the diagnosis and various prognostic factors; - correlations between the tumor immune infiltrate of the biopsies performed at the moment of the diagnosis and tumor regression, according to TNM staging (ctnm vs. yptnm); - correlations between the tumor immune infiltrate of the biopsies performed at the moment of the diagnosis and tumor regression, according to Dworak score. 28

30 Material and methods I evaluated biopsies made preteraputic for 55 cases. The other cases could not be assessed, because the tumor biopsy block was either not found (the patient was diagnosed and biopsied in another hospital), or it was insufficient for analysis. A.The marks of the CD3+ and CD8+ T cells of the rectal biopsies before treatment I performed the automated immunohistochemistry technique using the BENCHMARK XT Roche-Ventana machine and the protocols used and validated by the 15 INSERM team, Laboratory of Immunology, European Hospital Georges Pompidou, Paris. I analysed, quantitatively and qualitatively, the degree of infiltration of CD3 and CD8 T cells in the biopsied tumor tissue. The monoclonal antibodies used were as follows: - anti-cd3 rabbit monoclonal antibodies CONFIRM anti-cd3, clone 2GV6, IgG, ROCHE-VENTANA producer, which detects the expression of CD3 antigen in the cytoplasmic membrane of normal and tumor cells; - anti-cd8 mouse monoclonal antibodies clone C8/144B, IgG1, DAKO producer, which identifies the cytotoxic T cells / suppressor. The secondary antibody used was poly VMS. The acquisition of the electronic slides was performed using the acquisition and automated processing system HAMAMATSU, NanoZoomer Digital Pathology (virtual microscopy) into the Batch mode with x 20 resolution. I got the image of virtual microscopy and tissue labeled with CD3, respectively, CD8, then I selected, with the anatomopathologist, the tumor tissue area that I later analysed (Fig. 10.9). Fig Virtual microscopy images - the tumor zone to be analyzed (x5) I analysed the scanned images using the DEFINIENS Developer XD program, and got various qualitative and quantitative pieces of information. Basically, this analysis involves the 29

31 quantification of the absolute number of tumor and normal cells with positive immunohistochemical marker. The final results (quantitative and qualitative) are organised in an Excel table, which contains the following data, for each analysed quadrant: - the presence of tumor cells in the measured surface; - the number and density of labeled cells in the tumor tissue, such as intraglandular and tumoral stroma; - the number and density of labeled cells in healthy tissue; - the detected and analysed tissue area (tumoral and healthy); - the intensity s distribution of the cellular markings. I extracted, for each case, the quadrants in which the tumor glands were not detected, and I calculated the average density of the labeled cells CD3+ and CD8+ for the quadrans in which the tumor was detected and analysed. I have obtained, for each case of biopsy, an average density of imunomarked cells for each marker (CD3 and CD8). I have calculated, in the same way, the density score for CD3+, respectively CD8+ cells, for the quadrans containing more than 40% of the examined tissue, as well as the most infiltrated 3 quadrans, for each case. B. The assessment of anatomopathological tumor response to the neoadjuvant radio(chemo)therapy For the 55 patients for whom I performed IHC, I selected the hematoxylin-eosin stain slades made from the surgical resection piece, from the Department of Pathology and Prosecture of St. Spiridon Hospital, Iasi. I evaluated these slades microscopically, with the anatomopathologist, and have found the following factors: - the evaluation of the degree of fibrosis; - the evaluation of the degree of inflammation; -the evaluation of vascular status (the presence of arteriopathy). I performed the anatomopathological evaluation of the tumor response to the neoadjuvant treatment under Dworak score, which semiquantitatively classifies the tumor response to the neoadjuvant therapy. Between 4 and 10 hematoxylin-eosin staining slides were analysed for each case, with an average of 5.7 slides / case. No node response to radiochemotherapy was evaluated. Statistical analysis The obtained results were entered into a database, Microsoft Excel software (Microsoft Corporation, Redmond, WA) 2007 version. The data used were of two types: categorical and quantitative. In reporting, the expression of the quantitative data was 30

32 under average form (+ Standard Deviation), possibly presenting the extreme values. For the univariate analysis, the quantitative variables were transformed into qualitative variables, with a cut-off value determined by the median, winning two categories: higher or lower than the median. To evaluate the correlation between the efficiency of the neoadjuvant treatment and the cellular density of CD3+ and CD8+ lymphocytes, as well as the relationship between the degree of fibrosis and other clinical and anatomopathological parameters, the T-student test and the Wilcoxon-Mann-Whitney (ANOVA) test have been used. The limit of statistical significance in this study was considered to be 5% (p <0,05). Results A. Evaluation of CD3+ and CD8+ T lymphocytes in the rectal tumor biopsies taken before treatment I evaluated the density and distribution of CD3+ and CD8+ T cells in the biopsies performed before the neoadjuvant treatment using the automatic immunohistochemistry technique. In the figure it is noted that the CD3 and CD8 mark was performed both in the tumor tissue area (intraglandular and stroma), and in the normal tissue area. limphnode Normal tissue Tumor glande Fig Immunohistochemistry in rectal biopsies before neoadjuvant therapy for:a- lymphocytes T CD3+ (x 5) ; B- lymphocytes T CD8+ (x5) Analysing each quadrant, the cell density in the tumor tissue ranged from very high to light infiltrated, both for the CD3+ and for the CD8+ (fig.10.18). 31

33 Fig CD3 + cell density variation on the zone examined (x5); in red, CD3 + lymphocytes The average density of marked cells in each case, for all quadrants, showed the following values: - for CD3+ - cell density ranging from 0.11 to 2483 cells/mm2 - for CD8+ - cell density ranging from 13.4 to cells/mm2 B. The tumor regression score (Dworak) evaluated after surgery in rectal cancer treated with radiochemotherapy The degree of tumor regression appreciated by postradic fibrosis was as follows: - TRG 0 present for 4 patients (fig A)4 - TRG 1 present for 4 patients (fig B) - TRG 2 present for 18 patients (Fig C) - TRG 3 present for 25 patients (Fig D) - TRG 4 present for 4 patients (fig E) A B HE ob x4, TRG- 0 HE ob x4, TRG-1, inflammation + 32

34 C HE ob x4, TRG- 2 HE ob x4, TRG- 3, inflammation ++ D E Fig The tumor regression grade after neoadjuvant treatment (Dworak score) A. TRG 0; B. TRG 1; C. TRG 2; D.TRG 3; E.TRG 4 HE-hematoxylin-eosin,TRGtumor regression grade HE ob x4, TRG- 4, inflammation ++ In 4 cases no tumor cells were identified throughout the area examined, and in 4 cases there was no postradic fibrosis identified (Fig ). Fig The fibrosis grade, according to Dworak score The imagistic evaluation of tumor response to the neoadjuvant therapy (before treatment-ct versus anatomopathological-pt) is shown in Table 10.II. 33

35 Table 10.II. ct (endorectal ultrasound/ct/rmn) compared to pt after neoadjuvant treatment c T(CT/RMN) p T0 p T1 p T2 p T3 p T4 TOTAL T T T TOTAL The response to the neoadjuvant treatment according to Dworak score and pt status is shown in Table 10.III Table 10.III. pt stade and Dworak score p T TRG 0 TRG 1 TRG 2 TRG 3 TRG 4 pt p T p T p T p T4 1 (MTS) 1 1 (MTS) 1 0 The response to the neoadjuvant treatment according to Dworak score and the time between the end of the neoadjuvant treatment and surgery is presented in Table 10.IV. Table 10.IV. Dworak score and the time after neoadjuvant treatment TIME CRT/ OPERATION (WEEKS) TRG 0 TRG 1 TRG 2 TRG 3 TRG It was also appreciated the degree of inflammation and the presence of postradic arteriopathy. In 49 of the cases, the postradic arteriopathy was present, and the inflammation was assessed as 34

36 being present in the majority of cases (54 cases), being of various degrees of intensity, noted with a,,+'' for 24 cases, with '+ +' for 23 cases and with '+ + +' for 7 cases, the degree of inflammation being assessed by the anatomopathologist. C. Correlation between immune infiltrate (CD3+ and CD8+) and tumor regression after the intratumoral neoadjuvant treatment for the locally advanced rectal cancer The neoadjuvant treatment response was assessed by: - down-stage of the tumors, defined as the reduction of the ctnm stage after radiotherapy (ctnm vs. ptnm). The tumour response was considered as: total (complete response - CR) when no tumor cells were found after surgery, N0; partial (partial response - PR), when there was a reduction in the T or N stage; lack of response (nor), when the ptnm stage is stationary from ctnm. - the degree of tumor regression according to the Dworak score (TRG). The 55 patients, for whom I analysed the density of CD3 and CD8 cells in the biopsies taken before surgery, were divided, according to the average value of cell density in patients well infiltrated (Hi) and patients poorly infiltrated (Lo). The results were expressed as the average cell density for the most infiltrated quadrans. I applied the T-Student test, respectively, the Wilcoxon test to evaluate the correlations between various prognostic factors (clinical, anatomopathological) and the immune status. I analysed the following factors: age, sex, ct, cn, ptnm, pt, pn, pm, T tumor regression, tumor differentiation, tumor inflammation and vascular invasion (assessed on the piece of tumor biopsy), the Dworak score. The density of CD3+ and CD8+ T cells assessed on the tumor biopsies vary with age, meaning that younger patients have a higher cell density. This difference is significant for the three age groups studied. Assessing the immune status by sex, grade ct, pt, pn, ptnm, tumor differentiation, tumor inflammation and vascular invasion, I observed the differences between the groups of patients well infiltrated and the populations with low immune infiltration, but these differences were not statistically significant. 35

37 Statistically significant differences were obtained in order to assess the immune status by cn, for CD3+ lymphocytes (Fig ). Fig Correlation between the density of CD3 + lymphocytes and pn; left T-student test ; right Wilcoxon Mann-Whitney test; the statistic test value is referred in red. Assessing the tumor regression in terms of tumoral downstage, dichotomised high (Hi) and low infiltrated patients (Lo) have differences in the density of the imunomarked cells, depending on the response to the neoadjuvant treatment. Thus, for the subset of patients who are considered non-responders, prevails a low density in CD3+ (70% of cases, the Fisher Exact Test p = 0.018). The same tendency was observed for CD8+, but without statistical significance (Fig ). Fig Correlation between tumor regression and immune infiltrate CR-complete response, PR partial response, nor-non-responder 36

38 Regarding the Dworak score, the cellular density reported to TRG is shown in Table 10.V. Table 10.V. Dworak score classification and immune infiltration; TRG- tumor regression grade, SD- standard deviation I have not found statistically significant differences between the groups of tumor regression (TRG) and the density of immune cells (Fig ). Fig Correlation between tumor regression grade according to Dworak Score and CD8 + immune infiltrate 37