Primary antibodies used in the multiplex panels for the detection of the different immune populations

Change in GrzB expression min paired samples post-NACT

Immune cells distribution in post-placebo and post-BIBF tumors

Patients and diseases baseline characteristics in the CHIVA neoadjuvant trial

Feature plots of relevant immune populations in pre- and post-NACT clusters

Purpose: This study investigates changes in CD8+ cells, CD8+/Foxp3 ratio, HLA I expression, and immune coregulator density at diagnosis and upon neoadjuvant chemotherapy (NACT), correlating changes with clinical outcomes.Experimental Design: Multiplexed immune profiling and cell clustering analysis were performed on paired matched ovarian cancer samples to characterize the immune tumor microenvironment (iTME) at diagnosis and under NACT in patients enrolled in the CHIVA trial (NCT01583322).Results: Several immune cell (IC) subsets and immune coregulators were quantified pre/post-NACT. At diagnosis, patients with higher CD8+ T cells and HLA I+-enriched tumors were associated with a better outcome. The CD8+/Foxp3+ ratio increased significantly post-NACT in favor of increased immune surveillance, and the influx of CD8+ T cells predicted better outcomes. Clustering analysis stratified pre-NACT tumors into four subsets: high Binf, enriched in B clusters; high Tinf and low Tinf, according to their CD8+ density; and desert clusters. At baseline, these clusters were not correlated with patient outcomes. Under NACT, tumors were segregated into three clusters: high BinfTinf, low Tinf, and desert. The high BinfTinf, more diverse in IC composition encompassing T, B, and NK cells, correlated with improved survival. PDL1 was rarely expressed, whereas TIM3, LAG3, and IDO1 were more prevalent.Conclusions: Several iTMEs exist during tumor evolution, and the NACT impact on iTME is heterogeneous. Clustering analysis of patients unravels several IC subsets within ovarian cancer and can guide future personalized approaches. Targeting different checkpoints such as TIM3, LAG3, and IDO1, more prevalent than PDL1, could more effectively harness antitumor immunity in this anti-PDL1-resistant malignancy.

OBJECTIVE:To evaluate the prognostic impact of circulating tumor DNA (ctDNA) detection at diagnosis (T0) and its early decrease after one cycle (T1) of neoadjuvant chemotherapy (NACT) in patients with advanced epithelial ovarian cancer (EOC) included in the CHIVA trial (NCT01583322). METHODS:Blood samples were collected at T0 and before each administration of NACT. Circulating tumor DNA detection was performed by next-generation sequencing. Multivariate analysis was performed. A p-value of 0.05 was considered significant. Progression-free survival (PFS) and overall survival (OS) were compared between groups defined by ctDNA kinetic profile. Cox survival model was used to search variables associated with PFS and OS. Kaplan-Mayer curve was used to graphically express the differences in PFS and OS. A log-rank test compared the two curves. RESULTS:188 patients were included. Blood samples were available for 168 patients at T0 and for 160 patients at T0 and T1 to assess ctDNA ratio kinetics. At T0, 107 patients (63.7 %) had detectable ctDNA. At T1, 137 (85.6 %) patients had negative ctDNA or a decrease of more than 80 %. There was a significant benefit in either PFS (p = 0.0017) or OS (p = 0.0036) in favor of early decrease of ctDNA ratio. A favorable decrease was associated with a greater likelihood of being able to perform CRS (OR: 3.94 (CI95 % 1.45-10.70), p = 0.0074). CONCLUSIONS:Early decrease of ctDNA ratio can provide prognostic information early in the management of patients, allowing a more accurate information to patients and an early preparation for CRS (prehabilitation).

Positivity of immune coregulators in pre- and post-NACT clusters

Change in ieCD8+ and sCD8+ populations in paired samples with NACT

Background:Despite advances in radiation techniques, radiation cystitis (RC) remains a significant cause of morbidity from pelvic radiotherapy, which may affect patients' quality of life (QoL). The pathophysiology of RC is not well understood, which limits the development of effective treatments. Objective:The Radiotoxicity Bladder Biomarkers study aims to investigate the correlation between blood and urinary biomarkers and the intensity of acute RC symptoms and QoL in patients undergoing localized prostate cancer radiotherapy. Methods:This study included patients with low- or intermediate-risk localized prostate cancer who were eligible for localized radiotherapy. Blood and urinary biomarkers were analyzed before radiotherapy was initiated and at weeks 4 and 12 of radiation therapy. Patients completed questionnaires related to RC symptoms and QoL (International Prostate Symptom Score and Functional Assessment of Cancer Therapy-Prostate [FACT-P]) using a digital remote monitoring platform. The information was processed by means of an algorithm, which classified patients according to the severity of symptoms and adverse events reported. Levels of blood and urinary biomarkers were tested with the severity of acute RC symptoms and patient-reported QoL. Results:A total of 401 adverse events questionnaires were collected over the duration of this study from 20 patients. The most frequently reported adverse events at week 4 were pollakiuria, constipation, and diarrhea. In comparison with baseline, the mean FACT-P score decreased at week 4. A significant increase in the proportion of M2 phenotype cells (CD206+, CD163+, CD204+) at W12 compared to W0 was observed. An increase in serum and urine levels of macrophage colony-stimulating factor (M-CSF), hepatocyte growth factor, and macrophagic inflammatory protein was observed at week 12 compared to baseline levels. Baseline serum and urine M-CSF concentrations showed a significant negative correlation with FACT-P scores at weeks 4 and 12 (r=-0.65, P=.04, and r=-0.76, P=.02, respectively). Conclusions:The Radiotoxicity Bladder Biomarkers study is the first to explore the overexpression of inflammatory proteins in blood and urine of patients with symptoms of acute RC. These preliminary findings suggest that serum and urine levels of hepatocyte growth factor, M-CSF, and macrophagic inflammatory protein, as well as macrophage polarization, are mobilized after prostate radiotherapy. The elevated M-CSF levels in serum and urine at baseline were associated with the deterioration of QoL during radiotherapy. The results of this study may help to develop mitigation strategies to limit radiation damage to the bladder.

BACKGROUND:Ovarian cancer (OC) remains one of the most challenging and deadly malignancies facing women today. While PARP inhibitors (PARPis) have transformed the treatment landscape for women with advanced OC, many patients will relapse and the PARPi-resistant setting is an area of unmet medical need. Traditional immunotherapies targeting PD-1/PD-L1 have failed to show any benefit in OC. The CD47/TSP-1 axis may be relevant in OC. We aimed to describe changes in CD47 expression with platinum therapy and their relationship with immune features and prognosis. METHODS:Tumor and blood samples collected from OC patients in the CHIVA trial were assessed for CD47 and TSP-1 before and after neoadjuvant chemotherapy (NACT) and multiplex analysis was used to investigate immune markers. Considering the therapeutic relevance of targeting the CD47/TSP-1 axis, we used the CD47-derived TAX2 peptide to selectively antagonize it in a preclinical model of aggressive ovarian carcinoma. RESULTS:Significant reductions in CD47 expression were observed post NACT. Tumor patients having the highest CD47 expression profile at baseline showed the greatest CD4+ and CD8+ T-cell influx post NACT and displayed a better prognosis. In addition, TSP-1 plasma levels decreased significantly under NACT, and high TSP-1 was associated with a worse prognosis. We demonstrated that TAX2 exhibited a selective and favorable biodistribution profile in mice, localizing at the tumor sites. Using a relevant peritoneal carcinomatosis model displaying PARPi resistance, we demonstrated that post-olaparib (post-PARPi) administration of TAX2 significantly reduced tumor burden and prolonged survival. Remarkably, TAX2 used sequentially was also able to increase animal survival even under treatment conditions allowing olaparib efficacy. CONCLUSIONS:Our study thus (1) proposes a CD47-based stratification of patients who may be most likely to benefit from postoperative immunotherapy, and (2) suggests that TAX2 is a potential alternative therapy for patients relapsing on PARP inhibitors.

Primary antibodies used for IHC monomarker detection

Change in immune coregulators expression in intraepithelial (ie+) and stromal compartment (s+) in pre- and post-NACT tumors

Abstract Objective Normofractionated (NF) radiation therapy (RT) is still standard for loco-regional early breast cancer (EBC) in many countries. HypoG-01, a UNICANCER, non-inferiority, open-label, multicenter, randomized phase III trial (NCT03127995), conducted in parallel with the DBCG Skagen trial 1 (NCT02384733), evaluated hypofractionated (HF) RT with 40 Gy/15 fr (2.67 Gy/fr) versus NF RT 50 Gy/25 fr (2.0 Gy/fr). This is the first report of oncological outcomes, secondary endpoints of the trial. Methods Patients (pts) ≥18 years old were operated for T1-3, N0-3, M0 breast cancer. All pts received nodal and thoracic wall or breast RT. Tumor-bed boost (sequential or simultaneous) and nodal levels treated were decided according to local guidelines. Target volumes were delineated according to the ESTRO consensus. RT technique was left at the investigator’s discretion. Stratification factors included treating center, type of surgery, number of positive nodes and body mass index. The primary endpoint, reported previously, was time to occurrence of arm lymphedema. Oncological outcomes included locoregional relapse free survival (LRFS), invasive disease-free survival (IDFS), distant disease-free survival (DDFS), breast cancer specific survival (BCSS) and overall survival (OS) as defined per DATECAN guidelines. All time to cancer related endpoints were defined as starting from the date of randomization until the event. The primary statistic test was stratified one-sided logrank test: 5% significance level in per-protocol population (PPP) with a pre-specified non-inferiority margin of 1.545. Stratified Cox model was used for calculating hazard ratios of oncological outcomes. Results In total, 1265 pts were randomized to HF versus NF RT from Sep 2016 to Mar 2020 with 1221 in the PPP (HF group 614 pts (50.3%); NF group 607 (49.7%)), 5 consent withdrawn and 39 major deviations. Median age was 58 years (range 23-91), surgery included mastectomy (501 pts; 45%) and axillary clearance (921 pts; 82.8%). Sequential (67.8%) or simultaneous integrated (32.2%) tumor-bed boost was used in 596 pts (48.8%). With a median follow-up for OS in PP analysis of 3.8 years (95% CI 3.3 to 3.9), HF was non-inferior to NF RT in terms of lymphedema (HR=1.07; 90% CI 0.86-1.34, non-inferiority p=0.003). Table 1 reports the hazard ratio and 3-year survival rates for the oncological endpoints LRFS, IDFS, DDFS, BCCS and OS. There was no sign of disadvantage for any of these endpoints comparing HF to NF. Additional analyses will be presented at the meeting. Conclusion Moderately HF loco-regional RT is non-inferior to NF RT in terms of lymphedema risk in EBC and does not show a disadvantage in terms of LRFS, IDFS, DDFS, BCCS and OS with a median follow-up of 3.8 years. Together with the Skagen 1 trial, this study provides level 1A evidence supporting the use of 40 Gy/15 fr for loco-regional radiation therapy in EBC with respect to arm lymphedema risk; Table 1: Oncological outcomes according to treatment arm in the per protocol analysis HF: Moderately hypofractionated radiation therapy; NF: Normofractionated radiation therapy; n:number of patients; N: number of events Citation Format: Sofia Rivera, Eleni Karamouza, Youlia Kirova, Séverine Racadot, Mohamed Benchalal, Jean-Baptiste Clavier, Claire Charra-Brunaud, Marie-Eve Chand-Fouche, Delphine Argo-Leignel, Karine Peignaux, Ahmed Benyoucef, David Pasquier, Philippe Guilbert, Julien Blanchecotte, Agnès Tallet, Adeline Petit, Guillemette Bernadou, Xavier Zasadny, Claire Lemanski, Jacques Fourquet, Emmanuelle Malaurie, Honorine Kouto, Carole Massabeau, Alexandre Henni, Pauline Regnault, Aurélie Belliere, Yazid Belkacemi, Magali Le Blanc-Onfroy, Julien Geffrelot, Jean-Briac Prevost, Marie Bergeaud, Assia Lamrani-Ghaouti, Amandine Ruffier, Naima Bonnet, Stephanie Wong, Christine Rossier, Thomas Brion, Pierre Maroun, Claire Petit, Guillaume Auzac, Stefan Michiels. First oncological outcomes of HypoG-01: a UNICANCER phase III trial comparing loco-regional hypo vs normo fractionated radiation therapy in early breast cancer patients [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-19-10.

Correlation among the different immune coregulators in pre- and post-NACT tumors

Abstract Introduction Cosmetic evaluation after breast cancer treatment is a clinical indicator of toxicity. User bias and inter-subject variability hamper this objective. To address this limitation, a deep learning approach was developed on the basis of the HYPOG-01 trial (NCT03127995), a phase III trial comparing hypo to normo-fractionated radiotherapy (RT) in breast cancer patients requiring nodal irradiation. Material and Methods Cosmetic outcomes using the Harris scale were assessed by a radiation oncologist using a 4-level rating system from excellent to poor. This evaluation involved photographs from 581 female patients included in the intention-to-treat population of the HYPOG-01 study analysis (mastectomy/pamectomy and non-usable cases excluded). Front images were taken with the arms along the body at baseline, 3-weeks after radiotherapy start, end of treatment, then 6 months and every year after randomization up to 5 years. Comparing manual landmark annotation with the semi-automated software BCCT.core©, the agreement rate was moderate, with an intra-class correlation coefficient (ICC) of 0.66 (95%CI 0.57-0.73). The dataset consisted of 2,348 images, which were divided into exclusive patient-based training (1,661), validation (308), and testing (377) datasets. The distribution of Harris scores in the dataset was highly imbalanced: 7% excellent, 33% good, 45% fair, and 15% poor. Nipple landmarks were used as landmarks to address picture acquisition variations by cropping and resizing images to 224 × 224 resolution. Feature extraction was performed using a Swin-TransformerV2, an advanced attention-based vision model initially trained on ImageNet. Newly integrated fully connected layers categorized the extracted features. The model was trained for 300 epochs, and the highest F1-score model was selected. Asymmetry in texture, marks, and breast geometry played a crucial role in Harris scoring. To improve the model's performance, we generated symmetrical images from the region of interest, averaging them with the original images, and we incorporated the timestamps from image captures as an additional influencing factor. We employed techniques such as contrast modulation, lighting adjustments, and geometric transformations to augment the dataset, introducing additional variations and enhancing the model's generalization and accuracy. Results The performance of our model was evaluated using balanced binary classification, multi-class accuracy, and F1-score. Comparatively, our model performed similarly to BCCT in terms of overall accuracy but demonstrated better performance in separating multiple classes, as indicated in Table 1. In Table 2, we present a confusion matrix that provides insights into the model's performance Conclusion The proposed solution simplifies and accelerates the evaluation process by utilizing only two nipple landmarks, surpassing manual and semi-automated tools. This advancement opens doors for automated, large-scale cosmetic toxicity evaluation. Continuous improvement and validation contribute to its robustness and reinforce its significant impact in assessing cosmetic outcomes after breast cancer treatment. Table. Evaluation of performance on the test set (only on cases with evaluation by BCCT (327 images)). Table. Confusion matrix between our predictions and the labels on the test set (only on cases with evaluation by BCCT (327 images)). Citation Format: Alexandre Cafaro, Amandine Ruffier, Gabriele Bielinyte, Youlia Kirova, Séverine Racadot, Mohamed Benchalal, Jean-Baptiste Clavier, Claire Charra-Brunaud, Marie-Eve Chand-Fouche, Delphine Argo-Leignel, Karine Peignaux, Ahmed Benyoucef, David Pasquier, Philippe Guilbert, Julien Blanchecotte, Agnès Tallet, Adeline Petit, Guillemette Bernadou, Xavier Zasadny, Claire Lemanski, Jacques Fourquet, Emmanuelle Malaurie, Honorine Kouto, Carole Massabeau, Alexandre Henni, Pauline Regnault, Aurélie Belliere, Yazid Belkacemi, Magali Le Blanc-Onfroy, Julien Geffrelot, Jean-Briac Prevost, Eleni Karamouza, Stefan Michiels, Marie Bergeaud, Assia Lamrani-Ghaouti, Sami Romdhani, Alexis Bombezin-Domino, Nikos Paragios, Sofia Rivera. Cosmetic assessment in the UNICANCER HypoG-01 trial: a deep learning approach [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-21-03.