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The lack of multi-omics cancer datasets with extensive follow-up information hinders the identification of accurate biomarkers of clinical outcome. In this cohort study, we performed comprehensive genomic analyses on fresh-frozen samples from 348 patients affected by primary colon cancer, encompassing RNA, whole-exome, deep T cell receptor and 16S bacterial rRNA gene sequencing on tumor and matched healthy colon tissue, complemented with tumor whole-genome sequencing for further microbiome characterization. A type 1 helper T cell, cytotoxic, gene expression signature, called Immunologic Constant of Rejection, captured the presence of clonally expanded, tumor-enriched T cell clones and outperformed conventional prognostic molecular biomarkers, such as the consensus molecular subtype and the microsatellite instability classifications. Quantification of genetic immunoediting, defined as a lower number of neoantigens than expected, further refined its prognostic value. We identified a microbiome signature, driven by Ruminococcus bromii, associated with a favorable outcome. By combining microbiome signature and Immunologic Constant of Rejection, we developed and validated a composite score (mICRoScore), which identifies a group of patients with excellent survival probability. The publicly available multi-omics dataset provides a resource for better understanding colon cancer biology that could facilitate the discovery of personalized therapeutic approaches.

Background: The detection of breast lesions through self-examination or during screening tests is a frequent finding. Breast biopsy is required in case of radiologically suspect lesions, bestowing a high burden on both patients and national healthcare system, since only one every four biopsies is breast cancer (BC). To date, the assessment of circulating biomarkers failed to demonstrate clinical utility in the early diagnosis of BC, for its suboptimal accuracy and difficult transferability to clinical practice. The combination of novel cutting-edge methods for the assessment of circulating analytes in an integrated multiomic classifier may overcome such limitations, possibly allowing liquid biopsy to become a novel noninvasive procedure for the differential diagnosis of BC. Design: In the RENOVATE trial (NCT04781062), women with suspect (BI-RADS-4/5) breast lesions ≤ 2 cm (cT1) are asked, before biopsy, to donate ~ 35 mL of blood collected in four dedicated tubes and ~ 50 mL of urine at the Diagnostic Senology Unit of Ospedale Policlinico San Martino (Genoa, IT). Plasma cell-free DNA methylation and copy number alterations are assessed in a cohort of patients diagnosed with early BC and a matched set of patients with histologically proven benign lesions through cell-free methylated DNA immunoprecipitation and high throughput sequencing (cfMeDIPseq), as well as ultra-low pass whole genome sequencing (ULP-WGS). Thanks to the volume and quality of our sample set, other experimental techniques will be tested as well. Results from cfMeDIP-seq and ULP-WGS, possibly in combination with other findings, will be integrated in a unique classifier for the noninvasive differential diagnosis of suspect breast lesions. Eligibility criteria: Patients with radiologically suspect breast lesions ≤ 2 cm (i.e. BIRADS 4/5) are eligible. Patients with previous history of cancer, or diagnosed with autoimmune or active allergic diseases, acute or chronic hepatic, renal, or cardiac diseases, or acute or chronic infectious diseases are excluded from the present trial. Specific aims: The primary aim of the present trial is to develop a noninvasive classifier for the differential diagnosis of suspect breast lesions detected through mammography and/or ultrasound. For such purpose we will assess the performance of plasma cfMeDIPseq, ULP-WGS, and other promising techniques for the differential diagnosis of BC. Such techniques will be integrated in a unique classifier in order to reach the maximum possible accuracy. Statistical methods: Sample size was calculated with a semi-parametric simulation-based approach from beta-distributions of PBMC datasets: assuming to test 20,000 CpG regions, with 300 differentially methylated target CpGs, a target maximal difference in DNA methylation of 0.2 between groups and an FDR of 0.05, 1 – beta ~ 0.90 would be achieved with an overall sample size of 150 samples split in a 1:2 ratio. Target accrual and present accrual: Minimum target accrual is set at 49 patients with BC and 98 patients with benign lesions. To date, we have collected plasma samples from 74 eligible patients with BC and 115 eligible patients with benign lesions. A validation cohort accounting for ~30% of our sample set will be recruited at Istituto Nazionale dei Tumori (Milan, IT). Contact information: For further information, please contact Gabriele Zoppoli at gabriele.zoppoli@unige.it. Citation Format: Francesco Ravera, Martina Dameri, Isabella Lombardo, Mario Stabile, Alberto Tagliafico, Massimo Calabrese, Alberto Ballestrero, Lorenzo Ferrando, Gabriele Zoppoli. Development of a hoRizontal data intEgration classifier for NOn-invasive early diAgnosis of breasT cancEr: the RENOVATE trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT1-23-01.

Identifying innovative molecules involved in the tumor immune escape process could help refine the survival stratification of colorectal cancer (CRC) patients. HLA-G, a non-classical HLA molecule, physiologically involved in tolerogenic mechanisms, has recently emerged as a relevant prognostic marker in other tumor types, but ambiguous data are reported in the CRC setting. This study aims to evaluate the HLA-G expression and prognostic potential in a series of stage II/III CRCs. HLA-G expression was evaluated in 100 pT3 CRC cases by means of immunohistochemistry using the 4H84 and MEM-G/2 monoclonal antibodies. We observed heterogeneous expression of HLA-G showing different ranges: 4H84 expression ranged from > 1 to 40%—median 7%; MEM-G/2 expression ranged from 20 to 90%—median 50%. HLA-G positivity (any intensity > 1%) varied according to the antibody employed, identifying: 8 4H84 positive, 34 MEM-G/2 positive, 6 double-positive and 52 negative cases. Correlation with clinico-pathologic data showed a significant association with a poor tumor differentiation in stage III right-sided CRC subgroup (p = 0.043), while no other pathologic variable was significantly associated. Survival analysis revealed a reduced disease-free survival rate (HR 4.304613; p = 0.031) in the subgroup of CRC-related death cases, while no correlations were observed considering the whole series and the overall survival. In conclusion, HLA-G is a promising CRC prognostic marker however much work is still required regarding technical aspects and evaluation of expression.

A complex relationship between adipose tissue and malignancy, involving an inflammatory response, has been reported. The goal of this work was to assess the prevalence of white adipose tissue (WAT) inflammation in patients with endometrial cancer (EC), and the association with circulating inflammation markers. Furthermore, the aim was to characterize the pathways activated in and the cell type composition of adipose tissue in patients with EC.Adipose tissue and blood samples were prospectively collected from 101 patients with EC at initial surgery. WAT inflammation was determined based on adipocytes surrounded by macrophages forming crown-like structures. Circulating levels of metabolic syndrome-associated and inflammatory markers were quantified. RNA-sequencing was performed on adipose samples (n = 55); differential gene expression, pathway, and cellular decomposition analyses were performed using state-of-the-art bioinformatics methods.WAT inflammation was identified in 46 (45.5%) of 101 EC patients. Dyslipidemia, hypertension, and diabetes mellitus were significantly associated with WAT inflammation (p .05). WAT inflammation was associated with greater body mass index (p .001) and higher circulating levels of leptin, high-sensitivity C-reactive protein, and interleukin-6, as well as lower levels of adiponectin and sex hormone-binding globulin (p .05). Transcriptomic analysis demonstrated increased levels of proinflammatory and pro-neoplastic-related gene expression in inflamed omental adipose tissue.WAT inflammation is associated with metabolic syndrome, obesity, and inflammatory markers, as well as increased expression of proinflammatory and proneoplastic genes.

Background:Lack of Schlafen family member 11 (SLFN11) expression has been recently identified as a dominant genomic determinant of response to DNA damaging agents in numerous cancer types. Thus, strategies aimed at increasing SLFN11 could be used to restore chemosensitivity of refractory cancers.As oncogenic downregulation is often driven by methylation of the promotor region, we explore the demethylation effect of 5-aza-2’-deoxycytidine (decitabine), on the SLFN11 gene methylation. Since SLFN11 has been reported as an interferon inducible gene, and interferon is secreted during an active anti-tumor immune response, we investigated the in vitro effect of IFN-γ on SLFN11 expression in breast cancer cell lines. A second broader approach to show cross talk between immune cells and SLFN11 expression is indirect co-culture of breast cancer cells with activated PBMCs and evaluate if this can drive SLFN11 upregulation. Finally, as a definitive and specific way to modulate SLFN11 expression we implemented SLFN11 dCas9 (dead CRISPR associated protein 9) systems to specifically increase or decrease SLFN11 expression.Results:We first confirmed a correlation previously reported between methylation of SLFN11 promoter and its expression across multiple cell lines. We showedin-vitrothat decitabine and IFN-γ could increase moderately the expression of SLFN11 in both BT- 549 and T47D cell lines, but not in strongly methylated cell lines such as MDA-MB-231. Though,in-vitro, the co-culture of the same cell lines with CD8-CD25 activated PBMC failed to increase SLFN11 expression. On the one hand, the use of a CRISPR-dCas9 UNISAM system could increase SLFN11 expression significantly (up to 5-fold), stably and specifically in BT-549 and T47D cancer cell lines. Though, this system also failed to force a strong expression of SLFN11 in cell lines with robust SLFN11 promoter methylation such as MDA-MB-231. On the other hand, the use of CRISPR-dCas9 KRAB could significantly reduce the expression of SLFN11 in BT-549 and T47D. We then used the modified cell lines to confirm the alteration in chemo sensitivity of those cells to treatment with DNA Damaging Agents (DDAs) such as Cisplatin and Epirubicin or DNA Damage Response (DDRs) drugs like Olaparib. RNAseq was used to elucidate the mechanisms of action affected by the alteration in SLFN11 expression.Conclusion:To our knowledge this is the first report of the stable non-lethal increase of SLFN11 expression in a cancer cell line. Our results show that induction of SLFN11 expression can enhance DDA and DDR sensitivity in breast cancer cells and dCas9 systems may represent a novel approach to increase SLFN11 and achieve higher sensitivity to chemotherapeutic agents, improving outcome or decreasing required drug concentrations. SLFN11-targeting therapies might be explored pre-clinically to develop personalized approaches.

AURORA aims to study the processes of relapse in metastatic breast cancer (MBC) by performing multi-omics profiling on paired primary tumors and early-course metastases. Among 381 patients (primary tumor and metastasis pairs: 252 targeted gene sequencing, 152 RNA sequencing, 67 single nucleotide polymorphism arrays), we found a driver role for GATA1 and MEN1 somatic mutations. Metastases were enriched in ESR1, PTEN, CDH1, PIK3CA, and RB1 mutations; MDM4 and MYC amplifications; and ARID1A deletions. An increase in clonality was observed in driver genes such as ERBB2 and RB1. Intrinsic subtype switching occurred in 36% of cases. Luminal A/B to HER2-enriched switching was associated with TP53 and/or PIK3CA mutations. Metastases had lower immune score and increased immune-permissive cells. High tumor mutational burden correlated to shorter time to relapse in HR+/HER2− cancers. ESCAT tier I/II alterations were detected in 51% of patients and matched therapy was used in 7%. Integration of multi-omics analyses in clinical practice could affect treatment strategies in MBC.Significance:The AURORA program, through the genomic and transcriptomic analyses of matched primary and metastatic samples from 381 patients with breast cancer, coupled with prospectively collected clinical data, identified genomic alterations enriched in metastases and prognostic biomarkers. ESCAT tier I/II alterations were detected in more than half of the patients.This article is highlighted in the In This Issue feature, p. 2659

File with the supplementary material & methods, supplementary tables and supplementary figure.

Supplementary file with the listing of the liver genes excluded from transcriptomic analyses.

OBJECTIVE: To determine the effect of hyperthermic intraperitoneal chemotherapy (HIPEC) with carboplatin on the transcriptomic profiles of normal and ovarian cancer (OC) tissues. METHODS: Normal and tumor samples from four OCs were prospectively collected pre- and immediately post-HIPEC treatment and subjected to RNA-sequencing. Differential gene expression, gene ontology enrichment and pathway analyses were performed. Heat shock protein and immune-response protein expression was assessed using protein arrays and western blotting. RESULTS: RNA-sequencing revealed 4,231 and 322 genes significantly differentially expressed between pre- and post-treatment normal and OC tissues, respectively (both adjusted p-value

Background: As the development and use of oral anticancer agents increases, it is critical to understand patient adherence to both standard and investigational agents. The open label, phase 3 multicenter PALLAS trial investigates whether adding 2 years of the CDK4/6 inhibitor palbociclib (P) to adjuvant endocrine therapy (ET) improves invasive disease-free survival (iDFS) over adjuvant ET alone in patients (pts) with HR- positive, HR2-negative, stage II-III breast cancer. Pts were randomly assigned to either Palbociclib (125 mg/day, 3 weeks on, 1 week off, in a 28-day cycle) plus ongoing provider/patient-choice adjuvant ET (P+ET) versus ET alone. We examined patient-reported adherence to ET +/- P during the first two years of study treatment. Methods: Adherence outcomes were measured in English-speaking pts in the U.S., UK, Ireland and Australia, Spanish-speaking pts in Spain and Mexico, and German-speaking pts in Germany and Austria. Adherence measures included drug diaries completed at each cycle, pill counts (for P only) collected at each study visit, and the Morisky Medication Adherence Scale-4 item and the McHorney Adherence Estimator questionnaires completed at cycles 2, 3, 6, 12, 18, and 24 (22 months). Mean adherence for each cycle was defined as the average proportion of prescribed pills taken (via drug diary) across all patients who initiated that cycle. Persistence was defined as the duration of drug initiation to treatment cessation (via drug diary). Generalized estimating equations were used to model the “most adherent” pts on the Morisky (score = 5 vs score 0) to compare the average difference between arms over time for ET, adjusting for baseline demographic and clinical variables. Results: 81% (N=4688) of PALLAS pts were included in this analysis. Median persistence to ET was 23.6 months in P + ET (n=2169), 23.7 months in ET alone (2136) and 20.4 months for P (n=2194). The number of pts who initiated each cycle for ET declined over time and was similar between arms; the decline was more marked for P (Table 1). Mean adherence range as measured by drug diary was 98.2-99.3% for ET in P+ET and 98.0 - 99.4% in ET alone; and for P, ranged from 93.4 - 98.8%. The adherence and persistence results were nearly identical whether measured by drug diary or pill count for P. The observed percent “most adherent” for P measured by the Morisky scale ranged from 71.9% - 79.6% and the percent “low risk” for adherence problems measured by the McHorney scale, 64.0% - 73.4%. The percent of pts “most adherent” and “low risk” for adherence problems to ET was higher, on average over time, in the P+ET group compared to ET alone (75% vs 68%, p Table 1.Adherence and persistence in PALLAS over the 24 month treatment periodPalbocicilb + ETETPalbociclibETETTreatment cycleMean Adherence (SD)N*Mean Adherence (SD)NMean Adherence (SD)N193.4 (13.8)229098.7 (6.1)230999.0 (5.2)2343294.7 (13.3)218198.8 (6.4)220398.4 (7.1)2206397.4 (10.1)211298.7 (7.3)214298.8 (6.3)2168497.6 (10.2)203598.6 (7.8)211099.0 (5.7)2154597.6 (11.1)197598.3 (7.9)209398.1 (8.3)2148698.1 (8.7)189998.7 (6.6)203798.5 (8.2)2116798.1 (9.3)185599.0 (6.3)201298.6 (7.3)2102898.0 (9.1)181098.2 (8.2)199798.1 (8.2)2092998.4 (7.4)173399.0 (6.4)196298.8 (6.8)20511098.3 (8.6)171499.2 (4.9)194699.0 (5.5)20381198.3 (8.0)169198.5 (7.3)193798.2 (8.2)20311298.7 (6.2)161299.1 (5.3)191098.7 (7.5)19861398.5 (7.7)159698.9 (6.4)189398.8 (7.1)19691497.9 (9.3)157698.4 (7.3)188298.0 (9.1)19611598.5 (6.9)151499.2 (4.6)185499.0 (6.0)19281698.2 (8.7)148699.1 (4.6)183599.1 (5.7)19131798.1 (9.8)147998.3 (7.8)182698.5 (6.4)19031898.7 (6.8)141699.3 (4.1)176899.2 (5.3)18601998.7 (7.3)140899.3 (3.8)175699.4 (3.1)18482098.4 (8.9)139798.9 (5.3)174298.7 (5.8)18382198.5 (7.5)130199.0 (6.1)167698.8 (7.1)17842297.8 (9.7)124698.8 (6.2)164799.2 (5.7)17692398.1 (8.8)118098.2 (8.5)161998.4 (8.0)17552498.8 (7.2)108699.0 (6.3)146898.9 (7.1)16192598.8 (6.7)99699.2 (5.2)143298.8 (7.1)15892697.9 (11.2)84599.1 (5.7)140998.6 (8.8)1578ET=Endocrine therapy * Number of pts who initiated the treatment cycle. Citation Format: Eileen Shinn, David Zahrieh, Angela DeMichele, Nick Zdenkowski, Julie Lemieux, Jun Mao, Vesna Bjelic-Radisic, Michelle Naughton, Georg Pfeiler, Karen Gelmon, Ingrid Mayer, Daniel Egle, Gabriele Zoppoli, Tiffany Traina, Miguel Martin Jiménez, Silvia Antolin Novoa, Tufia Haddad, Arlene Chan, Alistair Edward Ring, Antonio Wolff, Jose JuanPonce Lorenzo, Dhanusha Sabanathan, Hal Burstein, Zbigniew Ireneusz Nowecki, Gunda Pristauz-Telsnigg, Adam Brufsky, Meritxell Bellet-Ezquerra, Theodoros Foukakis, Yelena Novik, Gabor Rubovszky, Karoline Muehlbacher, Otto Metzger, Theodora Goulioti, Ernest Law, Ann Partridge, Lisa Carey, Alex Zoroufy, Dominik Hlauschek, Christian Fesl, Erica Mayer, Michael Gnant. Adherence with adjuvant endocrine therapy with or without Palbociclib in the PALLAS trial [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-12-01.

Background/Aim: Clinicopathological features of patients undergoing margin enlargement after lumpectomy for early breast cancer with positive/close excision margins were analyzed in order to define whether a re-operative procedure could have been avoided. Furthermore, a standardized protocol of specimen orientation was adopted in order to optimize both the widening procedure as well as the oncologic outcome. Patients and Methods: A retrospective analysis was performed including pre-, peri-, and post-operative parameters, and a predictive score by means of a multivariate model was developed using all clinically and statistically significant variables associated with residual disease (RD). Results: RD was significantly related to positive tumor margins, hormone receptor negative, HER2-positive, and tumors with high Ki67 proliferation index (p3, only 2 patients (2.0%) had RD, while in 81 patients with a score ≤3, 55 patients (67.9%) had RD (p

Although several studies have explored the molecular landscape of metastatic melanoma, the genetic determinants of therapy resistance are still largely unknown. Here, we aimed to determine the contribution of whole-exome sequencing and circulating free DNA (cfDNA) analysis in predicting response to therapy in a consecutive real-world cohort of 36 patients, undergoing fresh tissue biopsy and followed during treatment. Although the underpowered sample size limited statistical analysis, samples from non-responders had higher copy number variations and mutations in melanoma driver genes compared to responders in the BRAF V600+ subset. In the BRAF V600− subset, Tumor Mutational Burden (TMB) was twice that in responders vs. non-responders. Genomic layout revealed commonly known and novel potential intrinsic/acquired resistance driver gene variants. Among these, RAC1, FBXW7, GNAQ mutations, and BRAF/PTEN amplification/deletion were present in 42% and 67% of patients, respectively. Both Loss of Heterozygosity (LOH) load and tumor ploidy were inversely associated with TMB. In immunotherapy-treated patients, samples from responders showed higher TMB and lower LOH and were more frequently diploid compared to non-responders. Secondary germline testing and cfDNA analysis proved their efficacy in finding germline predisposing variants carriers (8.3%) and following dynamic changes during treatment as a surrogate of tissue biopsy, respectively.

ATM germline pathogenic variants were recently found enriched in high-risk melanoma patients. However, ATM loss of heterozygosity (LOH) has never been investigated in melanoma and, therefore, a causal association with melanoma development has not been established yet. The purpose of this study was to functionally characterize 13 germline ATM variants found in high-risk melanoma patients—and classified by in silico tools as pathogenic, uncertain significance, or benign—using multiple assays evaluating ATM/pATM expression and/or LOH in melanoma tissues and cell lines. We assessed ATM status by Immunohistochemistry (IHC), Western Blot, Whole-Exome Sequencing/Copy Number Variation analysis, and RNA sequencing, supported by Sanger sequencing and microsatellite analyses. For most variants, IHC results matched those obtained with in silico classification and LOH analysis. Two pathogenic variants (p.Ser1135_Lys1192del and p.Ser1993ArgfsTer23) showed LOH and complete loss of ATM activation in melanoma. Two variants of unknown significance (p.Asn358Ile and p.Asn796His) showed reduced expression and LOH, suggestive of a deleterious effect. This study, showing a classic two-hit scenario in a well-known tumor suppressor gene, supports the inclusion of melanoma in the ATM-related cancer spectrum.

Background: Total mesorectal excision (TME) is the gold standard to treat locally advanced rectal cancer. This monocentric retrospective study evaluates the results of laparotomic, laparoscopic and robotic surgery in “COMRE GROUP” (REctalCOMmittee). Methods: 327 selected stage I-II-III patients (pts) underwent TME between November 2005 and April 2020 for low or middle rectal cancer; 91 pts underwent open, 200 laparoscopic and 36 robotic TME. Of these, we analyzed the anthropomorphic, intraoperative, anatomopathological parameters and outcome during the follow up. Results: The length of hospital stay was significantly different between robotic TME and the other two groups (8.47 ± 3.54 days robotic vs. 11.93 ± 5.71 laparotomic, p < 0.001; 8.47 ± 3.54 robotic vs. 11.10 ± 7.99 laparoscopic, p < 0.05). The mean number of harvested nodes was higher in the laparotomic group compared to the other two groups (19 ± 9 laparotomic vs. 15 ± 8 laparoscopic, p < 0.001; 19 ± 9 laparotomic vs. 15 ± 7 robotic, p < 0.05). Median follow-up was 52 months (range: 1–169). Overall survival was significantly shorter in the open TME group compared with the laparoscopic one (Chi2 = 13.36, p < 0.001). Conclusions: In the experience of the “COMRE” group, laparoscopic TME for rectal cancer is a better choice than laparotomy in a multidisciplinary context. Robotic TME has a significant difference in terms of hospital stay compared to the other two groups.

Background: Inflammatory breast cancer (IBC) is a rare (1-5%), but aggressive form of breast cancer (BC), accounting for ~10% of BC mortality. In early setting (M0), standard of care is neoadjuvant chemotherapy (NACT), followed by surgery. Nevertheless, outcome is still relatively poor. Pathological complete response (pCR) after NACT is prognostic in BC in general, and can be predicted by a high percentage of stromal tumor infiltrating lymphocytes (sTIL) in the primary tumor. The predictive value of sTIL in IBC has only been sporadically investigated, often in smaller series. Our aim was to determine which variables, including sTIL, are associated with pCR and to determine the prognostic value of pCR in IBC in a large multicentric, retrospective cohort. Patients & Methods: We included patients with IBC treated with NACT+/- anti-Human Epidermal growth factor Receptor 2 (HER2) therapy, followed by surgery from 10/1996 to 10/2021 in 7 different European hospitals. Clinicopathological variables were collected and central pathological review was performed, including sTIL scoring. This study focused on M0 cases. Considered clinicopathological variables were: age, histology, tumor grade, estrogen receptor status (ER), HER2 status, focality (unifocal vs not), and baseline locoregional nodal status (Table 1). Associations between pCR, clinicopathological variables and sTIL were assessed using Firth’s logistic regression models: Model 1 was adjusted for center, Model 2 additionally included all variables of interest. Similarly, linear regression was used to investigate the association between sTIL and clinicopathological features. Univariable and multivariable Cox regression was used to evaluate the role of pCR on disease free survival (DFS), distant recurrence free survival (DRFS) and overall survival (OS). DFS and DRFS were analyzed considering death without the respective event as competing risk. Results: 494 patients were included. The distribution according to receptor status was: ER-/HER2- (24.3%), ER+/HER2- (34.4%), ER+/HER2+ (13%) and ER-/HER2+ (20.2%). pCR rate was 26% and per receptor status: ER-/HER2- (28%), ER+/HER2- (10%), ER+/HER2+ (42%) and ER-/HER2+ (45%). pCR was associated with grade (G3 vs G1/2, OR =2.79 (1.70 − 4.74), p < .001), ER-status (positive vs negative, OR = 0.39 (0.26 − 0.60), p < .001) and HER2 status (positive vs negative, OR = 3.74 (2.43 − 5.81), p < .001) in Model 1. Only the association with HER2 status remained significant in Model 2 (OR = 5.34 (2.83 − 10.47), p < .001). sTIL was scored for 385 patients. Median sTIL was 5.3% [IQR 2.0%;16.7%] and according to receptor status: ER-/HER2- (10%), ER+/HER2- (2.5%), ER+/HER2+ (6.7%) and ER-/HER2+ (8.3%). Higher sTIL was associated with NST (p = .032), grade 3 (p = .015), and ER-negativity (p = .007) in Model 1. This was no longer significant in Model 2, but the direction of the trends was preserved. sTIL was associated with pCR (5% increment, OR = 1.13 (1.05 − 1.22), p = .002), but no longer after adjustment. No association between pCR and sTIL was found stratifying by receptor status. The median FU was 9.4 years and multivariable Cox regression models revealed that ER+ and HER2+ status and achieving pCR were significantly associated with better DFS, DRFS, and OS (Table 1). Conclusion: Our results indicate that patients with HER2+ tumors have a higher probability of achieving pCR and that pCR has an independent prognostic role in IBC. This is the largest IBC study with centrally scored sTIL, demonstrating that sTIL is associated with pCR but its role as an independent predictor of pCR is still not certain. Citation Format: Maxim De Schepper, Ha-Linh Nguyen, François Richard, Florence Lerebours, Roman Vion, Florian Clatot, Anca Berghian, Marion Maetens, Sophia Leduc, Edoardo Isnaldi, Chiara Molinelli, Matteo Lambertini, Frederica Grillo, Gabriele Zoppoli, Luc Dirix, Hilde Wuyts, Kevin Punie, Hans Wildiers, Chantal Remmerie, Ann Smeets, Ines Nevelsteen, Patrick Neven, Anne Salomon, Denis Larsimont, Caroline Duhem, Patrice Viens, François BERTUCCI, Elia Biganzoli, Peter Vermeulen, Giuseppe Floris, Christine Desmedt. Stromal tumor infiltrating lymphocytes and pathological complete response in patients with inflammatory breast cancer treated with neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-01-47.

Background With 8.7 years follow-up, the prospective phase III randomized MINDACT trial (EORTC 10041/BIG3-04) continues to meet its primary objective, i.e. 95.1% (95%CI 93.1-96.6), 5-year distant metastasis-free survival (DMFS) in clinical high (C-High)/genomic low (G-Low) risk patients who did not receive adjuvant chemotherapy (ACT) (Cardoso et al., ASCO 2020). In addition, about half of the MINDACT patients had a low clinical risk (C-Low) defined by pre-specified clinical-pathological characteristics. Here, we evaluated the outcome of this C-Low population stratified by the 70-gene signature (MammaPrint®) (G-Low or G-High) for outcome considering age, and present data on the total G-low population (C-Low and C-High combined). Methods Of 6693 patients enrolled in the MINDACT trial between 2007 and 2011, 3337 were C-Low, characterized as mainly T1, grade 1 or 2, and node negative. We evaluated the pre-specified DMFS, distant metastasis free interval (DMFI), and overall survival (OS) rates at 5 and 8 years in the C-Low population: i) in patients with genomic low risk (C-Low/G-Low, n=2744) who were recommended to receive endocrine therapy only (for 99% HR+), and ii) in C-Low/G-High who received ACT or not following randomization (ITT, n=690, 81% HR+). Exploratory analyses by age, ≤50 and >50, were conducted for ACT vs no ACT received in C-Low/G-High. In parallel we estimated survival rates for all G-low patients if all would have followed the genomic low risk assignment and received no ACT (C-Low/G-Low, and C-High/G-Low randomized to no ACT double weighted, n=4130). We used Kaplan-Meier estimates for time to event endpoints and hazard ratios with 95%CI from Cox-regression models adjusted for stratification factors used for the randomization. Results C-low/G-low patients who were recommended endocrine therapy only (compliance > 79%, based on local guidelines) have excellent 5 and 8 year survival rates for all endpoints (Table 1). The estimated survival rates for all G-Low patients, if all would have followed the genomic low risk assignment and received no ACT, is excellent as well (Table 1), albeit this population includes both C-Low and C-High patients. The survival estimates for C-Low/G-High patients are for all endpoints a few percentage points lower than for the C-Low/G-Low group (Table 1). At 8 years of follow-up, in the relatively small subset of 690 patients with C-Low/G-High tumors assigned to ACT or not by randomization (ITT), a 1.5% (SE ±2.3%) higher DMFS is seen in the ACT group, and a 2.9% (SE ±2.0%) higher DMFI. This suggested benefit is mostly seen in patients under 50 years of age (absolute Δ in DMFS for ACT vs no ACT at 8 years: 5.4% for age ≤50 vs -0.3% for age >50). Conclusion Patients with a 70-gene G-Low risk tumor have an excellent 8 year outcome in the context of C-Low characteristics when recommended for endocrine therapy only, very close to the outcome in the larger group of all G-Low patients regardless of clinical risk. Stratification of C-Low patients in to G-Low and G-high provides meaningful information. The benefit of ACT in C-Low patients with a 70-gene G-High risk tumor needs further confirmation, especially relevant in younger women. Table 1All Patients PopulationPatientsObserved events% at 5 years (95% CI)% at 8 years (95% CI)DMFSC-Low / G-Low274417097.3 (96.6-97.9)94.7 (93.8-95.6)C-Low / G-High5936194.2 (92.0-95.9)91.1 (88.4-93.3)DMFIC-Low / G-Low274410398.5 (97.9-98.9)96.7 (95.9-97.3)C-Low / G-High5934695.8 (93.8-97.2)93.5 (91.0-95.3)OSC-Low / G-Low274412298.2 (97.6-98.7)96.5 (95.7-97.2)C-Low / G-High5934496.8 (94.9-98.0)93.1 (90.5-95.0)Patients G-low (C-Low & C-High)PatientsEstimated events% at 5 years% at 8 yearsDMFSAll G-Low - no ACT413033996.492.8DMFIAll G-Low - no ACT413024497.494.6OSAll G-Low - no ACT413022397.995.7 Citation Format: Laura J van 't Veer, Fatima Cardoso, Coralie Poncet, Josephine Lopes Cardozo, Suzette Delaloge, Jean-Yves Pierga, Peter Vuylsteke, Etienne Brain, Giuseppe Viale, Sherko Kümmel, Isabel T Rubio, Gabriele Zoppoli, Alistair Thompson, Erika Matos, Khalil Zaman, Susan Knox, Florentine Hilbers, Aleksandra Peric, Bart Meulemans, Martine Picccart, Emiel J Th Rutgers. How low is low risk: MINDACT updated outcome and treatment benefit in patients considered clinical low risk and stratified by genomic signature, age and nodal status [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr GS4-11.

Background/aim The perspective validation of a selective approach in patients undergoing breast cancer surgery was performed in order to assess whether patients as well as Health Care Workers (HCWs) were exposed to any undue risk of COVD-19 infection. Patients and methods From March 9th to June 9th 2020, 207 patients were phone-triaged by a dedicated Breast Care Nurse; a patient-tailored program was adopted with the aim of avoiding hospitalization of SARS-CoV-2 symptomatic patients, with a careful prioritization of surgical procedures according to specific disease features. Results Two hundred and three out of 207 patients underwent operation; seven patients were temporarily excluded because they tested positive at phone triage (n=3), or in-hospital triage (n=3); another asymptomatic patient with negative NP swab tested IgM Ab-positive so that surgery was re-scheduled two weeks later. Four patients had no surgery; one of them was reconsidered for neoadjuvant chemotherapy (NAC) after testing positive at phone triage; three patients were excluded because they were already hospitalized for COVID-19. Overall, mean in-hospital stay was 2.2 days (±SD, 0.7) and, after hospital discharge, no patient required readmission. Conclusion This preventive program avoided any COVID-19 infection among patients and HCWs, so that an elective breast cancer surgical procedure can be safely and timely pursued without affecting the oncologic outcome.

PURPOSE The study of plasma cell-free DNA integrity (cfDI) has shown potential for providing useful information in neoplastic patients. The aim of this study is to estimate the accuracy of an electrophoresis-based method for cfDI evaluation in the assessment of pathologic complete response (pCR) in patients with breast cancer (BC) undergoing neoadjuvant chemotherapy (NACT). PATIENTS AND METHODS Fifty-one patients with BC undergoing anthracycline-/taxane-based NACT were recruited. Plasma samples were collected from each patient at diagnosis (t0), after anthracycline administration (t1), and after NACT completion (t2). The concentration of differently sized cell-free DNA fragments was assessed by automated electrophoresis. cfDI, expressed as cfDI index, was calculated as the ratio of 321-1,000 bp sized fragment concentration to 150-220 bp sized fragment concentration assessed at t2. cfDI index was then used to build an exploratory classifier for BC response to NACT, directly comparing its sensitivity and specificity with magnetic resonance imaging (MRI), through bootstrapped logistic regression. RESULTS cfDI index was assessed on 38 plasma samples collected from as many patients at t2, maintaining a 30/70 ratio between pCR and non-pCR patients. cfDI index showed an area under the receiver operating characteristic curve in predicting the achievement of pCR of 81.6, with a cutoff above 2.71 showing sensitivity = 81.8 and specificity = 81.5. The combination of cfDI index and MRI showed, in case of concordance, an area under the receiver operating characteristic curve of 92.6 with a predictive value of complete response of 87.5 and a predictive value of absence of complete response of 94.7. CONCLUSION cfDI index measured after NACT completion shows great potential in the assessment of pCR in patients with BC. The evaluation of its use in combination with MRI is strongly warranted in prospective studies.

BackgroundIt is becoming clear that tumor immune T cell infiltration and its functional orientation have substantial effect on cancer progression, influencing both response to therapy and prognosis. In this pan-cancer study, the previously described Immunologic Constant of Rejection (ICR) signature is used to define opposing immune phenotypes (i.e., immuneactive and immune-silent) across 31 different histologies. We systematically analyze the interconnections between the genetic programming of neoplasms and their immune orientation across different histologies, and the prognostic impact of such interplay. Moreover, we investigated the predictive value of ICR classification across various public datasets of immune checkpoint inhibition therapy.MethodsRNA-seq data of samples from a total of 9,282 patient tumor samples representing 31 cancer types were obtained from The Cancer Genome Atlas (TCGA). We classified each cancer type based on the expression of the ICR gene signature. Oncogenic pathway gene set enrichment and mutational status were analyzed in relation to ICR phenotypes. To explore whether tumorintrinsic attributes associate with the prognostic value of ICR across cancers, we compared mutational load, oncogenic alterations and expression of oncogenic pathways between cancer types using an integrative bioinformatic pipeline.ResultsOur analyses identified a distinct prognostic connotation of ICR depending on cancer histology. We identified several oncogenic pathways whose enrichment inversely correlated with ICR in multiple tumor types. We found several cancer specific pathways that were differentially enriched between tumors in which ICR had a prognostic impact versus the ones in which ICR did not bear any prognostic connotation such as proliferation and TGF-beta signaling. Importantly, this conditional impact of ICR was also validated in the context of immune checkpoint inhibition treatment.ConclusionsWe identified tumor-intrinsic attributes that correlate with immune phenotypes and potentially influence their development. In addition, a relationship was observed between the enrichment of oncogenic pathways and the prognostic significance of the ICR and its predictive value for patients treated with anti-CTLA4 immune checkpoint inhibition. Such information can be used to prioritize potential candidates for therapies aimed at converting immune-silent into immuneactive tumors and to refine stratification algorithms.

Sterol synthesis is a highly complex and integrated pathway in mammals. In the present review, we briefly summarize the main steps of this pathway, especially concerning its main rate-limiting enzymes, HMG-CoA reductase (HMGCR) and squalene epoxidase (SQLE), in relation with cancer. We focus on studies reporting key findings linking cholesterol with cancer. The inhibition of HMGCR and SQLE to prevent and inhibit cancer are reviewed. Finally, a pan-cancer review of publicly available data on genomic aberrations in the main enzymes involved in sterol biosynthesis and their transcription factors is reported, providing hitherto unexplored findings that may be the subject of future research in cancer metabolomics and tumor targeted treatment.

Histologic special types of breast cancer (BC) account for ~20% of BCs. Large sequencing studies of metastatic BC have focused on invasive ductal carcinomas of no special type (IDC-NSTs). We sought to define the repertoire of somatic genetic alterations of metastatic histologic special types of BC. We reanalyzed targeted capture sequencing data of 309 special types of BC, including metastatic and primary invasive lobular carcinomas (ILCs; n = 132 and n = 127, respectively), mixed mucinous (n = 5 metastatic and n = 14 primary), micropapillary (n = 12 metastatic and n = 8 primary), and metaplastic BCs (n = 6 metastatic and n = 5 primary), and compared metastatic histologic special types of BC to metastatic IDC-NSTs matched according to clinicopathologic characteristics and to primary special type BCs. The genomic profiles of metastatic and primary special types of BC were similar. Important differences, however, were noted: metastatic ILCs harbored a higher frequency of genetic alterations in TP53, ESR1, FAT1, RFWD2, and NF1 than primary ILCs, and in CDH1, PIK3CA, ERBB2, TBX3, NCOR1, and RFWD2 than metastatic IDC-NSTs. Metastatic ILCs displayed a higher mutational burden, and more frequently dominant APOBEC mutational signatures than primary ILCs and matched metastatic IDC-NSTs. ESR1 and NCOR mutations were frequently detected in metastatic mixed mucinous BCs, whereas PIK3CA and TP53 were the most frequently altered genes in metastatic micropapillary and metaplastic BCs, respectively. Taken together, primary and metastatic BCs histologic special types have remarkably similar repertoires of somatic genetic alterations. Metastatic ILCs more frequently harbor APOBEC mutational signatures than primary ILCs and metastatic IDC-NSTs.

Adipocytes and cancer-associated adipocytes (CAAs) are poorly investigated cells in the tumor microenvironment. Different image analysis software exist for identifying and measuring these cells using scanned hematoxylin and eosin (H&E)-stained slides. It is however unclear which one is the most appropriate for breast cancer (BC) samples. Here, we compared three software (AdipoCount, Adiposoft, and HALO®). HALO® outperformed the other methods with regard to adipocyte identification, (> 96% sensitivity and specificity). All software performed equally good with regard to area and diameter measurement (concordance correlation coefficients > 0.97 and > 0.96, respectively). We then analyzed a series of 10 BCE samples (n = 51 H&E slides) with HALO®. Distant adipocytes were defined >2 mm away from cancer cells or fibrotic region, whereas CAAs as the first three lines of adipocytes close to the invasive front. Intra-mammary heterogeneity was limited, implying that measuring a single region of ∼500 adipocytes provides a reliable estimation of the distribution of their size features. CAAs had smaller areas (median fold-change: 2.62) and diameters (median fold-change: 1.64) as compared to distant adipocytes in the same breast (both p = 0.002). The size of CAAs and distant adipocytes was associated with the body mass index (BMI) of the patient (area: rho = 0.89, p = 0.001; rho = 0.71, p = 0.027, diameter: rho = 0.87 p = 0.002; rho = 0.65 p = 0.049, respectively). To conclude, we demonstrate that quantifying adipocytes in BC sections is feasible by digital pathology using H&E sections, setting the basis for a standardized analysis of mammary adiposity in larger series of patients., info:eu-repo/semantics/published

BACKGROUND/AIM A retrospective study was performed in 246 breast cancer patients to define whether tumor-to-nipple distance (TND) assessment by breast MRI may select patients eligible to nipple-sparing mastectomy (NSM) as compared to permanent section assessment of retroareolar margin. PATIENTS AND METHODS Pre- and post-operative parameters including imaging data, histology of the primary tumor, biologic prognostic factors, and adjuvant regimens were retrieved; patients with close/positive retroareolar margins underwent nipple or NAC excision. The primary endpoint was loco-regional recurrence (LRR). RESULTS Patients with TND ≤2 cm had a significantly higher rate of invasive ductal carcinoma (p