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Introduction: Breast cancer (BC) during pregnancy (PrBC) is an uncommon malignancy characterized by a more aggressive clinical course compared to pregnancy-unrelated BC. Specific patterns of tumor-infiltrating lymphocytes (TILs) subpopulations have been observed in these patients, with significant prognostic roles. Previous studies demonstrated the varying histopathologic and prognostic profiles of PrBC by gestational age. However, the underlying immune landscape dynamics has never been investigated. Here, we sought to provide comprehensive insights into the association between gestational age at breast cancer diagnosis and tumor immune microenvironment (TIME) composition. Materials and Methods: A total of 110 PrBC were selected from our Institutional registry and categorized based on the trimester in which they were diagnosed. All cases were subjected to TILs profiling according to the International TILs Working Group recommendations. Immunohistochemistry for CD4, CD8, forkhead box P3 (FOXP3), and PD-L1 (clone 22C3) on a Dako Omnis platform was performed. Fisher’s and Chi-squared tests, multinomial logistic regression models, ROC curve, and survival analyses were performed. Results: The proportion of patients with high histologic grades incremented with the increase in gestational age (1st, n=24, 53%; 2nd, n=27, 69.2%; 3rd trimester, n=20, 87.0%; p=0.02). Neither breast cancer subtypes nor the hormone receptor (HR) and HER2 status changed significantly according to the pregnancy trimester. In HR+/HER2- subtype, the proportion of TILs+ tumors were higher in the early phases of pregnancy (1st, n=29, 100%; 2nd, n=17, 89.5%; 3rd trimester, n=9, 81.8%; p=0.04) imprinted by FOXP3 positivity where more FOXP3+ TILs were seen in the first months and decreased progressively (1st, n=10, 55.6%; 2nd, n=2, 11.8%; 3rd trimester, n=0, 0%; p< 0.01)). While in the triple negative breast cancer (TNBC) population, the proportion of PD-L1+ tumors (i.e. CPS>1) was significantly higher in the later stages of pregnancy (1st, n=2, 16.7%; 2nd, n=2, 18.2%; 3rd trimester, n=5, 71.4%; p=0.03). Patients who relapsed after a BC diagnosis during the 1st and 2nd trimesters lacked more frequently FOXP3+ and CD8+ cells, unlike those with no disease recurrence (n=21, 77.8% vs. n=17, 48.6%; p=0.02 and n=18, 66.7% vs. n=10, 28.6%; p< 0.01, respectively). Conclusions: TIME dynamics of PrBC are different according to the gestational age in both HR+ and TNBC PrBC. Our results suggest that immune tolerance events are likely to involve PrBC at later gestational age. Specific escape mechanisms (i.e., TILs and FOXP3 decrease in HR+ and PD-L1 expression in TNBC) might explain the aggressiveness of PrBC diagnosed during the later gestational age. Citation Format: Elham Sajjadi, Konstantinos Venetis, Mariia Ivanova, Marianna Noale, Concetta Blundo, Giovanna Scarfone, Eugenia Di Loreto, Stefano Ferrero, Stefania Maggi, Paolo Veronesi, Viviana Enrica Galimbreti, Giuseppe Viale, Fedro Alessandro A. Peccatori, Elena Guerini-Rocco, Nicola Fusco. The tumor immune microenvironment composition and prognostic value in breast cancer during pregnancy is dynamic during the gestation period [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 P2-21-04.

Background: Breast cancer is the most common malignancy occurring during gestation and should be managed with the same protocols as for young non-pregnant women. In early-stage breast cancer during pregnancy (PrBC), breast-conserving surgery (BCS) with delayed radiation therapy (RT) is a reasonable alternative to radical mastectomy. Regrettably, no specific and widely adopted guidelines on the surgical management of these patients are available. In this study, we investigated the feasibility and safety of BCS during the first trimester of pregnancy in women with early-stage PrBC. Method: The first trimester was defined as 12 weeks and 6 days after the first day of the last menstruation. As for internal protocols, the surgical strategy for PrBC followed the same conservative-oriented schemes applied for nonpregnant patients. Only women with early-stage PrBC treated with BCS during pregnancy followed by planned RT to the whole breast after delivery were included. Exclusion criteria were i) clinical diagnosis or suspicion of inflammatory breast cancer; ii) multicentric disease; iii) diffuse malignant microcalcifications on mammography; iv) previous; v) connective tissue disease, and vi) syndromic breast cancers. All cases underwent central pathological review at the Pathology Department of the European Institute of Oncology. The primary outcome was the isolated local recurrence (ILR). Results: Among 168 PrBC patients, 67 (39.9%) were diagnosed during the first trimester of gestation. Of these, 30 patients (age range, 23-43 years; median=36 years; gestational age, 2-12 weeks; median=7 weeks; median follow-up time=6.5 years) met the inclusion criteria. None of the patients included in this study experienced perioperative surgical complications. No ILR was observed within three months (n=30), 1 year (n=27), and 5 years (n=18) after surgery. The 5-year overall survival rate for all patients was 97% (n=29/30). Four patients experienced ILR or new carcinomas after 6-13 years. These patients are still alive and disease-free after 14-17 years of follow-up. Discussion: In our study, both adjuvant endocrine therapy and anti-HER2 treatment were postponed after delivery. Local recurrences/second primary tumors were observed in 4 out of 30 patients treated with BCS. Given that patient #4 did not receive postoperative RT, but an after-delivery mastectomy for preoperative diagnostic underestimation during pregnancy, this case does not represent a post-BCS recurrence. On the other hand, cases #1-3 could be considered real relapses. Two of these tumors occurred in patients that underwent CT during pregnancy, in which the interval between the end of systemic therapies and the onset of RT was not influenced by the pregnancy. In a single patient (not eligible for systemic treatment in pregnancy), the RT was performed with a longer interval than the usual one of the non-pregnant patients. Survival was not affected by local relapse, underlining the efficacy of salvage treatment.Conclusion: The findings of this study suggest that BCS in the first trimester of pregnancy can be considered reasonably safe for both mother and the child. Mammography should be mandatory in all patients for whom conservative surgery is recommended. Given that the availability of data concerning PrBC and its treatment is scarce and heterogeneous, multicentric studies are warranted. Citation Format: Nicola Fusco, Concetta Blundo, Massimo Giroda, Elham Sajjadi, Konstantinos Venetis, M. Cristina Leonardi, Elisa Vicini, Luca Despini, Claudia F. Rossi, Letterio Runza, Eugenia Di Loreto, Giovanna Scarfone, Elena Guerini-Rocco, Giuseppe Viale, Paolo Veronesi, Barbara Buonomo, Fedro A. Peccatori, Viviana E. Galimberti. Feasibility and safety of breast-conserving surgery in early-stage breast cancers during the first trimester of pregnancy [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 P3-18-08.

Background: In HER2-positive (HER2+) early breast cancer (eBC) treated with neoadjuvant therapy (NAT), HER2 loss on residual disease (RD) might be correlated with a dampen survival. Recent data from the KATHERINE trial showed a maintained benefit from post-neoadjuvant trastuzumab emtansine (T-DM1) in patients with HER2 loss on RD, even if clinicopathological variables associated with HER2 loss have not been reported. We aimed to characterize the clinicopathological variables and clinical outcomes associated with HER2 loss after NAT in patients with HER2+ eBC.Methods: We retrieved data from a prospectively collected database including all consecutive HER2+ eBC patients treated with NAT at European Institute of Oncology from September 1999 to May 2018. We collected data on age, menopausal status, NAT regimen, clinical and pathological stage, as well as histological subtype and grade, hormone receptor (HR), HER2, and Ki67 status before and after NAT. HR and HER2 status were re-assessed according to the latest ASCO/CAP guidelines. Invasive disease-free survival (IDFS), distant relapse-free survival (DRFS), and overall survival (OS) were estimated using the Kaplan-Meier method and compared using the log-rank test. Univariate (UVA) and multivariate analyses (MVA) were performed to identify variables associated with survival outcomes. Variables considered in UVA included: clinical tumor stage (cT: 1-2 vs 3-4) and nodal status (cN: 0 vs 1-2-3), pre-and post-NAT HR status (neg vs pos), NAT (anti-HER2 agents: yes vs no; endocrine therapy: yes vs no), pathological tumor stage (ypT: is-0-1-2 vs 3-4) and nodal status (ypN: 0 vs 1-2-3). Variables with a p value Citation Format: Stefania Morganti, Antonio Marra, Giulia Viale, Paola Zagami, Elham Sajjadi, Chiara Corti, Giuseppe Curigliano, Nicola Fusco, Carmen Criscitiello. Loss of HER2 on residual disease after neoadjuvant therapy in HER2-positive early breast cancer: Clinicopathological characteristics and association with outcomes [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 P4-07-01.

Introduction: Breast cancer during pregnancy (PrBC) accounts for ~4% of breast cancer cases in young women and its intrinsic biology is still largely undetermined. Tumor microenvironment (TME) of PrBC has been recently characterized with low levels of stromal tumor-infiltrating lymphocytes (TILs) and high relative expression of programmed death-ligand 1 (PD-L1), suggesting an increased immune evasion. The underlying immune landscape, however, has not been unveiled. Given the significant alterations of the immune system during gestation, we hypothesized that the TME of PrBC might have distinct biological traits. Here, we sought to evaluate the outcome of PrBC according to the TME characteristics and to assess whether pathogenic mechanisms of immune evasion are involved. Methods: Representative formalin-fixed paraffin-embedded tissue blocks of 83 consecutive PrBC and 89 age-matched early-onset pregnancy-unrelated breast cancers (controls) were subjected to immunohistochemistry (IHC) using antibodies against CD4, CD8, forkhead box P3 (FOXP3), and PD-L1 (clone 22C3) on a Dako Omnis platform. For all cases, TILs were evaluated according to the International TILs Working Group recommendations. Next-generation sequencing gene expression of 395 genes involved in tumor-immune interactions (Oncomine™ Immune Response Research Assay) was performed on RNA extracted from PrBC (n=20) and controls (n=16). Samples with mapped reads >1,000,000 and valid reads >800,000 were considered adequate. Fisher’s and Chi-squared tests, multinomial logistic regression models, ROC curve, and survival analyses were performed. Results: The fraction of tumors with CD8+TILs was significantly higher in PrBC than in the controls (n=71(85.0%) vs. n=61(68.5%); p=0.02), being mirrored by less cases with CD4+TILs (n=27 (32.5%) vs. n=43 (48.3%); p=0.03). Even higher differences were observed in hormone receptor (HR)+/HER2-negative tumors (CD8: n=39 (88.6%) vs. n=39 (66.1%); p=0.01). After a median follow-up of 78 (range, 1-247), 66/83 women (79.5%) with PrBC were alive and 53/83 (63.8%) relapse-free. Overall, PrBC with CD8+TILs had a better outcome compared to CD8-negative PrBC (OS 81 vs 69 months p=0.05) and CD8 expression was associated with better outcomes in HR+/HER2-negative tumors (OS p=0.02; DFS p=0.04). The overall comparison of immune-related genes in the 34 cases (PrBC, n=18; controls, n=16) that reached the quality parameters revealed significant differences in the expression of 63 immune-related genes. Of these, 4 genes (IFNA17, IFNB1, FUT4, and PECAM1) were upregulated, while 59 genes were downregulated in PrBC compared to the controls. Interestingly, IFNA17, IFNB1, and FUT4 remained upregulated in HR+ PrBC, where a slightly reduced number of differentially expressed genes was observed (n=60). In HR-/HER2- PrBC, only 25 genes were differentially expressed, of which 9, including IFNA17 and PECAM1, were significantly upregulated. Discussion: These data have the potential of improving our knowledge of the immunobiology that characterizes PrBC, suggesting that in these tumors the higher frequency of CD8+TILs might be related to an enhanced anti-tumor immune response, as CD8 expression was associated with better outcomes in PrBC. On the other hand, given that interferons (IFNs) may also trigger immune suppressive mechanisms in cancer cells, the activation of type I IFNs encoded by IFNA17 and IFNB1 seen in our RNA-seq analysis, combined with the lower frequency of CD4+TILs observed, suggest CD4+ cell suppression as a possible mechanism of immune evasion. Conclusion: PrBC TME is characterized by specific patterns of TILs subpopulations due to the possible activation of type I IFNs and its assessment might help in identifying women at high risk of death and recurrence. Citation Format: Nicola Fusco, Elham Sajjadi, Konstantinos Venetis, Barbara Buonomo, Concetta Blundo, Massimo Giroda, Eugenia Di Loreto, Giovanna Scarfone, Stefano Ferrero, Paolo Veronesi, Viviana E. Galimberti, Massimo Barberis, Giuseppe Viale, Elena Guerini-Rocco, Fedro A. Peccatori. Tumor microenvironment characteristics and prognosis in breast cancer during pregnancy: The role of differentially expressed immune-related genes [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 P4-07-02.

Background: The activity impairment of Phosphatase and tensin homolog (PTEN), a tumor suppressor and negative regulator of the PI3K/Akt pathway, is associated with tumorigenesis, tumor progression, and therapy resistance in breast cancer. However, the clinical value of PTEN as a biomarker in these patients is controversial. We sought to determine whether the benefit of traditional biomarkers testing for the identification of high-risk breast cancer is improved by the analysis of PTEN status.Methods: The study group consisted of 608 breast cancers (group A) extracted from our tissue microarray biobank and a total of 4,265 patients (group B) from the METABRIC and MSK studies available at cBioPortal. Cases from the group A were classified as PTEN-low (PTEN-L) or PTEN-retained (PTEN-WT) according to the PTEN expression levels by immunohistochemistry (IHC) and scored based on the ratio between normal and tumor tissue. Clinical and genomic data from group B were also analyzed, and, together with those from group A, relationships between PTEN and the clinicopathologic and molecular features were assessed using Fisher’s exact test, Chi-squared test, or Shapiro-Wilk test for continuous variables. Odds ratio (OR) and corresponding 95% confidence interval (CI) were calculated for each variable. To identify factors associated with PTEN expression, multinomial logistic regression models were defined considering a stepwise selection procedure. Survival curves were built according to the Kaplan-Meier method and compared using the log-rank test.Results: Alteration in PTEN status was significantly different at protein and gene levels, where the reduced protein expression was observed in 280/608 cases (46.1%) from group A, while genetic aberrations in only 315/4,265 (7.4%) cases of group B. PTEN-L tumors were significantly enriched for hormone receptors (HR) and HER2 negativity (n=48, 17.1%) compared to PTEN-WT tumors (n=22, 6.7%; p=0.0008). Lack of HR with or without HER2 overexpression/amplification was significantly associated with worse overall survival but not disease-free survival in PTEN-L but not in PTEN-WT breast cancers (p Citation Format: Nicola Fusco, Elham Sajjadi, Konstantinos Venetis, Marco Invernizzi, Donatella Gambini, Letterio Runza, Stefano Ferrero, Elena Guerini-Rocco. Combined analysis of PTEN and routinely assessed biomarkers reveals new high-risk subgroups of HR- or HER2+ breast cancers [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 P4-10-23.

BACKGROUND. Aromatase inhibitors (AIs) represent a cornerstone of hormone receptor (HR)-positive breast cancers management. However, AIs-induced musculoskeletal symptoms (AIMSS) might be frequently reported during estrogen depletion, with a crucial impact on cancer treatment adherence [1-2]. To date, physical exercise has been proposed as a promising therapeutic intervention to improve symptoms in AIMSS breast cancer patients [3] and recently, also whole-body vibration (WBV) showed promising results in these women. WBV is an effective neuromuscular training proposed to enhance muscle function and proprioception through various frequencies of mechanical vibration inducing repetitive muscle contractions. However, at present, data about tailored and effective multimodal rehabilitative exercise protocols to treat AIMSS are still controversial [3]. Therefore, this pilot randomized controlled study aimed at assessing the safety and efficacy of WBV in breast cancer women suffering from AIMSS. METHODS: In this study we recruited adults women undergoing AIs treatment for breast cancer and suffering from AIMSS. Twentytwo patients were enrolled and subsequently randomly assigned 1:1 to two groups. Group A received physical exercise and WBV, while Group B received physical exercise combined with sham WBV. The patients were assessed at the baseline (T0) and after 4 weeks of treatment (T1). The primary endpoint was the numerical pain rating scale (NPRS) for pain assessment; the secondary endpoints were the handgrip strength (HGS) test to assess appendicular muscle strength, the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) to assess pain, joint stiffness, and function; 10-meter walking test (10MWT) and 6MWT to evaluate physical performance; the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-C30) to assess function, symptoms, and global health. RESULTS: Group A (n=11; mean age: 51.73±10.73 years; body mass index (BMI): 25.56±5.17 kg/m2) showed a statistically significant improvement of pain (NPRS: 6.82±1.17 vs 5.73±1.01; p=0.031). In contrast, patients in Group B, control group, (n=11; mean age: 58.55±9.71 years; BMI: 27.31±3.84 kg/m2), did not significantly improved in terms of pain (NPRS: 6.91±2.02 vs 5.91±2.51; p=0.07). Moreover, significant differences between groups at T1 were found in terms of WOMAC physical functioning (Group A: 77.56±9.853; Group B: 65.63±14.27; p=0.044). Furthermore, a significant improvement in terms of muscle strength, physical performance and quality of life (EORTC QLQ-C30) were reported in intragroup analysis in both groups, without significant differences between groups. We did not register dropouts or side effects in both groups. Lastly, both patients and the physical therapist reported a high level of satisfaction about the rehabilitative intervention performed. CONCLUSION: These findings showed that physical exercise and WBV combination therapy might be safe, well-tolerated and effective in patients with AIMSS. However, further studies are warranted to assess long terms effects of this combined rehabilitative treatment. REFERENCES: 1. Molehin D, Rasha F, Rahman RL, Pruitt K. Regulation of aromatase in cancer. Mol Cell Biochem. 2021. 2. Murphy CC, Bartholomew LK, Carpentier MY, Bluethmann SM, Vernon SW. Adherence to adjuvant hormonal therapy among breast cancer survivors in clinical practice: a systematic review. Breast Cancer Res Treat. 2012;134(2):459-78. 3. Roberts KE, Rickett K, Feng S, Vagenas D, Woodward NE. Exercise therapies for preventing or treating aromatase inhibitor-induced musculoskeletal symptoms in early breast cancer. Cochrane Database Syst Rev. 2020;1:CD012988. Citation Format: Lorenzo Lippi, Alessandro de Sire, Antonio Ammendolia, Carlo Cisari, Konstantinos Venetis, Elham Sajjadi, Nicola Fusco, Marco Invernizzi. Whole-body vibration combined with physical exercise to treat aromatase inhibitor-induced musculoskeletal symptoms in breast cancer women: Results of a pilot randomized controlled study [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 P4-10-15.

The mismatch repair (MMR) system has a key role in supporting the DNA polymerase proofreading function and in maintaining genome stability. Alterations in the MMR genes are driving events of tumorigenesis, tumor progression, and resistance to therapy. These genetic scars may occur in either hereditary or sporadic settings, with different frequencies across tumor types. Appropriate characterization of the MMR status is a crucial task in oncologic pathology because it allows for both the tailored clinical management of cancer patients and surveillance of individuals at risk. The currently available MMR testing methods have specific strengths and weaknesses, and their application across different tumor types would require a tailored approach. This article highlights the indications and challenges in MMR status assessment for molecular pathologists, focusing on the possible strategies to overcome analytical and pre-analytical issues.

Background. Breast cancer fatigue (BCF) is a complex and multidimensional condition characterized by a persistent sense of physical and/or mental stiffness, resulting in a substantial impairment of health-related quality of life in breast cancer patients and survivors. Several therapeutic approaches have been proposed for BCF. Among them, supervised exercise therapy is a valuable non-pharmacologic option. However, the optimal exercise scheme (i.e. type, combination, frequency, intensity, and duration) remains controversial. Here, we sought to evaluate the effects of a specific therapeutic exercise protocol on BCF and muscle performance. Methods. This is a still-recruiting pilot prospective cohort study including women with a diagnosis of BCF up to two months after breast surgery. Exclusion criteria: Hb 2 days of rest for 4 weeks, under the supervision of an experienced physical therapist. Primary outcome evaluation: brief fatigue inventory (BFI). Secondary outcomes: the European organization for research and treatment of cancer quality of life questionnaire (EORTC QLQ-C30); hand grip strength test (HGS); short physical performance battery (SPPB); 10 meter walking test (10MWT); 6 minute walking test (6MWT). All outcomes were assessed at baseline (T0), after 1 month (T1), and after 3 months (T2). Results. Of the 102 patients assessed, 48 did not meet the inclusion and exclusion criteria and 18 refused to sign the informed consent. Finally, 36 BC women (mean age: 55.17 ±7.76 years; body mass index: 25.15 ±5.52 kg/m2) were enrolled. BFI showed a statistically significant reduction both at T1 (5.4 ±1.6 vs 4.2 ±1.7; p=0.004) and T2 (5.4 ±1.6 vs 4.4 ±1.6; p=0.004). Furthermore, we found significant differences at T1 in terms of HGS (20.1 ± 5.8 vs 22.5 ± 5.2: p Citation Format: Marco Invernizzi, Alessandro de Sire, Elham Sajjadi, Konstantinos Venetis, Alessandra Gennari, Nicola Fusco. Exercise therapy to reduce breast cancer fatigue: Results from the EXPECT study [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 PS9-36.

Alterations in the mismatch repair (MMR) system result in genomic instability, neoantigen production, and immune response in cancer. There is evidence that gastroesophageal tumors with MMR deficiency may be susceptible to immune-checkpoint inhibitors treatment, especially in those presenting at advanced-stage disease. Although a number of biomarkers have been developed in histology-agnostic settings to assess MMR status, there is evidence that a tumor-specific testing approach would improve the selection of patients for immunotherapy. However, no testing methods have been developed specifically for gastroesophageal cancers so far. Here, we discuss the state of the art, current advances, and future perspectives of MMR-related biomarkers’ biologic and clinical role in gastroesophageal cancers.

Metaplastic breast cancer (MBC) and triple-negative (TN) BC of no special type are often confounded with each other in terms of survival and prognosis. In this systematic study and meta-analysis, we evaluated the prognosis of each of these two different diagnoses.We conducted a systematic literature search and review using the MOOSE guidelines, through PUBMED database, the Ovid MEDLINE database, and the ISI Web of Science Citation Index Expanded (SCI Expanded). Overall survival (OS) and disease-free survival (DFS) were the main outcomes considered.Our review eventually selected six independent studies, with a total of more than 59 519 patients. MBC was found to associate with worse OS compared to TNBC of no special type, with a significant 40% increased risk of death [summary hazard ratio (SHR) = 1.40, 95% confidence interval (CI): 1.30-1.50]. We found neither heterogeneity ( I2 = 0%) nor evidence of publication bias ( P = 0.82 and P = 0.49 by Begg's and Egger's test, respectively) between studies. No statistically significant difference was found between MBC and TNBC of no special type in terms of DFS (SHR = 1.17, 95% CI: 0.80-1.71).This study demonstrates that TNBC of no special type and MBC have comparable DFS, although the latter presents a significantly worse prognosis in terms of OS. Despite DFS being similar in both subtypes, this did not result in significant OS benefits, with MBC score being the worse of the two diseases.

Immune Checkpoint Inhibitors (ICIs) have remarkably modified the way solid tumors are managed, including breast cancer. Unfortunately, only a relatively small number of breast cancer patients significantly respond to these treatments. To maximize the immunotherapy benefit in breast cancer, several efforts are currently being put forward for the identification of i) the best therapeutic strategy (i.e. ICI monotherapy or in association with chemotherapy, radiotherapy, or other drugs); ii) optimal timing for administration (e.g. early/advanced stage of disease; adjuvant/ neoadjuvant setting); iii) most effective and reliable predictive biomarkers of response (e.g. tumor-infiltrating lymphocytes, programmed death-ligand 1, microsatellite instability associated with mismatch repair deficiency, and tumor mutational burden). In this article, we review the impacts and gaps in the characterization of immune-related biomarkers raised by clinical and translational research studies with immunotherapy treatments. Particular emphasis has been put on the documented evidence of significant clinical benefits of ICI in different randomized clinical trials, along with preanalytical and analytical issues in predictive biomarkers pathological assessment.

Introduction: Based on the SOLAR-1 study, the PI3Kα-specific inhibitor alpelisib has been approved in combination with fulvestrant for postmenopausal women, and men, with HR+/HER2-, PIK3CA-mutated, advanced or metastatic breast cancer (BC). Despite PIK3CA mutations occurring in ⁓40% of HR+/HER2- BC, PIK3CA molecular testing is a new task to be carried out in clinical practice. Adopting the most appropriate testing strategy (RT-PCR vs. NGS) on the most appropriate biological sample is of capital significance in oncologic pathology. Here, we sought to assess the concordance rate for PIK3CA molecular analysis between different technical platforms in both metastatic and matched primary tumors. Methods: From our Institutional registry, n=16 HR+/HER2- metastatic BC, which were found to harbor PIK3CA mutations via next-generation sequencing (NGS) assays [custom panel and Oncomine Comprehensive Assay (OCA) v3 (Thermo Fisher Scientific, Waltham, MA, USA)], were selected. In half of the cases (n=8), matched primary tumors were available and were subjected to PIK3CA testing with NGS. The analytical performance between NGS and a semi-closed RT-PCR (EasyPGX®, Diatech Pharmacogenetics, Italy) was assessed in 13/16 (81.3%) metastatic samples for which archival slides and blocks and/or residual extracted DNA were available, and all the primary tumors. Results: Overall, upfront testing of PIK3CA mutational status with NGS detected genetic alterations in exons 7, 9, and 20. A concordance rate of 100.0% was observed between primary and metastatic tumors. On the other hand, the analysis of primary tumors (n=8) and 13/16 (81.3%) metastatic samples with RT-PCR revealed a concordance of 42.9%. Analytical performance comparison showed that the two technologies were concordant in 7/13 metastatic cases (53.8%). In two of the discordant cases (15.4%), RT-PCR did not identify the PIK3CA mutations due to their absence from its reference range. Interestingly, visual inspection of the RT-PCR raw data increased the concordance to 76.9%. In primary tumors, consensus between the two testing methods was observed in 5 (62.5%) cases. Discussion: The concordance rate analysis shows that upfront PIK3CA molecular testing with NGS appears to be more efficacious compared with RT-PCR. Our data suggest that primary tissues reflect the PIK3CA mutational status when tested with NGS. RT-PCR is simpler with a shorter turnaround time, and less expensive than NGS approaches, however, trained personnel are required for accurate results interpretation. In addition, the limited reference range of this testing method should be taken into account for potential false negative results. Conclusion: In terms of PIK3CA molecular testing, NGS should be the preferred method in comparison with RT-PCR. Primary tumors represent a valid biospecimen to be used for this analysis in the absence of the metastatic sample. Citation Format: Konstantinos Venetis, Francesco Pepe, Elham Sajjadi, Giuseppina Bonizzi, Mariia Ivanova, Davide Vacirca, Alessandra Rappa, Massimo Barberis, Giuseppe Viale, Elena Guerini-Rocco, Elisabetta Munzone, Umberto Malapelle, Nicola Fusco. Comparison of next-generation sequencing and real-time PCR for PIK3CA testing in hormone receptor-positive/HER2-negative breast cancer on metastatic and matched primary tumor samples [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 P6-04-14.

Celiac disease (CD) is a multiorgan autoimmune disorder of the chronic intestinal disease group characterized by duodenal inflammation in genetically predisposed individuals, precipitated by gluten ingestion. The pathogenesis of celiac disease is now widely studied, overcoming the limits of the purely autoimmune concept and explaining its hereditability. The genomic profiling of this condition has led to the discovery of numerous genes involved in interleukin signaling and immune-related pathways. The spectrum of disease manifestations is not limited to the gastrointestinal tract, and a significant number of studies have considered the possible association between CD and neoplasms. Patients with CD are found to be at increased risk of developing malignancies, with a particular predisposition of certain types of intestinal cancer, lymphomas, and oropharyngeal cancers. This can be partially explained by common cancer hallmarks present in these patients. The study of gut microbiota, microRNAs, and DNA methylation is evolving to find the any possible missing links between CD and cancer incidence in these patients. However, the literature is extremely mixed and, therefore, our understanding of the biological interplay between CD and cancer remains limited, with significant implications in terms of clinical management and screening protocols. In this review article, we seek to provide a comprehensive overview of the genomics, epigenomics, and transcriptomics data on CD and its relation to the most frequent types of neoplasms that may occur in these patients.

HER2 status in breast cancer is assessed to select patients eligible for targeted therapy with anti-HER2 therapies. According to the American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP), the HER2 test positivity is defined by protein overexpression (score 3+) at immunohistochemistry (IHC) and/or gene amplification at

Introduction: Breast cancer (BC) occurring during gestation or lactation is rare, yet highly challenging under both biological and clinical standpoints. This condition, referred to as pregnancy-associated (PA) BC (PABC), shows enrichment in mismatch repair (MMR) deficiency mutational signature, higher expression of immune-checkpoint genes, and less tumor-infiltrating lymphocytes (TILs) compared to non-PA BCs. Despite these insights, no comprehensive data on MMR protein status, immune checkpoints, and immune microenvironment are currently available for these tumors. The aim of this study was to characterize the MMR status and immunologic milieu of PABC. Methods: Among a multi-Institutional database comprising 142 PABCs, we conducted a comparative analysis of a cohort of PABC (n=29) and a control group of age-matched non-PA BCs (n=74). Distinct areas of each tumor and the corresponding normal breast tissue were incorporated into a tissue microarray (4-6 cores per case, mean 4.5). For all cases, both stromal and intratumoral TILs were quantified according to the International TILs Working Group recommendations. Representative slides were subjected to immunohistochemical (IHC) analysis of the MMR proteins (i.e. MLH1, MSH2, MSH6, and PMS2), programmed death-ligand 1 (PD-L1), CD4, and CD8. Cases were classified as MMR-proficient (pMMR), MMR-deficient (dMMR), and, when the protein was expressed only in a part of the tumor, MMR-heterogeneous (hMMR). PD-L1 expression was evaluated separately in the tumor cells, stromal TILs, and intratumoral TILs. Finally, the relative proportion of CD4+ and CD8+ cells was assessed in both stromal and intratumoral TILs. Results: The study group included 4 (14%) Luminal A, 10 (35%) Luminal B, 4 (14%) non-luminal HER2+, and 11 (37%) triple-negative (TN) PABCs. Taken together, both the dMMR and hMMR status were more common in PABCs than in non-PA BC (n=3/29, 19% vs. n=6/74, 8% and n=7/29, 24% vs. 14/74, 19%, respectively). Specifically, dMMR was seen in Luminal A (n=2, 50%) and in TN (n=1, 9%) PABCs. Conversely, in non-PA BCs, all Luminal A (n=15, 100%) were pMMR, while 5/45(11%) Luminal B and 1/13 (7%) TNBC were dMMR. Despite no differences were observed in intratumoral TILs, PABCs showed significantly higher levels of stromal TILs (p=0.01). Compared to the control group, PD-L1 expression in PABCs was significantly higher in the tumor cells and in stromal TILs (p Discussion: This study is the first to investigate the immune response alongside the MMR status by IHC in PABCs. Our findings broaden the understanding of the immunobiology underpinning PABC, suggesting that in these tumors i) MMR protein alterations occur at higher frequency than in non-PA BCs; ii) the tumor cells and tumor microenvironment may be capable to suppress the adaptive arm of immune system through the expression of PD-L1; and iii) lymphocytes located at the periphery rather than those inside of the tumor are likely to be implicated in the immune modulation. Conclusion: The MMR system and immune microenvironment may play a consistent role in the natural history of PABCs. An intimate knowledge of the multifaceted interplay between tumor and tumor immune microenvironment is likely to unveil clinically relevant mechanisms that may have a positive net health impact for women with BC during gestation or lactation. Citation Format: Nicola Fusco, Elena Guerini-Rocco, Barbara Buonomo, Roberto Croci, Caterina Fumagalli, Gianluca Lopez, Giorgio A Croci, Letterio Runza, Elham Sajjadi, Concetta Blundo, Luca Despini, Massimo Giroda, Viviana E Galimberti, Paolo Veronesi, Massimo Barberis, Stefano Ferrero, Giovanna Scarfone, Silvano Bosari, Giuseppe Viale, Fedro Peccatori. Integrated analysis of mismatch repair, PD-L1, and immune microenvironment status in pregnancy-associated breast cancers [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-05-03.

Alterations to mismatch repair (MMR) pathways are a known cause of cancer, particularly colorectal and endometrial carcinomas. Recently, checkpoint inhibitors have been approved for use in MMR-deficient cancers of any type (Prasad et al. in JAMA Oncol 4:157-158, 2018). Functional studies in breast cancer have shown associations between MMR loss, resistance to aromatase inhibitors and sensitivity to palbociclib (Haricharan et al. in Cancer Discov 7:1168-1183, 2017). Herein, we investigate the clinical meaning of MMR deficiency in breast cancer by immunohistochemical assessment of MSH2, MSH6, MLH1 and PMS2 on a large series of breast cancers linked to detailed biomarker and long-term outcome data.Cases were classified as MMR intact when all four markers expressed nuclear reactivity, but MMR-deficient when at least one of the four biomarkers displayed loss of nuclear staining in the presence of positive internal stromal controls on the tissue microarray core.Among the 1635 cases with interpretable staining, we identified 31 (1.9%) as MMR-deficient. In our cohort, MMR deficiency was present across all major breast cancer subtypes, and was associated with high-grade, low-progesterone receptor expression and high tumor-infiltrating lymphocyte counts. MMR deficiency is significantly associated with inferior overall (HR 2.29, 95% CI 1.02-5.17, p = 0.040) and disease-specific survival (HR 2.71, 95% CI 1.00-7.35, p = 0.042) in the 431 estrogen receptor-positive patients who were uniformly treated with tamoxifen as their sole adjuvant systemic therapy.Overall, this study supports the concept that breast cancer patients with MMR deficiency as assessed by immunohistochemistry may be good candidates for alternative treatment approaches such as immune checkpoint or CDK4 inhibitors.

The clinical management of cancers has progressed rapidly into the immunopathology era, with the unprecedented histology-agnostic approval of pembrolizumab in mismatch repair (MMR) deficient tumors. Despite the significant recent achievements in the treatment of these patients, however, the identification of clinically relevant subclasses of cancers based on the MMR status remains a major challenge. Many investigations have assessed the role of different diagnostic tools, including immunohistochemistry, microsatellite instability, and tumor mutational burden in both prognostic and therapeutic settings, with heterogenous results. To date, there are no tumor-specific guidelines or companion diagnostic tests for MMR assessment, and this analysis is often performed with locally developed methods. In this review, we provide a comprehensive overview of the current state-of-knowledge of MMR alterations in syndromic and sporadic tumors and discuss the available armamentarium for MMR pathologic characterization, from morphology to high-throughput molecular tools.

Background Phosphatase and tensin homolog (PTEN) loss is associated with tumorigenesis, tumor progression, and therapy resistance in breast cancer. However, the clinical value of PTEN as a biomarker in these patients is controversial. We sought to determine whether the benefit of traditional biomarkers testing is improved by the analysis of PTEN status for the identification of high-risk breast cancer. Methods A cohort of 608 patients with breast cancer was included in this study. Based on the expression on the neoplastic cells compared to the normal internal controls by immunohistochemistry (IHC), cases were classified as PTEN-low (PTEN-L) or PTEN-retained (PTEN-WT). The former constituted the study group, while the latter the control group. Analysis of gene expression was performed on publicly available genomic data and included 4265 patients from the METABRIC and MSK cohorts retrieved from cBioPortal. The Shapiro-Wilk test was used to analyze the normal distributions of continuous variables. Relationships between PTEN status and the clinicopathologic and molecular features of the patient population were assessed using Fisher’s exact test or Chi-squared/Wilcoxon rank-sum test. Survival curves were built according to the Kaplan-Meier method. Results Alteration in PTEN status was significantly different at protein and gene levels, where the reduced protein expression was observed in 280/608 cases (46.1%) from our group, while genetic aberrations in only 315/4265 (7.4%) cases of the METABRIC and MSK cohorts. PTEN-L tumors were significantly enriched for hormone receptors (HR) and HER2 negativity (n = 48, 17.1%) compared to PTEN-WT tumors (n = 22, 6.7%; p = 0.0008). Lack of HR with or without HER2 overexpression/amplification was significantly associated with worse overall survival (OS) in PTEN-L but not in PTEN-WT breast cancers (p p = 0.002). Conclusions The combined analysis of PTEN, HER2, and HR status offers relevant information for a more precise risk assessment of patients with breast cancer.

Despite extensive research efforts, advanced gastric cancer still has a dismal prognosis with conventional treatment options. Immune checkpoint inhibitors have revolutionized the treatment landscape for many solid tumors. Amongst gastric cancer subtypes, tumors with microsatellite instability and Epstein Barr Virus positive tumors provide the strongest rationale for responding to immunotherapy. Various predictive biomarkers such as mismatch repair status, programmed death ligand 1 expression, tumor mutational burden, assessment of tumor infiltrating lymphocytes and circulating biomarkers have been evaluated. However, results have been inconsistent due to different methodologies and thresholds used. Clinical implementation therefore remains a challenge. The role of immune checkpoint inhibitors in gastric cancer is emerging with data from monotherapy in the heavily pre-treated population already available and studies in earlier disease settings with different combinatorial approaches in progress. Immune checkpoint inhibitor combinations with chemotherapy (CT), anti-angiogenics, tyrosine kinase inhibitors, anti-Her2 directed therapy, poly (ADP-ribose) polymerase inhibitors or dual checkpoint inhibitor strategies are being explored. Moreover, novel strategies including vaccines and CAR T cell therapy are also being trialed. Here we provide an update on predictive biomarkers for response to immunotherapy with an overview of their strengths and limitations. We discuss clinical trials that have been reported and trials in progress whilst providing an account of future steps needed to improve outcome in this lethal disease.

The clinical outcome of patients with a diagnosis of hormone receptor (HR)+ breast cancer has improved remarkably since the arrival of endocrine therapy. Yet, resistance to standard treatments is a major clinical challenge for breast cancer specialists and a life-threatening condition for the patients. In breast cancer, mismatch repair (MMR) status assessment has been demonstrated to be clinically relevant not only in terms of screening for inherited conditions such as Lynch syndrome, but also for prognostication, selection for immunotherapy, and early identification of therapy resistance. Peculiar traits characterize the MMR biology in HR+ breast cancers compared to other cancer types. In these tumors, MMR genetic alterations are relatively rare, occurring in ~3 % of cases. On the other hand, modifications at the protein level can be observed also in the absence of gene alterations and vice versa. In HR+ breast cancers, the prognostic role of MMR deficiency has been confirmed by several studies, but its predictive value remains a matter of controversy. The characterization of MMR status in these patients is troubled by the lack of tumor-specific guidelines and/or companion diagnostic tests. For this reason, precise identification of MMR-deficient breast cancers can be problematic. A deeper understanding of the MMR biology and clinical actionability in HR+ breast cancer may light the path to effective tumor-specific diagnostic tools. For a precise MMR status profiling, the specific strengths and limitations of the available technologies should be taken into consideration. This article aims at providing a comprehensive overview of the current state of knowledge of MMR alterations in HR+ breast cancer. The available armamentarium for MMR testing in these tumors is also examined along with possible strategies for a tailored pathological characterization.

Gastroesophageal adenocarcinoma (GEA) is a global health issue with a high fatality-to-case ratio and a 5-year overall survival that has only slightly improved. High-throughput molecular profiling has uncovered a profound complexity and heterogeneity in GEA biology, which limits considerably the treatment advances. Liquid biopsy with circulating tumor (ct)DNA analysis could elucidate GEA molecular heterogeneity and provide diagnostic, prognostic and predictive information to guide clinical decision-making. However, only a handful of studies have shown positive results for the application of ctDNA analysis in GEA clinical management. As a result, no comprehensive information is available to date on this continuously evolving topic. Here, we discuss the current state of knowledge, along with promises and challenges related to ctDNA analysis in GEA.

Malignant pleural mesothelioma (MPM) is a rare tumor with an extremely poor prognosis. Its pathogenesis is related to an immune response against asbestos fibers. The T-lymphocytes, including CD8POS and CD4POS cells, are an important part of the MPM immune microenvironment, and likely contribute to the therapy resistance observed in these tumors. Here, we sought to characterize the MPM-specific lymphocytes subpopulations within the tumor immune microenvironment to identify novel clinically relevant immunologic subtypes of tumors. Representative formalin-fixed, paraffin-embedded (FFPE) tissue blocks of 88 MPMs were included in tissue microarrays and subjected to tumor-infiltrating lymphocytes (TILs) quantification and subtyping by immunohistochemistry (IHC) with antibodies specific for CD4, CD8, and CD19. Further, PD-L1 (clone 22C3) expression was assessed by IHC as a combined positive score (CPS). Our data show that PD-L1 expression by tumor cells or the presence of a sarcomatoid component is related to increased stromal TILs presence in MPM. Survival analyses showed that low CD4POS and high CD8POS stromal TILs are associated with poor patients' survival. In MPMs with PD-L1 CPS > 1, stromal CD8HIGH was a poor prognostic factor, akin stromal CD4POS peritumoral TILs correlated with a worse prognosis. Furthermore, we demonstrated that a high CD4POS/CD8POS ratio in the tumor immune microenvironment is an independent prognostic factor for survival. Finally, we provided evidence that the characterization of the stromal immune landscape of MPM predicts responses to chemotherapy in subgroups of MPM. The results of this study provide novel insights into the clinical scenario of immune-related biomarkers in MPM.

Breast cancer is the most common malignancy in women worldwide, with a relatively high proportion of patients experiencing resistance to standard treatments. Cellular immunotherapy (CI), which is based on the extraction, modification, and re-infusion of the patient's immune cells, is showing promising results in these patients. Among CI possible approaches, adoptive cell therapy (ACT) and dendritic cell (DC) vaccination are the most comprehensively explored in both primary/translational research studies and clinical trials. ACT may include the use of tumor-infiltrating lymphocytes (TILs), T cell receptor (TCR)-, or chimeric antigen receptor (CAR)-engineered T-cells. There are indications suggesting that a biomarker-based approach might be beneficial in effectively selecting breast cancer patients for CI. Here, we sought to provide the current knowledge of CI in breast cancer, focusing on candidate biomarkers, ongoing clinical trials, limitations, and immediate future perspectives.

Alterations in the tumor suppressor phosphatase and tensin homolog (PTEN) occur in a substantial proportion of solid tumors. These events drive tumorigenesis and tumor progression. Given its central role as a downregulator of the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway, PTEN is deeply involved in cell growth, proliferation, and survival. This gene is also implicated in the modulation of the DNA damage response and in tumor immune microenvironment modeling. Despite the actionability of PTEN alterations, their role as biomarkers remains controversial in clinical practice. To date, there is still a substantial lack of validated guidelines and/or recommendations for PTEN testing. Here, we provide an update on the current state of knowledge on biologic and genetic alterations of PTEN across the most frequent solid tumors, as well as on their actual and/or possible clinical applications. We focus on possible tailored schemes for cancer patients’ clinical management, including risk assessment, diagnosis, prognostication, and treatment.

Triple-negative breast cancers (TNBC) comprise biologically and clinically heterogeneous diseases characterized by the lack of hormone receptors (HR) and HER2 expression. This subset of tumors accounts for 15-20% of all breast cancers and pursues an ominous clinical course. However, there is a spectrum of low-risk TNBCs with no/minimal metastatic potential, including the salivary gland-type tumors, those with extensive apocrine differentiation and/or high tumor-infiltrating lymphocytes, and small-sized, early-stage (pT1a/bN0M0) TNBCs. De-escalating the treatment in low-risk TNBC, however, is not trivial because of the substantial lack of dedicated randomized clinical trials and cancer registries. The development of new diagnostic and/or prognostic biomarkers based on clinical and molecular aspects of low-risk TNBCs would lead to improved clinical treatment. Here, we sought to provide a portrait of the clinicopathological and molecular features of low-risk TNBC, with a focus on the diagnostic challenges along with the most important biological characteristics underpinning their favorable clinical course.