UBC9, the sole E2-conjugating enzyme required for SUMOylation, is a key regulator of essential cellular functions and, as such, is frequently altered in cancers. Along these lines, we recently ...reported that its expression gradually increases during early stages of human papillomavirus (HPV)-mediated cervical lesions transformation. However, a better understanding of how UBC9 is exploited by transforming viral oncoproteins is still needed. In the present study, we show that in human samples HPV drives UBC9 up-regulation also in very early steps of head and neck tumorigenesis, pointing to the important role for UBC9 in the HPV-mediated carcinogenic program. Moreover, using HPV-infected pre-cancerous tissues and primary human keratinocytes as the natural host of the virus, we investigate the pathological meaning and the cellular mechanisms responsible for UBC9 de-regulation in an oncoviral context. Our results show that UBC9 overexpression is promoted by transforming viral proteins to increase host cells' resistance to apoptosis. In addition, ultrastuctural, pharmacological and genetic approaches crucially unveil that UBC9 is physiologically targeted by autophagy in human cells. However, the presence of HPV E6/E7 oncoproteins negatively impacts the autophagic process through selective inhibition of autophagosome-lysosome fusion, finally leading to p53 dependent UBC9 accumulation during viral-induced cellular transformation. Therefore, our study elucidates how UBC9 is manipulated by HPV oncoproteins, details the physiological mechanism by which UBC9 is degraded in cells, and identifies how HPV E6/E7 impact on autophagy. These findings point to UBC9 and autophagy as novel hallmarks of HPV oncogenesis, and open innovative avenues towards the treatment of HPV-related malignancies.
Background
Breast cancer during pregnancy (PrBC) is a rare condition known for its aggressive clinical behavior. The presence of tumor-infiltrating lymphocytes (TILs) has been shown to have a ...significant impact on the prognosis of these patients. Despite some biological characteristics of the tumor that may differ depending on the gestational age, little is known about the dynamics of the immune landscape within the tumor microenvironment (TME) in PrBC. Therefore, in this study, our objective was to gain comprehensive insights into the relationship between gestational age at breast cancer diagnosis and the composition of the TME.
Methods
n
= 108 PrBC were selected from our institutional registry and categorized based on the gestational age by trimester. For all cases, TILs were profiled according to the International TILs Working Group recommendations, and subtyped by CD4, CD8, and forkhead box P3 (FOXP3) immunohistochemistry. PD-L1 was tested according to the combined positive score (CPS) using the IHC 22C3 pharmDx assay, with a cutoff value of ≥10 for positivity. The statistical approach encompassed Fisher’s and Chi-squared tests, with appropriate adjustments for multiple comparisons, logistic regression models, and survival analyses based on the Kaplan–Meier method.
Results
The proportion of patients with poorly differentiated (G3) neoplasms increased as the gestational age advanced (first trimester,
n
= 25, 56.8%; second trimester,
n
= 27, 69.2%; third trimester,
n
= 21, 87.5%;
p
= 0.03). The histologic subtypes as well as the hormone receptor (HR) and HER2 status did not show significant changes across different pregnancy trimesters. In the HR+/HER2– subtype, there was a higher proportion of tumors with high/moderate TILs in the early phases of pregnancy, similar to FOXP3 expression (TILs: first trimester,
n
= 10, 35.7%; second trimester,
n
= 2, 10.5%; third trimester,
n
= 0;
p
= 0.02; FOXP3: first trimester,
n
= 10, 40%; second trimester,
n
= 3, 15.8%; third trimester,
n
= 0;
p
= 0.03). The median follow-up for our cohort was 81 months. Patients who relapsed after a breast cancer diagnosis during the first trimester were more frequently PD-L1-negative, unlike those with no disease recurrence (
n
= 9, 100% vs.
n
= 9, 56.3%;
p
= 0.03; hormone therapy and
n
= 9, 100% vs.
n
= 7, 53.9%;
p
= 0.02; chemotherapy). No statistically significant differences were seen among the three trimesters in terms of survival outcome.
Conclusion
The TME dynamics of HR+/HER2− PrBC vary based on gestational age, suggesting that immune tolerance expression during later gestational age could explain the increased aggressiveness of tumors diagnosed at that stage.
Breast cancer is the most common malignancy occurring during gestation. In early-stage breast cancer during pregnancy (PrBC), breast-conserving surgery (BCS) with delayed RT is a rational alternative ...to mastectomy, for long considered the standard-of-care. Regrettably, no specific 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. All patients with a diagnosis of PrBC during the first trimester of pregnancy jointly managed in two PrBC-specialized Centers were included in this study. All patients underwent BCS followed by adjuvant radiotherapy to the ipsilateral breast after delivery. Histopathological features and biomarkers were first profiled on pre-surgical biopsies. The primary outcome was the isolated local recurrence (ILR). 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. The patients that were subjected to radical surgery (n=14) served as controls. None of the patients experienced perioperative surgical complications. No ILR were observed within three months (n=30), 1 year (n=27), and 5 years (n=18) after surgery. Among the study group, 4 (12.3%) patients experienced ILR or new carcinomas after 6-13 years, the same number (n=4) had metastatic dissemination after 3-7 years. These patients are still alive and disease-free after 14-17 years of follow-up. The rate of recurrences and metastasis in the controls were not significantly different. The findings provide evidence that BCS in the first trimester PrBC is feasible and reasonably safe for both the mother and the baby.
Breast cancer during pregnancy (PrBC) is a rare tumor with only a little information on its immune landscape. Here, we sought to characterize the cellular composition of the tumor microenvironment ...(TME) of PrBC and identify its differences from early-onset breast cancer (EOBC) in non-pregnant women. A total of 83 PrBC and 89 EOBC were selected from our Institutional registry and subjected to tumor-infiltrating lymphocytes (TILs) profiling and immunohistochemistry for CD4, CD8, forkhead box P3 (FOXP3), and programmed death-ligand 1 (PD-L1) (clone 22C3). A significantly lower frequency of hormone receptor (HR)-positive tumors was observed in PrBC. The prevalence of low/null PD-L1 and CD8+TILs was higher in PrBC than in the controls, specifically in HR+/HER2– breast cancers. PrBC had a significantly higher risk of relapse and disease-related death, compared to EOBC. The presence of TILs and each TIL subpopulation were significantly associated with disease relapse. Moreover, the death rate was higher in PrBC with CD8+ TILs. The TME of PrBC is characterized by specific patterns of TIL subpopulations with significant biological and prognostic roles. Routine assessment of TILs and TILs subtyping in these patients would be a valid addition to the pathology report that might help identify clinically relevant subsets of women with PrBC.
Vorinostat (suberoylanilide hydroxamic acid; SAHA) is a histone deacetylase inhibitor (HDACi) approved in the clinics for the treatment of T-cell lymphoma and with the potential to be effective also ...in breast cancer. We investigated the responsiveness to SAHA in human breast primary tumors and cancer cell lines.
We observed a differential response to drug treatment in both human breast primary tumors and cancer cell lines. Gene expression analysis of the breast cancer cell lines revealed that genes involved in cell adhesion and redox pathways, especially glutathione metabolism, were differentially expressed in the cell lines resistant to SAHA compared with the sensitive ones, indicating their possible association with drug resistance mechanisms. Notably, such an association was also observed in breast primary tumors. Indeed, addition of buthionine sulfoximine (BSO), a compound capable of depleting cellular glutathione, significantly enhanced the cytotoxicity of SAHA in both breast cancer cell lines and primary breast tumors.
We identify and validate transcriptional differences in genes involved in redox pathways, which include potential predictive markers of sensitivity to SAHA.
In breast cancer, it could be relevant to evaluate the expression of antioxidant genes that may favor tumor resistance as a factor to consider for potential clinical application and treatment with epigenetic drugs (HDACis).
Abstract
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.
Abstract
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.
Abstract
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<0.01) but not in intratumoral TILs. Hence, 9 (31%) PABCs were PD-L1+, with tumor proportion scores (TPS) ranging from 2 to 10 (mean 5), while only 4 (5%) non-PA BCs were PD-L1+ (TPS 2-20, mean 8)(p=0.01). The expression of PD-L1 in stromal TILs was higher in PABCs (n=9, 31%) than in the controls (n=19, 26%)(p=0.005). In Luminal tumors, both the CD4+ and CD8+ populations were more represented than in non-PA BCs (p=0.01), while in TNBC only the relative proportion of CD4+ cells was significantly higher (p=0.02).
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.
The biological antagonism between Notch and Numb controls the proliferative/differentiative balance in development and homeostasis. Although altered Notch signaling has been linked to human diseases, ...including cancer, evidence for a substantial involvement of Notch in human tumors has remained elusive. Here, we show that Numb-mediated control on Notch signaling is lost in ∼50% of human mammary carcinomas, due to specific Numb ubiquitination and proteasomal degradation. Mechanistically, Numb operates as an oncosuppressor, as its ectopic expression in Numb-negative, but not in Numb-positive, tumor cells inhibits proliferation. Increased Notch signaling is observed in Numb-negative tumors, but reverts to basal levels after enforced expression of Numb. Conversely, Numb silencing increases Notch signaling in normal breast cells and in Numb-positive breast tumors. Finally, growth suppression of Numb-negative, but not Numb-positive, breast tumors can be achieved by pharmacological inhibition of Notch. Thus, the Numb/Notch biological antagonism is relevant to the homeostasis of the normal mammary parenchyma and its subversion contributes to human mammary carcinogenesis.
The 14th St Gallen International Breast Cancer Conference (2015) reviewed substantial new evidence on locoregional and systemic therapies for early breast cancer. Further experience has supported the ...adequacy of tumor margins defined as ‘no ink on invasive tumor or DCIS’ and the safety of omitting axillary dissection in specific cohorts. Radiotherapy trials support irradiation of regional nodes in node-positive disease. Considering subdivisions within luminal disease, the Panel was more concerned with indications for the use of specific therapies, rather than surrogate identification of intrinsic subtypes as measured by multiparameter molecular tests. For the treatment of HER2-positive disease in patients with node-negative cancers up to 1 cm, the Panel endorsed a simplified regimen comprising paclitaxel and trastuzumab without anthracycline as adjuvant therapy. For premenopausal patients with endocrine responsive disease, the Panel endorsed the role of ovarian function suppression with either tamoxifen or exemestane for patients at higher risk. The Panel noted the value of an LHRH agonist given during chemotherapy for premenopausal women with ER-negative disease in protecting against premature ovarian failure and preserving fertility. The Panel noted increasing evidence for the prognostic value of commonly used multiparameter molecular markers, some of which also carried prognostic information for late relapse. The Panel noted that the results of such tests, where available, were frequently used to assist decisions about the inclusion of cytotoxic chemotherapy in the treatment of patients with luminal disease, but noted that threshold values had not been established for this purpose for any of these tests. Multiparameter molecular assays are expensive and therefore unavailable in much of the world. The majority of new breast cancer cases and breast cancer deaths now occur in less developed regions of the world. In these areas, less expensive pathology tests may provide valuable information. The Panel recommendations on treatment are not intended to apply to all patients, but rather to establish norms appropriate for the majority. Again, economic considerations may require that less expensive and only marginally less effective therapies may be necessary in less resourced areas. Panel recommendations do not imply unanimous agreement among Panel members. Indeed, very few of the 200 questions received 100% agreement from the Panel. In the text below, wording is intended to convey the strength of Panel support for each recommendation, while details of Panel voting on each question are available in supplementary Appendix S2, available at Annals of Oncology online.
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