PD-L1 intrinsically promotes the proliferation of breast cancer cells through the SKP2-p27/p21 axis.

BACKGROUND: PD-L1 intrinsically promotes tumor progression through multiple mechanisms, which potentially leads to resistance to anti-PD-1/PD-L1 therapies. The intrinsic effect of PD-L1 on breast cancer (BC) cell proliferation has not been fully elucidated. METHODS: we used proteomics, gene expression knockdown (KD), quantitative immunofluorescence (qIF), western blots, functional assays including colony-forming assay (CFA) and real-time cell analyzer (RTCA), and in vivo data using immunohistochemistry in breast cancer patients. RESULTS: PD-L1 promoted BC cell proliferation by accelerating cell cycle entry at the G1-to-S phase transition. Global proteomic analysis of the differentially expressed nuclear proteins indicated the involvement of several proliferation-related molecules, including p21CIP1/WAF1. Western blotting and qIF demonstrated the higher expression of SKP2 and the lower expression of p21CIP1/WAF1 and p27Kip1 in PD-L1 expressing (PD-L1pos) cells as compared to PD-L1 KD (PD-L1KD) cells. Xenograft-derived cells and the TCGA BC dataset confirmed this relationship in vivo. Functionally, CFA and RTCA demonstrated the central role of SKP2 in promoting PD-L1-mediated proliferation. Finally, immunohistochemistry in 74 breast cancer patients confirmed PD-L1 and SKP-p21/p27 axis relationship, as it showed a highly statistically significant correlation between SKP2 and PD-L1 expression (p < 0.001), and both correlated significantly with the proliferation marker Ki-67 (p < 0.001). On the other hand, there was a statistically significant inverse relationship between PD-L1 and p21CIP1/WAF1 expression (p = 0.005). Importantly, double negativity for p21CIP1/WAF1 and p27Kip1 correlated significantly with PD-L1 (p < 0.001), SKP2 (p = 0.002), and Ki-67 (p = 0.002). CONCLUSIONS: we have demonstrated the role of the SKP2-p27/p21 axis in intrinsic PD-L1-enhanced cell cycle progression. Inhibitors of SKP2 expression can alleviate resistance to ICPIs.

Expanding the phenotypic and genotypic spectrum of GGPS1 related congenital muscular dystrophy.

Congenital muscular dystrophies are a group of progressive disorders with wide range of symptoms associated with diverse cellular mechanisms. Recently, biallelic variants in GGPS1 were linked to a distinct autosomal recessive form of muscular dystrophy associated with hearing loss and ovarian insufficiency. In this report, we present a case of a young patient with a homozygous variant in GGPS1. The patient presented with only proximal muscle weakness, and elevated liver transaminases with spared hearing function. The hepatic involvement in this patient caused by a novel deleterious variant in the gene extends the phenotypic and genotypic spectrum of GGPS1 related muscular dystrophy.

Fascin is essential for mammary gland lactogenesis.

Fascin expression has commonly been observed in certain subtypes of breast cancer, where its expression is associated with poor clinical outcome. However, its role in normal mammary gland development has not been elucidated. Here, we used a fascin knockout mouse model to assess its role in normal mammary gland morphogenesis and lactation. Fascin knockout was not embryonically lethal, and its effect on the litter size or condition at birth was minimal. However, litter survival until the weaning stage significantly depended on fascin expression solely in the nursing dams. Accordingly, pups that nursed from fascin-/- dams had smaller milk spots in their abdomen, suggesting a lactation defect in the nursing dams. Mammary gland whole-mounts of pregnant and lactating fascin-/- mice showed significantly reduced side branching and alveologenesis. Despite a typical composition of basal, luminal, and stromal subsets of mammary cells and normal ductal architecture of myoepithelial and luminal layers, the percentage of alveolar progenitors (ALDH+) in fascin-/- epithelial fraction was significantly reduced. Further in-depth analyses of fascin-/- mammary glands showed a significant reduction in the expression of Elf5, the master regulator of alveologenesis, and a decrease in the activity of its downstream target p-STAT5. In agreement, there was a significant reduction in the expression of the milk proteins, whey acidic protein (WAP), and ß-casein in fascin-/- mammary glands. Collectively, our data demonstrate, for the first time, the physiological role of fascin in normal mammary gland lactogenesis, an addition that could reveal its contribution to breast cancer initiation and progression.

Bi-allelic variants in HOPS complex subunit VPS41 cause cerebellar ataxia and abnormal membrane trafficking.

Membrane trafficking is a complex, essential process in eukaryotic cells responsible for protein transport and processing. Deficiencies in vacuolar protein sorting (VPS) proteins, key regulators of trafficking, cause abnormal intracellular segregation of macromolecules and organelles and are linked to human disease. VPS proteins function as part of complexes such as the homotypic fusion and vacuole protein sorting (HOPS) tethering complex, composed of VPS11, VPS16, VPS18, VPS33A, VPS39 and VPS41. The HOPS-specific subunit VPS41 has been reported to promote viability of dopaminergic neurons in Parkinson's disease but to date has not been linked to human disease. Here, we describe five unrelated families with nine affected individuals, all carrying homozygous variants in VPS41 that we show impact protein function. All affected individuals presented with a progressive neurodevelopmental disorder consisting of cognitive impairment, cerebellar atrophy/hypoplasia, motor dysfunction with ataxia and dystonia, and nystagmus. Zebrafish disease modelling supports the involvement of VPS41 dysfunction in the disorder, indicating lysosomal dysregulation throughout the brain and providing support for cerebellar and microglial abnormalities when vps41 was mutated. This provides the first example of human disease linked to the HOPS-specific subunit VPS41 and suggests the importance of HOPS complex activity for cerebellar function.

Tocilizumab potentiates cisplatin cytotoxicity and targets cancer stem cells in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a very aggressive subtype with high recurrence rate and no molecular targets for therapies. This subtype is characterized by high expression/secretion of the proinvasive/metastatic interleukin-6 (IL-6) cytokine. In the present study, we have shown that tocilizumab inhibits the IL-6/STAT3 signaling and suppresses the cancer/inflammatory epigenetic IL-6/STAT3/NF-κB positive feedback loop. Furthermore, tocilizumab inhibited the proliferative and the migratory/invasiveness capacities as well as the epithelial-to-mesenchymal transition (EMT) process in TNBC cells. Importantly, tocilizumab suppressed the stemness-related characteristics of TNBC cells, through the inhibition of the Wnt/ß-catenin breast cancer stem cell-related pathway. Additionally, we have shown that tocilizumab suppresses the paracrine activation of normal breast stromal fibroblasts to myofibroblats. Moreover, tocilizumab sensitized TNBC cells to the cytotoxic effect of cisplatin in vitro. Furthermore, pharmacological inhibition of IL-6 by tocilizumab had great inhibitory effect on tumor growth and the EMT process in humanized orthotopic breast tumors in mice. In addition, tocilizumab potentiated the proapoptotic effect of cisplatin in humanized breast tumors. Together, these findings indicate that tocilizumab can suppress the prometastatic capacity of TNBC cells and enhances the cytotoxic effect of cisplatin against these cells. Therefore, tocilizumab could be of great therapeutic value for these hard-to-treat TNBC patients.

CD3+T-lymphocyte infiltration is an independent prognostic factor for advanced nasopharyngeal carcinoma.

BACKGROUND: Locally advanced nasopharyngeal carcinoma (LA-NPC) is a relatively rare disease in the west but more common in East Asia and areas of the Middle East like Saudi Arabia. Despite the advances in radiation therapy techniques, some patients relapse after treatment. In the coming era of cancer immunotherapy, prognostic factors for LA-NPC need to be further defined using immune-relevant markers. Several markers are available; however, the most robust and accessible/affordable marker is not well-defined. METHODS: Retrospectively, tumor-infiltrating lymphocytes (TIL), their subsets as well as tumoral PD-L1 expression were analyzed in tumor tissues from 63 LA-NPC patients treated with platinum-based concurrent chemo-radiotherapy (CCRT) in addition to 20 cases with metastatic (MET) disease. Immunostaining was done using a validated and fully automated system. Scoring was done by two independent pathologists and results were compared. RESULTS: There was no statistical difference between LA-NPC and MET disease in terms of CD3+, CD8+ TIL infiltration, or tumoral PD-L1 expression. In LA-NPC, low CD3+ TIL infiltration highly correlated with shorter disease-free survival (DFS, HR = 8.5, p = < 0.001) and overall survival (OS, HR = 13, p = 0.015) with substantial agreement between scoring pathologists. A similar correlation was found between low CD8+ TIL and survival. Correlation of total TIL was significant with DFS (HR = 4.0, p = 0.008), borderline with OS and the correlation was dependent on the scoring pathologist. Having histological WHO type I&II correlated significantly with shorter DFS (HR 4.03, p = 0.008) and low CD3+ TIL (p = 0.009). Subgroup analysis of LA-NPC that included undifferentiated type (WHO type III) cases only (n = 58), showed a strong correlation between low CD3+ TIL and shorter DFS (HR = 7.2, p = < 0.001) and OS (HR = 17.3, p = 0.008). PD-L1 was expressed in 72% of type III LA-NPC cases while lacking PD-L1 expression correlated with shorter OS (HR = 6.1, p = 0.031). Patients with a combination of low CD3+ TIL and lack of PD-L1 expression had the worst OS (p < 0.001). CONCLUSIONS: CD3+ TIL is promising as a robust and independent prognostic marker for DFS and OS of LA-NPC patients treated with platinum-based CCRT. We would suggest the use of CD3 + TIL as a stratifying factor for LA-NPC, which warrants further validation in prospective trials.

ß1 Integrin is essential for fascin-mediated breast cancer stem cell function and disease progression.

Breast cancer remains the second cause of tumor-related mortality in women worldwide mainly due to chemoresistance and metastasis. The chemoresistance and metastasis are attributed to a rare subpopulation with enriched stem-like characteristics, thus called Cancer Stem Cells (CSCs). We have previously reported aberrant expression of the actin-bundling protein (fascin) in breast cancer cells, which enhances their chemoresistance, metastasis and enriches CSC population. The intracellular mechanisms that link fascin with its downstream effectors are not fully elucidated. Here, loss and gain of function approaches in two different breast cancer models were used to understand how fascin promotes disease progression. Importantly, findings were aligned with expression data from actual breast cancer patients. Expression profiling of a large breast cancer dataset (TCGA, 530 patients) showed statistically significant correlation between fascin expression and a key adherence molecule, ß1 integrin (ITGB1). In vitro manipulation of fascin expression in breast cancer cells exhibited its direct effect on ITGB1 expression. Fascin-mediated regulation of ITGB1 was critical for several breast cancer cell functions including adhesion to different extracellular matrix, self-renewability and chemoresistance. Importantly, there was a significant relationship between fascin and ITGB1 co-expression and short disease-free as well as overall survival in chemo-treated breast cancer patients. This novel role of fascin effect on ITGB1 expression and its outcome on cell self-renewability and chemoresistance strongly encourages for dual targeting of fascin-ITGB1 axis as a therapeutic approach to halt breast cancer progression and eradicate it from the root.

PD-L1 promotes OCT4 and Nanog expression in breast cancer stem cells by sustaining PI3K/AKT pathway activation.

The expression of PD-L1 in breast cancer is associated with estrogen receptor negativity, chemoresistance and epithelial-to-mesenchymal transition (EMT), all of which are common features of a highly tumorigenic subpopulation of cancer cells termed cancer stem cells (CSCs). Hitherto, the expression and intrinsic role of PD-L1 in the dynamics of breast CSCs has not been investigated. To address this issue, we used transcriptomic datasets, proteomics and several in vitro and in vivo assays. Expression profiling of a large breast cancer dataset (530 patients) showed statistically significant correlation (p < 0.0001, r = 0.36) between PD-L1 expression and stemness score of breast cancer. Specific knockdown of PD-L1 using ShRNA revealed its critical role in the expression of the embryonic stem cell transcriptional factors: OCT-4A, Nanog and the stemness factor, BMI1. Conversely, these factors could be induced upon PD-L1 ectopic expression in cells that are normally PD-L1 negative. Global proteomic analysis hinted for the central role of AKT in the biology of PD-L1 expressing cells. Indeed, PD-L1 positive effect on OCT-4A and Nanog was dependent on AKT activation. Most importantly, downregulation of PD-L1 compromised the self-renewal capability of breast CSCs in vitro and in vivo as shown by tumorsphere formation assay and extreme limiting dilution assay, respectively. This study demonstrates a novel role for PD-L1 in sustaining stemness of breast cancer cells and identifies the subpopulation and its associated molecular pathways that would be targeted upon anti-PD-L1 therapy.

Fascin Is Critical for the Maintenance of Breast Cancer Stem Cell Pool Predominantly via the Activation of the Notch Self-Renewal Pathway.

An emerging dogma shows that tumors are initiated and maintained by a subpopulation of cancer cells that hijack some stem cell features and thus referred to as "cancer stem cells" (CSCs). The exact mechanism that regulates the maintenance of CSC pool remains largely unknown. Fascin is an actin-bundling protein that we have previously demonstrated to be a major regulator of breast cancer chemoresistance and metastasis, two cardinal features of CSCs. Here, we manipulated fascin expression in breast cancer cell lines and used several in vitro and in vivo approaches to examine the relationship between fascin expression and breast CSCs. Fascin knockdown significantly reduced stem cell-like phenotype (CD44hi /CD24lo and ALDH+ ) and reversal of epithelial to mesenchymal transition. Interestingly, expression of the embryonic stem cell transcriptional factors (Oct4, Nanog, Sox2, and Klf4) was significantly reduced when fascin expression was down-regulated. Functionally, fascin-knockdown cells were less competent in forming colonies and tumorspheres, consistent with lower basal self-renewal activity and higher susceptibility to chemotherapy. Fascin effect on CSC chemoresistance and self-renewability was associated with Notch signaling. Activation of Notch induced the relevant downstream targets predominantly in the fascin-positive cells. Limiting-dilution xenotransplantation assay showed higher frequency of tumor-initiating cells in the fascin-positive group. Collectively, our data demonstrated fascin as a critical regulator of breast CSC pool at least partially via activation of the Notch self-renewal signaling pathway and modification of the expression embryonic transcriptional factors. Targeting fascin may halt CSCs and thus presents a novel therapeutic approach for effective treatment of breast cancer. Stem Cells 2016;34:2799-2813 Video Highlight: https://youtu.be/GxS4fJ_Ow-o.

Bidirectional crosstalk between PD-L1 expression and epithelial to mesenchymal transition: significance in claudin-low breast cancer cells.

BACKGROUND: The T-cell inhibitory molecule PD-L1 (B7-H1, CD274) is expressed on tumor cells of a subset of breast cancer patients. However, the mechanism that regulates PD-L1 expression in this group of patients is still not well-identified. METHODS: We have used loss and gain of function gene manipulation approach, multi-parametric flow cytometry, large scale gene expression dataset analysis and immunohistochemistry of breast cancer tissue sections. RESULTS: Induction of epithelial to mesenchymal transition (EMT) in human mammary epithelial cells upregulated PD-L1 expression, which was dependent mainly on the activation of the PI3K/AKT pathway. Interestingly, gene expression signatures available from large cohort of breast tumors showed a significant correlation between EMT score and the PD-L1 mRNA level (p < 0.001). Strikingly, very strong association (p < 0.0001) was found between PD-L1 expression and claudin-low subset of breast cancer, which is known to have high EMT score. On the protein level, significant correlation was found between PD-L1 expression and standard markers of EMT (p = 0.005) in 67 breast cancer patients. Importantly, specific downregulation of PD-L1 in claudin-low breast cancer cells showed signs of EMT reversal as manifested by CD44 and Vimentin downregulation and CD24 upregulation. CONCLUSIONS: We have demonstrated a bidirectional effect between EMT status and PD-L1 expression especially in claudin-low subtype of breast cancer cells. Our findings highlights the potential dual benefit of anti-PD-L1 particularly in this subset of breast cancer patients that will likely benefit more from anti-PD-L1 targeted therapy as well as in monitoring biological changes upon treatment.

Metformin inhibits 7,12-dimethylbenz[a]anthracene-induced breast carcinogenesis and adduct formation in human breast cells by inhibiting the cytochrome P4501A1/aryl hydrocarbon receptor signaling pathway.

Recent studies have established that metformin (MET), an oral anti-diabetic drug, possesses antioxidant activity and is effective against different types of cancer in several carcinogen-induced animal models and cell lines. However, whether MET can protect against breast cancer has not been reported before. Therefore, the overall objectives of the present study are to elucidate the potential chemopreventive effect of MET in non-cancerous human breast MCF10A cells and explore the underlying mechanism involved, specifically the role of cytochrome P4501A1 (CYP1A1)/aryl hydrocarbon receptor (AhR) pathway. Transformation of the MCF10A cells into initiated breast cancer cells with DNA adduct formation was conducted using 7,12-dimethylbenz[a]anthracene (DMBA), an AhR ligand. The chemopreventive effect of MET against DMBA-induced breast carcinogenesis was evidenced by the capability of MET to restore the induction of the mRNA levels of basic excision repair genes, 8-oxoguanine DNA glycosylase (OGG1) and apurinic/apyrimidinic endonuclease1 (APE1), and the level of 8-hydroxy-2-deoxyguanosine (8-OHdG). Interestingly, the inhibition of DMBA-induced DNA adduct formation was associated with proportional decrease in CYP1A1 and in NAD(P)H: quinone oxidoreductase 1 (NQO1) gene expression. Mechanistically, the involvements of AhR and nuclear factor erythroid 2-related factor-2 (Nrf2) in the MET-mediated inhibition of DMBA-induced CYP1A1 and NQO1 gene expression were evidenced by the ability of MET to inhibit DMBA-induced xenobiotic responsive element and antioxidant responsive element luciferase reporter gene expression which suggests an AhR- and Nrf2-dependent transcriptional control. However, the inability of MET to bind to AhR suggests that MET is not an AhR ligand. In conclusion, the present work shows a strong evidence that MET inhibits the DMBA-mediated carcinogenicity and adduct formation by inhibiting the expression of CYP1A1 through an AhR ligand-independent mechanism.

Locally advanced nasopharyngeal carcinoma in adolescents treated with tomotherapy: Experience at King Faisal Specialist Hospital and Research Centre.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a rare disease worldwide; To the best of our knowledge, there is no established standard of care specifically tailored for the adolescent population. The majority of existing research relies on retrospective data analysis. OBJECTIVE: Evaluate clinical features, treatment results, prognostic factors and late toxicities of locally advanced NPC patients treated with tomotherapy. DESIGN: Retrospective. SETTINGS: Tertiary care hospital. PATIENTS AND METHODS: Between January 2007 and January 2020, we treated patients with NPC, aged between 14 and 21 years, with concomitant chemoradiotherapy using tomotherapy at our institution. We prospectively collected details of clinical characteristics, treatment modalities, outcomes and prognostic factors of these patients and then analysed them retrospectively. MAIN OUTCOME MEASURES: 3-5 years overall survival (OS), 3-5 years locoregional control rate, 3-5 years disease-free survival (DFS), prognostic factors. SAMPLE SIZE: 51 patients. RESULTS: There were 26 male and 25 female patients included in our study. The mean age was 16.5 years, 5 (9.8%) patients with stage III, and 46 (90.2%) with stage IVa according to the American Joint Committee on Cancer, 8th edition staging system. Most patients (98%) received two or more cycles of induction chemotherapy. All patients received concomitant chemoradiotherapy. The median total dose of radiotherapy delivered was 6600 cGy (range 4800-7000). With a median follow-up of 73 months (range 9-168 months), a 5-year locoregional control rate, 5-year OS and 5-year DFS rates were 100%, 86.8% and 71.7%, respectively. Five years later, disease control was 71.7%. Ten (19.6%) patients had disease recurrence in the form of distant metastases during the follow up. CONCLUSIONS: Helical tomotherapy has an excellent late toxicity profile without compromising clinical outcome for patients with NPC. Radiotherapy remains the mainstay of treatment of nasopharyngeal carcinoma to achieve remarkable local control rates. LIMITATIONS: Single institution experience, small number of patients, and retrospective design.

A unique STK4 mutation truncating only the C-terminal SARAH domain results in a mild clinical phenotype despite severe T cell lymphopenia: Case report.

Mutations in STK4 (MST1) are implicated in a form of autosomal recessive combined immunodeficiency, resulting in recurrent infections (especially Epstein-Barr virus viremia), autoimmunity, and cardiac malformations. Here we report a patient with an atypically mild presentation of this disease, initially presenting with severe T cell lymphopenia (< 500 per mm3) and intermittent neutropenia, but now surviving well on immunoglobulins and prophylactic antibacterial treatment. She harbors a unique STK4 mutation that lies further downstream than all others reported to date. Unlike other published cases, her mRNA transcript is not vulnerable to nonsense mediated decay (NMD) and yields a truncated protein that is expected to lose only the C-terminal SARAH domain. This domain is critical for autodimerization and autophosphorylation. While exhibiting significant differences from controls, this patient's T cell proliferation defects and susceptibility to apoptosis are not as severe as reported elsewhere. Expression of PD-1 is in line with healthy controls. Similarly, the dysregulation seen in immunophenotyping is not as pronounced as in other published cases. The nature of this mutation, enabling its evasion from NMD, provides a rare glimpse into the clinical and cellular features associated with the absence of a "null" phenotype of this protein.

Differential marker expression by cultures rich in mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells have properties that make them amenable to therapeutic use. However, the acceptance of mesenchymal stem cells in clinical practice requires standardized techniques for their specific isolation. To date, there are no conclusive marker (s) for the exclusive isolation of mesenchymal stem cells. Our aim was to identify markers differentially expressed between mesenchymal stem cell and non-stem cell mesenchymal cell cultures. We compared and contrasted the phenotype of tissue cultures in which mesenchymal stem cells are rich and rare. By initially assessing mesenchymal stem cell differentiation, we established that bone marrow and breast adipose cultures are rich in mesenchymal stem cells while, in our hands, foreskin fibroblast and olfactory tissue cultures contain rare mesenchymal stem cells. In particular, olfactory tissue cells represent non-stem cell mesenchymal cells. Subsequently, the phenotype of the tissue cultures were thoroughly assessed using immuno-fluorescence, flow-cytometry, proteomics, antibody arrays and qPCR. RESULTS: Our analysis revealed that all tissue cultures, regardless of differentiation potential, demonstrated remarkably similar phenotypes. Importantly, it was also observed that common mesenchymal stem cell markers, and fibroblast-associated markers, do not discriminate between mesenchymal stem cell and non-stem cell mesenchymal cell cultures. Examination and comparison of the phenotypes of mesenchymal stem cell and non-stem cell mesenchymal cell cultures revealed three differentially expressed markers - CD24, CD108 and CD40. CONCLUSION: We indicate the importance of establishing differential marker expression between mesenchymal stem cells and non-stem cell mesenchymal cells in order to determine stem cell specific markers.

Profiling of normal and malignant breast tissue show CD44high/CD24low phenotype as a predominant stem/progenitor marker when used in combination with Ep-CAM/CD49f markers.

BACKGROUND: Accumulating evidence supports cancer to initiate and develop from a small population of stem-like cells termed as cancer stem cells (CSC). The exact phenotype of CSC and their counterparts in normal mammary gland is not well characterized. In this study our aim was to evaluate the phenotype and function of stem/progenitor cells in normal mammary epithelial cell populations and their malignant counterparts. METHODS: Freshly isolated cells from both normal and malignant human breasts were sorted using 13 widely used stem/progenitor cell markers individually or in combination by multi-parametric (up to 9 colors) cell sorting. The sorted populations were functionally evaluated by their ability to form colonies and mammospheres, in vitro. RESULTS: We have compared, for the first time, the stem/progenitor markers of normal and malignant breasts side-by-side. Amongst all markers tested, we found CD44high/CD24low cell surface marker combination to be the most efficient at selecting normal epithelial progenitors. Further fractionation of CD44high/CD24low positive cells showed that this phenotype selects for luminal progenitors within Ep-CAMhigh/CD49f + cells, and enriches for basal progenitors within Ep-CAM-/low/CD49f + cells. On the other hand, primary breast cancer samples, which were mainly luminal Ep-CAMhigh, had CD44high/CD24low cells among both CD49fneg and CD49f + cancer cell fractions. However, functionally, CSC were predominantly CD49f + proposing the use of CD44high/CD24low in combination with Ep-CAM/CD49f cell surface markers to further enrich for CSC. CONCLUSION: Our study clearly demonstrates that both normal and malignant breast cells with the CD44high/CD24low phenotype have the highest stem/progenitor cell ability when used in combination with Ep-CAM/CD49f reference markers. We believe that this extensive characterization study will help in understanding breast cancer carcinogenesis, heterogeneity and drug resistance.

Peripheral blood eosinophil count is associated with response to chemoimmunotherapy in metastatic triple-negative breast cancer.

Introduction: There is evidence for an association between peripheral blood eosinophil count (PBEC) and response to cancer immunotherapy; however, such data is limited in metastatic triple-negative breast cancer (mTNBC). Patients & methods: This report presents patients (n = 14) who received a combination of durvalumab and paclitaxel for mTNBC (NCT02628132). Results: There was a statistically significant correlation (p = 0.028) between an increase in PBEC (>300/mm3) during treatment and response to the combination therapy. Survival analysis showed a statistically significant association between progression-free survival and increased PBEC, after therapy (p = 0.005). A similar trend existed for overall survival, although it did not reach statistical significance (p = 0.167). Conclusion: This is the first study to report on eosinophilia in mTNBC treated with chemoimmunotherapy and supports a role for eosinophils in immunotherapy for mTNBC.

Plain language summary Previous reports have shown that an increase in peripheral blood eosinophil count is associated with a good response to cancer immunotherapy; however, data on this association is limited in a subtype of breast cancer called metastatic triple-negative breast cancer (mTNBC). The eosinophil count in patients who received the combination of an immunotherapeutic agent, durvalumab and a chemotherapeutic agent, paclitaxel, in mTNBC was assessed. There was a statistically significant association between an increase in eosinophils during treatment and the response to therapy. The progression of the disease was slower or less likely to occur in patients with increased eosinophils. This study supports the role of eosinophils in immunotherapy for mTNBC.

Neoadjuvant concurrent chemoradiotherapy using infusional gemcitabine in locally advanced rectal cancer: A phase II trial.

INTRODUCTION: Gemcitabine is a well-known radiosensitizer. Herein, we tested the efficacy and toxicity of preoperative concurrent infusional gemcitabine and radiotherapy in locally advanced rectal cancer. PATIENTS AND METHODS: This was a phase II, single-arm trial. Eligible patients had a diagnosis of rectal adenocarcinoma with clinical stage T3-T4 and/or nodal involvement, age ≥18 years, and no prior chemotherapy or radiotherapy. Patients received preoperative radiation at a dose of 50.4-54 Gy over 28 days with concurrent infusional gemcitabine administered at a dose of 100 mg/m2 over the course of 24 h weekly for 6 weeks. The primary endpoint was pathological complete response (pCR). RESULTS: Forty patients were recruited. Only one patient did not complete therapy due to death. Eight patients did not undergo surgery, one died, two progressed to nonresectable disease, and five withdrew consent. Five patients progressed prior to surgery, with two having unresectable metastases and three having resectable liver metastases. One was found to have peritoneal metastasis during surgery. Out of the 32 patients who underwent surgery, seven achieved pCR at a rate of 20%. With a median follow-up of 30 months, four additional patients had a distant relapse (one had a subsequent local relapse). The 3-year event-free and overall survival rates were 70% and 85%, respectively. The commonest preoperative grade 3-4 toxicity included lymphopenia (50%), neutropenia (41%), anemia (15%), diarrhea (12%), abdominal pain (12%), and proctitis (8%). CONCLUSION: Concurrent preoperative chemoradiotherapy using infusional gemcitabine for locally advanced rectal cancer achieved an encouraging degree of local control with manageable toxicity.

Prognostic markers compared to CD3+TIL in locally advanced nasopharyngeal carcinoma.

ABSTRACT: Locally advanced nasopharyngeal carcinoma (LA-NPC) is more prevalent in some geographic regions, including Saudi Arabia. Typically, Tumor-Node-Metastasis (TNM) staging is used in NPC. However, it is inadequate to assess the prognosis of LA-NPC.Therefore, we analyzed and compared several previously reported prognostic factors in LA-NPC patients, retrospectively, including CD3+tumor-infiltrating lymphocytes (TIL) and peripheral blood hemoglobin, EBV DNA copy number, ratios of albumin-to-alkaline phosphatase ratio (AAPR), neutrophils, or platelets-to-lymphocytes (NLR, PLR). The studied cohort was 83 LA-NPC patients previously recruited for a randomized phase II trial with a different aim.Univariate cox regression analysis showed no significant correlation between any of the tested variables with disease-free survival (DFS) or overall survival (OS) with the exception of low CD3+ TIL infiltration, which correlated significantly with DFS (HR = 6.7, P = <.001) and OS (HR = 9.1, P = .043). Similarly, in a validated multivariate cox regression analysis, only low CD3+ TIL correlated significantly with DFS (HR = 7.0, P < .001 for TIL) and OS (HR = 9.4, P = .040).Among tested parameters, CD3+ TIL was the only independent prognostic marker for DFS and OS in LA-NPC patients treated with CCRT. This study supports the use of CD3+TIL, over other factors, as an independent prognostic factor in LA-NPC.

Osteoprotegerin (OPG) mediates the anti-carcinogenic effects of normal breast fibroblasts and targets cancer stem cells through inhibition of the ß-catenin pathway.

Normal breast fibroblasts (NBFs) support and maintain the architecture of the organ, and can also suppress tumorigenesis. However, the mechanisms involved are not fully understood. We have shown here that NBFs suppress breast carcinogenesis through secretion of osteoprotegerin (OPG), a soluble decoy receptor for the Receptor Activator of NF-κB ligand (RANKL). Indeed, NBFs and human recombinant OPG (rOPG), suppressed breast cancer cells proliferation and motility through inhibition of the epithelial-to-mesenchymal transition (EMT) process both in vitro and in vivo. Additionally, rOPG inhibited the IL-6/STAT3 and NF-κB pathways as well as the OPG gene, which turned out to be STAT3-regulated. This was confirmed using denosumab, a RANKL-targeted antibody, which also inhibited NF-κB, down-regulated OPG and repressed EMT in breast cancer cells grown in 2D and 3D. Importantly, both rOPG and denosumab targeted cancer stem cells (CSCs). This was mediated through inhibition of the CSC-related pathway ß-catenin. Moreover, rOPG reduced tumor growth and inhibited breast CSC biomarkers in orthotopic humanized breast tumors. Therefore, normal mammary fibroblasts can suppress carcinogenesis through OPG, which constitutes great potential as preventive and/or therapeutic molecule for breast carcinomas.

Weekly Paclitaxel given concurrently with Durvalumab has a favorable safety profile in triple-negative metastatic breast cancer.

Therapeutic anti-PD-L1 antibodies are safe as a monotherapy, albeit with minimal efficacy in triple-negative breast cancer (TNBC). This trial aimed to test the safety and efficacy of Durvalumab and Paclitaxel in metastatic TNBC. In this open-label, one-arm trial, five cycles of weekly paclitaxel were delivered intravenously (IV) concurrent with Durvalumab that was given IV every 2 weeks. The combination was preceded by one cycle of paclitaxel alone, for immunological priming, followed by Durvalumab solo until disease progression or unacceptable toxicity. Between 2017 and 2019, 14 patients received at least one cycle of the combination therapy. The therapy was safe with no-dose limiting toxicity, except one case of skin lesions. Adverse events (AEs) were reported in 71% of patients, and there was no death due to the combination therapy. Regardless of grade, the most common AEs were headache and peripheral neuropathy, as each happened in four patients (29%), followed by fatigue and skin rash in three patients (21%) each. Grade 3/4 AEs were experienced by three patients (21%), with the most common being headache and anemia, which happened in two patients (14%). The confirmed objective response rate (ORR) was observed in five patients with a median duration of 10.0 months. Median Progression-free survival (PFS) and overall survival (OS) were 5 and 20.7 months, respectively. The combination of Durvalumab and Paclitaxel is safe, leaving room for additional agents. This is the first report on the combination of Durvalumab and Paclitaxel in the treatment of TNBC (NCT02628132).