Real-World Evaluation of Disease Progression After CDK 4/6 Inhibitor Therapy in Patients With Hormone Receptor-Positive Metastatic Breast Cancer.

BACKGROUND: Cyclin-dependent kinase 4/6 inhibitors (CDKi) have changed the landscape for treatment of patients with hormone receptor positive, human epidermal growth factor receptor 2-negative (HR+/HER-) metastatic breast cancer (MBC). However, next-line treatment strategies after CDKi progression are not yet optimized. We report here the impact of clinical and genomic factors on post-CDKi outcomes in a single institution cohort of HR+/HER2- patients with MBC. METHODS: We retrospectively reviewed the medical records of patients with HR+/HER2- MBC that received a CDKi between April 1, 2014 and December 1, 2019 at our institution. Data were summarized using descriptive statistics, the Kaplan-Meier method, and regression models. RESULTS: We identified 140 patients with HR+/HER2- MBC that received a CDKi. Eighty percent of patients discontinued treatment due to disease progression, with a median progression-free survival (PFS) of 6.0 months (95% CI, 5.0-7.1), whereas those that discontinued CDKi for other reasons had a PFS of 11.3 months (95% CI, 4.6-19.4) (hazard ratio (HR) 2.53, 95% CI, 1.50-4.26 [P = .001]). The 6-month cumulative incidence of post-CDKi progression or death was 51% for the 112 patients who progressed on CDKi. Patients harboring PTEN mutations pre-CDKi treatment had poorer clinical outcomes compared to those with wild-type PTEN. CONCLUSION: This study highlights post-CDKi outcomes and the need for further molecular characterization and novel therapies to improve treatments for patients with HR+/HER2- MBC.

Cationic Polymer Modified Mesoporous Silica Nanoparticles for Targeted SiRNA Delivery to HER2+ Breast Cancer.

In vivo delivery of siRNAs designed to inhibit genes important in cancer and other diseases continues to be an important biomedical goal. We now describe a new nanoparticle construct that has been engineered for efficient delivery of siRNA to tumors. The construct is comprised of a 47-nm mesoporous silica nanoparticle (MSNP) core coated with a cross-linked PEI-PEG copolymer, carrying siRNA against the HER2 oncogene, and coupled to the anti-HER2 monoclonal antibody (trastuzumab). The construct has been engineered to increase siRNA blood half-life, enhance tumor-specific cellular uptake, and maximize siRNA knockdown efficacy. The optimized anti-HER2-nanoparticles produced apoptotic death in HER2 positive (HER2+) breast cancer cells grown in vitro, but not in HER2 negative (HER2-) cells. One dose of the siHER2-nanoparticles reduced HER2 protein levels by 60% in trastuzumab-resistant HCC1954 xenografts. Multiple doses administered intravenously over 3 weeks significantly inhibited tumor growth (p < 0.004). The siHER2-nanoparticles have an excellent safety profile in terms of blood compatibility and low cytokine induction, when exposed to human peripheral blood mononuclear cells. The construct can be produced with high batch-to-batch reproducibility and the production methods are suitable for large-scale production. These results suggest that this siHER2-nanoparticle is ready for clinical evaluation.

A dose response analysis of a specific bone marrow concentrate treatment protocol for knee osteoarthritis.

BACKGROUND: Prior studies describing the treatment of symptomatic knee osteoarthritis with injections of bone marrow concentrate have provided encouraging results. The relationship between the cellular dose contained within the bone marrow concentrate and efficacy of the treatment, however, is unclear. In the present study we describe clinical outcomes for symptomatic knee osteoarthritis in relation to higher and lower cell concentrations contained within a bone marrow concentrate treatment protocol. METHODS: Data from an ongoing patient registry was culled to identify 373 patients that received bone marrow concentrate injections for the treatment of 424 osteoarthritic knee joints. The clinical scales for these patients were assessed at baseline and then tracked post-procedure at 1, 3, 6 and 12 months, and annually thereafter. Tracked outcomes included the numeric pain scale; a lower extremity functional questionnaire; an International Knee Documentation Committee scale; and a subjective improvement rating scale. Using pain and functional outcome measures, a receiver operating characteristic analysis was used to define an optimal clinical outcome threshold at which bone marrow nucleated cell count could be divided into either a lower or higher cell count group within a treatment protocol. RESULTS: The lower and higher cell count groups were defined using a threshold of 4 × 10(8) cells. There were 224 and 185 knee joints treated in the lower (≤4 × 10(8)) and higher (>4 × 10(8)) cell count groups respectively. Most joints were diagnosed with early stage knee osteoarthritis. Both the lower and higher cell count groups demonstrated significant positive results with the treatment for all of the pain and functional metrics. The higher cell count group reported lower post treatment numeric pain scale values, in comparison with the lower cell count group (1.6 vs. 3.2; P < 0.001). No significant differences were detected for the other metrics, however. CONCLUSIONS: Improved function and reduced pain was observed in patients treated with a bone marrow concentrate protocol regardless of cellular dose; however, patients receiving a higher concentration of cells reported a better pain outcome in comparison with the lower dose group. These preliminary findings suggest that cell dose may be an important factor governing clinical outcomes in autologous bone marrow concentrate treatment of knee osteoarthritis. Further studies using a larger patient population may help elucidate these findings.

MicroRNA-21 regulates the self-renewal of mouse spermatogonial stem cells.

MicroRNAs (miRs) play a key role in the control of gene expression in a wide array of tissue systems, where their functions include the regulation of self-renewal, cellular differentiation, proliferation, and apoptosis. However, the functional importance of individual miRs in controlling spermatogonial stem cell (SSC) homeostasis has not been investigated. Using high-throughput sequencing, we profiled the expression of miRs in the Thy1(+) testis cell population, which is highly enriched for SSCs, and the Thy1(-) cell population, composed primarily of testis somatic cells. In addition, we profiled the global expression of miRs in cultured germ cells, also enriched for SSCs. Our results demonstrate that miR-21, along with miR-34c, -182, -183, and -146a, are preferentially expressed in the Thy1(+) SSC-enriched population, compared with Thy1(-) somatic cells. Importantly, we demonstrate that transient inhibition of miR-21 in SSC-enriched germ cell cultures increased the number of germ cells undergoing apoptosis and significantly reduced the number of donor-derived colonies of spermatogenesis formed from transplanted treated cells in recipient mouse testes, indicating that miR-21 is important in maintaining the SSC population. Moreover, we show that in SSC-enriched germ cell cultures, miR-21 is regulated by the transcription factor ETV5, known to be critical for SSC self-renewal.

PD-L1 expression in pancreaticobiliary adenosquamous carcinoma: a single-institution case series.

Background: The programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) pathway is a potent negative regulator of T-cell-mediated immune response that is upregulated in many neoplasms. Pancreaticobiliary adenosquamous carcinoma (PB-ASC) is an aggressive cancer that carries a poorer prognosis compared with pure pancreaticobiliary adenocarcinoma (PB-AC). To date, there is little published information regarding PD-L1 expression in PB-ASC. The aim of the study was to examine the relationship between PD-L1 expression and tumor-infiltrating lymphocytes in PB-ASC and PB-AC. Methods: We evaluated 15 PB-ASCs (10 pancreatic, 5 gallbladder) and 34 control PB-ACs (22 pancreatic ductal, and 12 gallbladder) for tumor expression of PD-L1 using anti-PD-L1 (E1L3N) antibody. All tumors were classified into three immune phenotypes: immune inflamed (II), immune excluded (IE), and immune desert (ID) according to the distribution of tumor-infiltrating lymphocytes in tumor tissues. Results: The frequency of PD-L1 expression was significantly higher in PB-ASC (10/15; 66.7%) than in PB-AC (3/34; 8.8%). In PB-ASC, PD-L1 expression occurred exclusively in the squamous component in six cases, exclusively in the glandular component in one case, and in both the squamous and the glandular components in three cases. PD-L1 expression in PB-ASC was irrespective of the tumor immune status, whereas its expression in PB-AC was observed only in tumors with the II or IE phenotype. The ID phenotype was relatively rare (4/15; 26.7%) in PB-ASC compared with PB-AC (22/34; 65%; P=0.02). Conclusions: PB-ASCs are notably enriched in inflammatory response and showed significantly higher PD-L1 expression than PB-AC (P<0.001), suggesting a potential therapeutic role for immune checkpoint inhibitors in managing patients with PB-ASC.

A Review of Circulating Tumor DNA as a Biomarker Guide for Total Neoadjuvant Therapy in Patients with Locally Advanced Rectal Cancer.

PURPOSE: Non-operative management of patients with locally advanced rectal cancer (LARC) is emerging as a popular approach for patients that have no evidence of disease following neoadjuvant therapy. However, high rates of local recurrence or distant metastases have highlighted the urgent need for robust biomarker strategies to aid clinical management of these patients. METHODS: This review summarizes recent advances in the utility of cell-free (cf) and circulating tumor (ct) DNA as potential biomarkers to help guide individualized non-operative management strategies for LARC patients receiving neoadjuvant therapy. RESULTS: Liquid biopsies and the detection of cfDNA/ctDNA is an emerging technology with the potential to provide a non-invasive approach to monitor disease response and improve the identification of patients with LARC that would best benefit from non-operative management. CONCLUSIONS: Substantial work is still needed before cfDNA/ctDNA monitoring can be widely adopted in the clinical setting. Studies reviewed herein highlight several areas of opportunity for improving the effectiveness and utility of cfDNA/ctDNA for managing patients with LARC.

Updated Management of Colorectal Cancer Liver Metastases: Scientific Advances Driving Modern Therapeutic Innovations.

Colorectal cancer is the second leading cause of cancer-related deaths in the United States and accounts for an estimated 1 million deaths annually worldwide. The liver is the most common site of metastatic spread from colorectal cancer, significantly driving both morbidity and mortality. Although remarkable advances have been made in recent years in the management for patients with colorectal cancer liver metastases, significant challenges remain in early detection, prevention of progression and recurrence, and in the development of more effective therapeutics. In 2017, our group held a multidisciplinary state-of-the-science symposium to discuss the rapidly evolving clinical and scientific advances in the field of colorectal liver metastases, including novel early detection and prognostic liquid biomarkers, identification of high-risk cohorts, advances in tumor-immune therapy, and different regional and systemic therapeutic strategies. Since that time, there have been scientific discoveries translating into therapeutic innovations addressing the current management challenges. These innovations are currently reshaping the treatment paradigms and spurring further scientific discovery. Herein, we present an updated discussion of both the scientific and clinical advances and future directions in the management of colorectal liver metastases, including adoptive T-cell therapies, novel blood-based biomarkers, and the role of the tumor microbiome. In addition, we provide a comprehensive overview detailing the role of modern multidisciplinary clinical approaches used in the management of patients with colorectal liver metastases, including considerations toward specific molecular tumor profiles identified on next generation sequencing, as well as quality of life implications for these innovative treatments.

Association between Gut Microbiota and Breast Cancer: Diet as a Potential Modulating Factor.

Breast cancer (BCa) has many well-known risk factors, including age, genetics, lifestyle, and diet; however, the influence of the gut microbiome on BCa remains an emerging area of investigation. This study explores the connection between the gut microbiome, dietary habits, and BCa risk. We enrolled newly diagnosed BCa patients and age-matched cancer-free controls in a case-control study. Comprehensive patient data was collected, including dietary habits assessed through the National Cancer Institute Diet History Questionnaire (DHQ). 16S rRNA amplicon sequencing was used to analyze gut microbiome composition and assess alpha and beta diversity. Microbiome analysis revealed differences in the gut microbiome composition between cases and controls, with reduced microbial diversity in BCa patients. The abundance of three specific microbial genera-Acidaminococus, Tyzzerella, and Hungatella-was enriched in the fecal samples taken from BCa patients. These genera were associated with distinct dietary patterns, revealing significant associations between the presence of these genera in the microbiome and specific HEI2015 components, such as vegetables and dairy for Hungatella, and whole fruits for Acidaminococus. Demographic characteristics were well-balanced between groups, with a significantly higher body mass index and lower physical activity observed in cases, underscoring the role of weight management in BCa risk. Associations between significant microbial genera identified from BCa cases and dietary intakes were identified, which highlights the potential of the gut microbiome as a source of biomarkers for BCa risk assessment. This study calls attention to the complex interplay between the gut microbiome, lifestyle factors including diet, and BCa risk.

c-Myc expression and MEK1-induced Erk2 nuclear localization are required for TGF-beta induced epithelial-mesenchymal transition and invasion in prostate cancer.

Understanding the initial mechanisms by which epithelial cells transform to an invasive phenotype is critical to the development of diagnostics that can identify the metastatic potential of cancers as well as therapeutic agents that can prevent metastases. Changes in cellular response to the transforming growth factor-beta (TGF-ß) cytokine are known to promote epithelial cell invasion and metastasis in part through induction of epithelial-mesenchymal transitions (EMTs). In this report, we demonstrate that non-metastatic human prostate cancer cell lines of increasing Gleason score can be induced to undergo EMT when treated with TGF-ß in combination with epidermal growth factor. Mechanistic studies revealed that in cells stably transfected with activated Ras, TGF-ß alone induced EMT and that a Ras-Raf-MEK1, but not MEK2, signaling cascade is necessary and sufficient for Erk2 nuclear localization that works in concert with TGF-ß to promote EMT. Furthermore, we show for the first time that expression of the transcription factor c-myc, which is phosphorlyated by Erk2, is required for EMT. Characteristically, EMT involved adoption of a spindle-shaped morphology, loss of E-cadherin and increased expression of Vimentin, Fibronectin and Fibroblast Specific Protein-1 (S100A4). Prostate cells undergoing EMT became invasive and expressed several genes associated with metastasis, including MT-MMP1, MMP-2/9, the MMP-9 homodimer, Slug and Twist2. In sum, we demonstrate a novel mechanism by which non-invasive primary prostate tumor cells transition to an invasive phenotype characteristic of malignant tumor cells in response to TGF-ß signaling.

Fertile offspring derived from mouse spermatogonial stem cells cryopreserved for more than 14 years.

BACKGROUND: Approximately 80% of childhood cancers can now be cured but a side effect of treatment results in about one-third of the surviving boys being infertile or severely subfertile when they reach reproductive age. Currently, more than 1 in 5000 men of reproductive age who are childhood cancer survivors suffer from this serious quality of life problem. It is possible to obtain a testicular biopsy before treatment to preserve the spermatogonial stem cells (SSCs) of the male by cryopreservation, but the results of long-term storage of SSCs on their subsequent functional ability to generate normal offspring has not been examined in any mammalian species. Moreover, it will be necessary to increase the number of these cryopreserved SSCs to remove any contaminating malignant cells and assure regeneration of spermatogenesis. METHODS AND RESULTS: In this report, we demonstrate that long-term cryopreservation (>14 years) of testis cells from mouse, rat, rabbit and baboon safeguards SSC viability, and that these cells can colonize the seminiferous tubules of recipient testes. Moreover, mouse and rat SSCs can be cultured and re-establish complete spermatogenesis, and fertile mouse progeny without apparent genetic or epigenetic errors were generated by the sperm produced. CONCLUSIONS: These findings provide a platform for fertility preservation in prepubertal boys undergoing gonadotoxic treatments.

Glial cell line-derived neurotrophic factor and endothelial cells promote self-renewal of rabbit germ cells with spermatogonial stem cell properties.

Previous studies suggest that exogenous factors crucial for spermatogonial stem cell (SSC) self-renewal are conserved among several mammalian species. Since glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2) are critical for rodent SSC self-renewal, we hypothesized that they might promote self-renewal of nonrodent SSCs. Therefore, we cultured testicular germ cells from prepubertal rabbits in the presence of GDNF and FGF2 and found they proliferated indefinitely as cellular clumps that displayed characteristics previously identified for rodent SSCs. The rabbit germ cells could not be maintained on mouse embryonic fibroblast (STO) feeders that support rodent SSC self-renewal in vitro but were rather supported on mouse yolk sac-derived endothelial cell (C166) feeder layers. Proliferation of rabbit germ cells was dependent on GDNF. Of critical importance was that clump-forming rabbit germ cells colonized seminiferous tubules of immunodeficient mice, proliferated for at least 6 mo, while retaining an SSC phenotype in the testes of recipient mice, indicating that they were rabbit SSCs. This study demonstrates that GDNF is a mitogenic factor promoting self-renewal that is conserved between rodent and rabbit SSCs; with an evolutionary separation of ∼ 60 million years. These findings provide a foundation to study the mechanisms governing SSC self-renewal in nonrodent species.

Impact of TP53 mutations in Triple Negative Breast Cancer.

Identifying triple negative breast cancer (TNBC) patients expected to have poor outcomes provides an opportunity to enhance clinical management. We applied an Evolutionary Action Score to functionally characterize TP53 mutations (EAp53) in 96 TNBC patients and observed that EAp53 stratification may identify TP53 mutations associated with worse outcomes. These findings merit further exploration in larger TNBC cohorts and in patients treated with neoadjuvant chemotherapy regimens.

Spermatogonial stem cell self-renewal requires ETV5-mediated downstream activation of Brachyury in mice.

Insight regarding mechanisms controlling gene expression in the spermatogonial stem cell (SSC) will improve our understanding of the processes regulating spermatogenesis and aid in treating problems associated with male infertility. In the present study, we explored the global gene expression profiles of the glial cell line-derived neurotrophic factor (GDNF)-regulated transcription factors Ets (E-twenty-six) variant gene 5 (Etv5); B-cell chronic lymphocytic leukemia (CLL)/lymphoma 6, member B (Bcl6b); and POU domain, class-3 transcription factor 1 (Pou3f1). We reasoned that these three factors may function as a core set of transcription factors, regulating genes responsible for maintaining the SSC population. Using transient siRNA oligonucleotides to individually target Etv5, Bcl6b, and Pou3f1 within mouse SSC cultures, we examined changes to the global gene expression profiles associated with these transcription factors. Only modest overlaps in the target genes regulated by the three factors were noted, but ETV5 was found to be a critical downstream regulator of GDNF signaling that mediated the expression of several known SSC self-renewal related genes, including Bcl6b and LIM homeobox 1 (Lhx1). Notably, ETV5 was identified as a regulator of Brachyury (T) and CXC chemokine receptor, type 4 (Cxcr4), and we showed that ETV5 binding to the Brachyury (T) gene promoter region is associated with an active state of transcription. Moreover, in vivo transplantation of SSCs following silencing of Brachyury (T) significantly reduced the number of donor cell-derived colonies formed within recipient mouse testes. These results suggest Brachyury is of biological importance and functions as part of GDNF/ETV5 signaling to promote self-renewal of mouse SSCs cultured in vitro.

Combination therapy with epigallocatechin-3-gallate and doxorubicin in human prostate tumor modeling studies: inhibition of metastatic tumor growth in severe combined immunodeficiency mice.

The polyphenol epigallocatechin-3-gallate (EGCG) in combination with doxorubicin (Dox) exhibits a synergistic activity in blocking the growth and colony-forming ability of human prostate cell lines in vitro. EGCG has been found to disrupt the mitochondrial membrane potential, induce vesiculation of mitochondria, and induce elevated poly (ADP-ribose) polymerase (PARP) cleavage and apoptosis. EGCG in combination with low levels of Dox had a synergistic effect in blocking tumor cell growth. In vivo tumor modeling studies with a highly metastatic tumor line, PC-3ML cells, revealed that EGCG (228 mg/kg or 200 µmol/L) appeared to sensitize tumors to Dox. EGCG combined with low levels of Dox (0.14 mg/kg or 2 µmol/L) blocked tumor growth by PC-3ML cells injected intraperitoneally (ie, in CB17 severe combined immunodeficiencies) and significantly increased mouse survival rates. Similarly, relatively low levels of EGCG (57 mg/kg or 50 µmol/L) plus Dox (0.07 mg/kg or 1 µmol/L) eradicated established tumors (ie, in nonobese diabetic-severe combined immunodeficiencies) that were derived from CD44(hi) tumor-initiating cells isolated from PCa-20a cells. Flow cytometry results showed that EGCG appeared to enhance retention of Dox by tumor cells to synergistically inhibit tumor growth and eradicate tumors. These data suggest that localized delivery of high dosages of EGCG combined with low levels of Dox may have significant clinical application in the treatment of metastatic prostate and/or eradication of primary tumors derived from tumor-initiating cells.

MoleMapper: an application for crowdsourcing mole images to advance melanoma early-detection research.

Advancements in smartphone technologies and the use of specialized health care applications offer an exciting new era to promote melanoma awareness to the public and improve education and prevention strategies. These applications also afford an opportunity to power meaningful research aimed at improving image diagnostics and early melanoma detection. Here, we summarize our experience associated with developing and managing the implementation of MoleMapper™, a research-based application that not only provides an efficient way for users to digitally track images of moles and facilitate skin self-examinations but also provides a platform to crowdsource research participants and the curation of mole images in efforts to advance melanoma research. Obtaining electronic consent, safeguarding participant data, and employing a framework to ensure collection of meaningful data represent a few of the inherent difficulties associated with orchestrating such a wide-scale research enterprise. In this review, we discuss strategies to overcome these and other challenges leading to the implementation of MoleMapper™.

Lanthanide-Loaded Nanoparticles as Potential Fluorescent and Mass Probes for High-Content Protein Analysis.

Multiparametric and high-content protein analysis of single cells or tissues cannot be accomplished with the currently available flow cytometry or imaging techniques utilizing fluorophore-labelled antibodies, because the number of spectrally resolvable fluorochromes is limited. In contrast, mass cytometry can resolve more signals by exploiting lanthanide-tagged antibodies; however, only about 100 metal reporters can be attached to an antibody molecule. This makes the sensitivity of lanthanide-tagged antibodies substantially lower than fluorescent reporters. A new probe that can carry more lanthanide molecules per antibody is a desirable way to enhance the sensitivity needed for the detection of protein with low cellular abundance. Herein, we report on the development of new probes utilizing mesoporous silica nanoparticles (MSNPs) with hydroxyl, amine, or phosphonate functional groups. The phosphonated MSNPs proved to be best at loading lanthanides for up to 1.4 × 106 molecules per particle, and could be loaded with various lanthanide elements (Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Yb, and Lu) at relatively similar molar extents. The modified MSNPs can also load a fluorescent dye, allowing bimodal mass and fluorescence-based detection. We achieved specificity of antibody-conjugated nanoparticles (at 1.4 × 10³ antibodies per nanoparticle) for targeting proteins on the cell surface. The new materials can potentially be used as mass cytometry probes and provide a method for simultaneous monitoring of a large host of factors comprising the tumor microenvironment (e.g., extracellular matrix, cancer cells, and immune cells). These novel probes may also benefit personalized medicine by allowing for high-throughput analysis of multiple proteins in the same specimen.

Isolation of the Spermatogonial Stem Cell-Containing Fraction from Testes.

A testis cell fraction enriched for spermatogonial stem cells (SSCs) compared with the unfractionated total testis cell population can be isolated from testes after birth and adult stages. This protocol involves dissociating the seminiferous tubules into a single-cell suspension, using enzymes and mechanical disruption. This is followed by the selection of the Thy1+ cell fraction that contains nearly the entire SSC population and is enriched for these cells compared with the total testis cell population. This isolated fraction can be used for molecular studies, transfer into culture to establish primary cultures, or transplantation into the testes of recipients for spermatogenesis. Typically, if the SSCs are destined for transplantation, a marker is included to visualize the clones of spermatogenesis (e.g., lacZ or green fluorescent protein [GFP] transgenes). It is important to note that the isolated Thy1+ cell fraction does not consist of pure SSCs and that most germ cells are non-stem-cell progenitors, and a smaller portion of the population is somatic cells.

Culture and Expansion of Primary Undifferentiated Spermatogonial Stem Cells.

This protocol describes a culture method for supporting the maintenance and expansion of a primary mouse undifferentiated spermatogonial population containing spermatogonial stem cells (SSCs). The doubling time for SSCs in culture is relatively slow. Once established, SSC lines are split 1:2 to 1:4 every 7 d. Therefore, the time required to generate sufficient SSCs for experimentation can be considerable and requires careful planning.

Spermatogonial Stem Cell Transplantation to the Testis.

Spermatogonial stem cells (SSCs) are located in the basal region of the seminiferous tubules and provide a self-renewing reservoir from which progenitor spermatogonia arise and transiently amplify in number before transition to a differentiating state. SSC transplantation involves grafting mouse testicular cells into the seminiferous tubules of an aspermic recipient testis that is generated either by chemical (e.g., busulfan) treatment or genetic mutations.