Histopathologic diagnosis of thin, invasive cutaneous melanoma (CM) is only 34-62% accurate. Therefore, we sought to develop a transcriptomic biomarker to distinguish benign from malignant melanocytic neoplasms. We generated a targeted RNA-Sequencing dataset (TempO-Seq) of benign nevi (BN; n = 50) and CM (Breslow depth ≤ 1.0 mm; n = 51) and demonstrated enrichment of immune-related pathways among the 450 differentially expressed genes. Next, we trained a putative transcriptomic biomarker in two datasets, including BN and CM, and one dataset with CM in association with a nevus, macrodissected into CM and nevus regions. We refer to the nevus portion of CM in association with a nevus as progressing nevi (PN), since these nevi progressed to CM. Principal component analysis showed that PN samples clustered in a component intermediate to BN and CM. Ordinal regularized regression selected PYGL, AP000845.1, PHYHIP, WSCD1, FBXO7, TRPM1, SLC4A4, NALCN, FRMD4B, HHATL, COL1A1, CRYM, EPOP, RGS1, KRT6C, IGHG1, CNTN1, MMP11, GZMM, AP001880.1, TTYH3, TMEM132A, and PRAME; these genes were consistently selected in 1000 models using data from bootstrap resamples and had a single model predictive accuracy of at least 0.90 (area under the receiver operator characteristics curve). Linear regression models fit with these 23 genes in the TempO-Seq data, and publicly available microarray datasets from BN, dysplastic nevi, and CM, showed high consistency in the magnitude and directionality of gene expression differences between nevi and CM. Furthermore, immunohistochemical staining showed consistent protein-level changes in MMP11 and PYGL. These results illuminate the potential for a transcriptomic biomarker to differentiate benign from malignant melanocytic neoplasms and improve the accuracy of melanoma diagnosis.

Background/Objectives: BMS-202, is a potent small molecule with demonstrated antitumor activity. The study aimed to comprehensively characterize the physical and chemical properties of BMS-202 and evaluate its suitability for topical formulation, focusing on uniformity, stability and safety profiles. Methods: A range of analytical techniques were employed to characterize BMS-202. Scanning Electron Microscopy (SEM) was used to assess morphology, Differential Scanning Calorimetry (DSC) provided insights of thermal behavior, and Hot-Stage Microscopy (HSM) corroborated these thermal behaviors. Molecular fingerprinting was conducted using Raman spectroscopy and Fourier Transform Infrared (FTIR) spectroscopy, with chemical uniformity of the batch further validated by mapping through FTIR and Raman microscopies. The residual water content was measured using Karl Fisher Coulometric titration, and vapor sorption isotherms examined moisture uptake across varying relative humidity levels. In vitro safety assessments involved testing with skin epithelial cell lines, such as HaCaT and NHEK, and Transepithelial Electrical Resistance (TEER) to evaluate barrier integrity. Results: SEM revealed a distinctive needle-like morphology, while DSC indicated a sharp melting point at 110.90 ± 0.54 ℃ with a high enthalpy of 84.41 ± 0.38 J/g. HSM confirmed the crystalline-to-amorphous transition at the melting point. Raman and FTIR spectroscopy, alongside chemical imaging, confirmed chemical uniformity as well as validated the batch consistency. A residual water content of 2.76 ± 1.37 % (w/w) and minimal moisture uptake across relative humidity levels demonstrated its low hygroscopicity and suitability for topical formulations. Cytotoxicity testing showed dose-dependent reduction in skin epithelial cell viability at high concentrations (100 µM and 500 µM), with lower doses (0.1 µM to 10 µM) demonstrating acceptable safety. TEER studies indicated that BMS-202 does not disrupt the HaCaT cell barrier function. Conclusions: The findings from this study establish that BMS-202 has promising physicochemical and in vitro characteristics at therapeutic concentrations for topical applications, providing a foundation for future formulation development focused on skin-related cancers or localized immune modulation.

Background: Obesity, with increasing worldwide prevalence, is associated with increased breast cancer burden. The association is attributed to multiple metabolic disturbances including chronic inflammation due to dysfunctional adipose tissue. Features of local breast adipose tissue inflammation such as macrophage infiltration and an enriched pro-inflammatory gene signature were recently reported in mouse models of obesity and in women with high adiposity or metabolic disorders. Strategies that can reduce obesity-induced chronic inflammation may lead to reduction of breast cancer risk. Metformin, a widely used anti-diabetic drug, exerts favorable effects on multiple metabolic disturbances. This study aims to evaluate the clinical effects of metformin on breast tissue inflammation. Methods: Macrophage infiltration and polarization were determined in breast core needle biopsies collected at baseline and 6 months after agent intervention from a Phase II randomized, double-blind, placebo-controlled trial of metformin in premenopausal women with components of metabolic syndrome. Macrophage infiltration was assessed using a pan macrophage marker (CD68) by immunohistochemistry (IHC). M1 and M2 macrophages were assessed using CD40 and CD206 surface markers, respectively, by IHC. The primary endpoint is the change in macrophage density in breast adipose tissue. Secondary endpoints include the change in macrophage density in breast stroma and epithelium tissues, the proportion of M1 (pro-inflammatory) and M2 (anti-inflammatory) macrophages in breast adipose, stroma, and epithelium tissues. Results: Baseline biopsies from 76 participants (40 in the metformin arm and 36 in the placebo arm) were available for CD68 analysis. The baseline CD68 density was 22.48 ± 3.25, 67.26 ± 9.68, 418.81± 71.02 (mean±SE) per mm2 in breast adipose, stroma, and epithelium tissues, respectively. Biopsies from 71 participants (34 in the metformin arm and 37 in the placebo arm) were available for CD68 analysis at 6 months. Comparing to placebo, metformin intervention led to a significant reduction in CD68 density in breast adipose tissue (p = 0.01) but did not change the CD68 density in breast epithelium and stroma tissues. Conclusion: Metformin intervention resulted in favorable changes in macrophage infiltration in breast adipose tissue in premenopausal women with component of metabolic syndrome. Studies are ongoing to evaluate the effects of metformin on macrophage polarization. Citation Format: Liane E. Pinto, Sara Centuori, Jose Guillen-Rodriguez, Denise J. Roe, Edgar Tapia, Pavani Chalasani, H-H. Sherry Chow. Effects of metformin on breast tissue inflammation in premenopausal women with components of metabolic syndrome. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P065.

Magnetic Resonance Percent Breast Density by Study Group at Baseline.

<p>Details on MRI Scanner information.</p>

Aromatase inhibitor-induced arthralgia (AIA) presents a major problem for patients with breast cancer but is poorly understood. This prospective study explored the inflammatory metabolomic changes in the development of AIA. This single-arm, prospective clinical trial enrolled 28 postmenopausal women with early-stage (0–3) ER+ breast cancer starting adjuvant anastrozole. Patients completed the Breast Cancer Prevention Trial (BCPT) Symptom Checklist and the Western Ontario and McMaster Universities Arthritis Index (WOMAC) at 0, 3, and 6 months. The plasma levels of four polyunsaturated fatty acids (PUFAs) and 48 oxylipins were quantified at each timepoint. The subscores for WOMAC-pain and stiffness as well as BCPT-total, hot flash, and musculoskeletal pain significantly increased from baseline to 6 months (all p < 0.05). PUFA and oxylipin levels were stable over time. The baseline levels of 8-HETE were positively associated with worsening BCPT-total, BCPT-hot flash, BCPT-musculoskeletal pain, WOMAC-pain, and WOMAC- stiffness at 6 months (all p < 0.05). Both 9-HOTrE and 13(S)-HOTrE were related to worsening hot flash, and 5-HETE was related to worsening stiffness (all p < 0.05). This is the first study to prospectively characterize oxylipin and PUFA levels in patients with breast cancer starting adjuvant anastrozole. The oxylipin 8-HETE should be investigated further as a potential biomarker for AIA.

Understanding early tumorigenic events have obvious diagnostic and prognostic implications, particularly in the skin since this is the site of more US cancer diagnoses than any other organ (Rogers et al., 2015Rogers HW, Weinstock MA, Feldman SR, Coldiron BM. Incidence Estimate of Nonmelanoma Skin Cancer (Keratinocyte Carcinomas) in the U.S. Population, 2012. JAMA Dermatol 2015;151(10):1081-1086.Google Scholar). Actinic keratosis (AK) are dysplastic keratinocyte lesions characterized by irregularly shaped scaly papules frequently manifesting on sun-damaged skin, and are considered precursor lesions which can develop into cutaneous squamous cell carcinoma (cSCC), the second most common keratinocyte cancer (Siegel et al., 2017Siegel RL, Miller KD, Jemal A. Cancer Statistics, 2017. CA Cancer J Clin 2017;67(1):7-30.Google Scholar). Delineating the molecular events that characterize transition from normal skin to AK and on to established skin cancer has been the focus of a growing number of studies employing various technologies to identify markers of both initiation and progression of AK and cSCC (Chitsazzadeh et al., 2016Chitsazzadeh V. Coarfa C. Drummond J.A. Nguyen T. Joseph A. Chilukuri S. et al.Cross-species identification of genomic drivers of squamous cell carcinoma development across preneoplastic intermediates.Nat Commun. 2016; 712601Crossref PubMed Scopus (91) Google Scholar, Hameetman et al., 2013Hameetman L. Commandeur S. Bavinck J.N. Wisgerhof H.C. de Gruijl F.R. Willemze R. et al.Molecular profiling of cutaneous squamous cell carcinomas and actinic keratoses from organ transplant recipients.BMC Cancer. 2013; 13: 58Crossref PubMed Scopus (67) Google Scholar, Kim et al., 2021Kim Y.S. Shin S. Jung S.H. Park Y.M. Park G.S. Lee S.H. et al.Genomic Progression of Precancerous Actinic Keratosis to Squamous Cell Carcinoma.J Invest Dermatol. 2021; Google Scholar, Lambert et al., 2014Lambert S.R. Mladkova N. Gulati A. Hamoudi R. Purdie K. Cerio R. et al.Key differences identified between actinic keratosis and cutaneous squamous cell carcinoma by transcriptome profiling.Br J Cancer. 2014; 110: 520-529Crossref PubMed Scopus (68) Google Scholar, Padilla et al., 2010Padilla R.S. Sebastian S. Jiang Z. Nindl I. Larson R. Gene expression patterns of normal human skin, actinic keratosis, and squamous cell carcinoma: a spectrum of disease progression.Arch Dermatol. 2010; 146: 288-293Crossref PubMed Scopus (101) Google Scholar, Ra et al., 2011Ra S.H. Li X. Binder S. Molecular discrimination of cutaneous squamous cell carcinoma from actinic keratosis and normal skin.Mod Pathol. 2011; 24: 963-973Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, Thomson et al., 2021Thomson J. Bewicke-Copley F. Anene C.A. Gulati A. Nagano A. Purdie K. et al.The Genomic Landscape of Actinic Keratosis.J Invest Dermatol. 2021; 141: 1664-16674 e7Abstract Full Text Full Text PDF Scopus (12) Google Scholar, Zheng et al., 2021Zheng Q. Capell B.C. Parekh V. O'Day C. Atillasoy C. Bashir H.M. et al.Whole-Exome and Transcriptome Analysis of UV-Exposed Epidermis and Carcinoma In Situ Reveals Early Drivers of Carcinogenesis.J Invest Dermatol. 2021; 141: 295-307 e13Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Traditionally, cancer biology focusses on change within the tumor compartment for diagnosis and staging. Recent genomic profiling of AK has been similar as multiple studies have focused on DNA analysis to identify clonal chromosomal abnormalities and somatic mutations (Kim et al., 2021Kim Y.S. Shin S. Jung S.H. Park Y.M. Park G.S. Lee S.H. et al.Genomic Progression of Precancerous Actinic Keratosis to Squamous Cell Carcinoma.J Invest Dermatol. 2021; Google Scholar, Thomson et al., 2021Thomson J. Bewicke-Copley F. Anene C.A. Gulati A. Nagano A. Purdie K. et al.The Genomic Landscape of Actinic Keratosis.J Invest Dermatol. 2021; 141: 1664-16674 e7Abstract Full Text Full Text PDF Scopus (12) Google Scholar), and transcriptional analysis has been limited to whole skin (Chitsazzadeh et al., 2016Chitsazzadeh V. Coarfa C. Drummond J.A. Nguyen T. Joseph A. Chilukuri S. et al.Cross-species identification of genomic drivers of squamous cell carcinoma development across preneoplastic intermediates.Nat Commun. 2016; 712601Crossref PubMed Scopus (91) Google Scholar, Hameetman et al., 2013Hameetman L. Commandeur S. Bavinck J.N. Wisgerhof H.C. de Gruijl F.R. Willemze R. et al.Molecular profiling of cutaneous squamous cell carcinomas and actinic keratoses from organ transplant recipients.BMC Cancer. 2013; 13: 58Crossref PubMed Scopus (67) Google Scholar, Padilla et al., 2010Padilla R.S. Sebastian S. Jiang Z. Nindl I. Larson R. Gene expression patterns of normal human skin, actinic keratosis, and squamous cell carcinoma: a spectrum of disease progression.Arch Dermatol. 2010; 146: 288-293Crossref PubMed Scopus (101) Google Scholar) or the epidermal compartment alone, isolated using laser capture microdissection (Lambert et al., 2014Lambert S.R. Mladkova N. Gulati A. Hamoudi R. Purdie K. Cerio R. et al.Key differences identified between actinic keratosis and cutaneous squamous cell carcinoma by transcriptome profiling.Br J Cancer. 2014; 110: 520-529Crossref PubMed Scopus (68) Google Scholar, Zheng et al., 2021Zheng Q. Capell B.C. Parekh V. O'Day C. Atillasoy C. Bashir H.M. et al.Whole-Exome and Transcriptome Analysis of UV-Exposed Epidermis and Carcinoma In Situ Reveals Early Drivers of Carcinogenesis.J Invest Dermatol. 2021; 141: 295-307 e13Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). This approach is in line with a mutation-centric view of cancer supported by the clear requirement for a tumor to harbor multiple somatic DNA changes, without consideration of the dermal compartment, populated predominantly by fibroblasts and, in the case of AK and cSCC, infiltrating immune cells. Since sun-damaged skin can harbor multiple so called tumor driver-gene mutations caused by prolonged exposure to UV radiation (Martincorena et al., 2015Martincorena I, Roshan A, Gerstung M, Ellis P, Van Loo P, McLaren S, et al. Tumor evolution. High burden and pervasive positive selection of somatic mutations in normal human skin. Science 2015;348(6237):880-886.Google Scholar, South et al., 2014South A.P. Purdie K.J. Watt S.A. Haldenby S. den Breems N. Dimon M. et al.NOTCH1 mutations occur early during cutaneous squamous cell carcinogenesis.J Invest Dermatol. 2014; 134: 2630-2638Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar) it becomes challenging to differentiate tumor from non-tumor and tumor from pre-cancerous lesions based on mutation burden alone. As part of a pilot study to evaluate current technologies available to assess a large cohort of archival matched patient samples, we employed the Nanostring Digital Spatial Profiler (DSP) to compare transcriptional change in sun-protected skin with different regions of matched AK from the six individuals. The Nanostring DSP allows for the selection of tissue compartments of interest for RNA analysis using photocleavable RNA probes isolated on the basis of antibody staining. Here we used a pan-cytokeratin antibody to differentiate the epidermal from dermal compartment and assayed over 18,000 RNA probes from multiple regions of interest (ROI) comparing sun protected skin (n=5-6 from 6 individuals for a total of 34 ROI), the center of an AK lesion (n=3 for a total of 18 ROI) and edge of the AK lesion (n=2-3 for a total of 17 ROI) representing sun-damaged skin, from six unrelated individuals (Figure 1A). Analysis of these DSP data identified the expected changes in the epidermal compartment comparing sun-protected skin to AK demonstrating an upregulation of gene expression associated with P38 MAPK, TP53, ERK and DNA damage associated pathways and in line with previous studies (Chitsazzadeh et al., 2016Chitsazzadeh V. Coarfa C. Drummond J.A. Nguyen T. Joseph A. Chilukuri S. et al.Cross-species identification of genomic drivers of squamous cell carcinoma development across preneoplastic intermediates.Nat Commun. 2016; 712601Crossref PubMed Scopus (91) Google Scholar, Zheng et al., 2021Zheng Q. Capell B.C. Parekh V. O'Day C. Atillasoy C. Bashir H.M. et al.Whole-Exome and Transcriptome Analysis of UV-Exposed Epidermis and Carcinoma In Situ Reveals Early Drivers of Carcinogenesis.J Invest Dermatol. 2021; 141: 295-307 e13Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) (Figure 2A and Supplemental Tables 1&2). Comparison of the dermal compartment revealed up-regulation of pathways associated with matrix metalloproteinases, cell adhesion and extracellular matrix (ECM) remodeling as well as multiple immune related pathways including innate immune system, MHC class II antigen presentation and toll-like receptor signaling (Supplemental Tables 3&4All participants provided written, informed consent and this protocol was approved by the Institutional Review Board at the University of Arizona (Protocol Title: Skin Cancer Prevention Program Biorepository; Protocol Number: 1200000229), Differential expression analysis of batch corrected, normalized read counts using a pairwise FDR adjusted p-value of less than 0.05, for those genes with an overall t-test statistic of 0.05 revealed the greatest change in transcriptional activity in the dermal compartment when comparing sun protected skin with the center of the AK lesion. The fewest transcriptional changes in our matched samples were evident from comparing AK edge with AK center, again with the greatest change being in the dermal compartment (Figure 1B and Supplemental Table 5). Interestingly, comparing sun protected skin with AK edge revealed more change in the epidermal compartment suggesting a progressive model where early transcriptional change from sun protected skin to AK edge (assumed to be sun damaged normal skin) is evident in the epidermal compartment which leads to activation of multiple signaling cascades within the dermal compartment and significant changes to ECM remodeling, presumably with a major contribution from infiltration of immune cells (Figure 2B). These data are intriguing since previous studies of AK have focused on the cell of origin, the keratinocyte, and while clear differences in transcription have been identified comparing AK with sun-protected or UV-exposed normal skin, fewer changes are observed comparing AK with cSCC (Chitsazzadeh et al., 2016Chitsazzadeh V. Coarfa C. Drummond J.A. Nguyen T. Joseph A. Chilukuri S. et al.Cross-species identification of genomic drivers of squamous cell carcinoma development across preneoplastic intermediates.Nat Commun. 2016; 712601Crossref PubMed Scopus (91) Google Scholar, Padilla et al., 2010Padilla R.S. Sebastian S. Jiang Z. Nindl I. Larson R. Gene expression patterns of normal human skin, actinic keratosis, and squamous cell carcinoma: a spectrum of disease progression.Arch Dermatol. 2010; 146: 288-293Crossref PubMed Scopus (101) Google Scholar, Zheng et al., 2021Zheng Q. Capell B.C. Parekh V. O'Day C. Atillasoy C. Bashir H.M. et al.Whole-Exome and Transcriptome Analysis of UV-Exposed Epidermis and Carcinoma In Situ Reveals Early Drivers of Carcinogenesis.J Invest Dermatol. 2021; 141: 295-307 e13Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Indeed, AK and cSCC keratinocytes are characterized by a high tumor mutation burden and gross chromosomal changes (Chitsazzadeh et al., 2016Chitsazzadeh V. Coarfa C. Drummond J.A. Nguyen T. Joseph A. Chilukuri S. et al.Cross-species identification of genomic drivers of squamous cell carcinoma development across preneoplastic intermediates.Nat Commun. 2016; 712601Crossref PubMed Scopus (91) Google Scholar, Ra et al., 2011Ra S.H. Li X. Binder S. Molecular discrimination of cutaneous squamous cell carcinoma from actinic keratosis and normal skin.Mod Pathol. 2011; 24: 963-973Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, Thomson et al., 2021Thomson J. Bewicke-Copley F. Anene C.A. Gulati A. Nagano A. Purdie K. et al.The Genomic Landscape of Actinic Keratosis.J Invest Dermatol. 2021; 141: 1664-16674 e7Abstract Full Text Full Text PDF Scopus (12) Google Scholar), and a compelling hypothesis supported by the data presented here is that key molecular changes which influence progression of AK are in fact found in the dermal compartment. Further work with a wider sample set and inclusion of matched cSCC samples will be needed to test this hypothesis and the DSP platform offers the ability to do so in archival, formalin fixed samples. A recent study combining single cell and spatial transcriptomics to compare 10 SCC samples with patient matched normal skin also included separation of the dermal compartment and highlighted intercellular communication between the stroma and the leading edge of invasive SCC (Ji et al., 2020Ji A.L. Rubin A.J. Thrane K. Jiang S. Reynolds D.L. Meyers R.M. et al.Multimodal Analysis of Composition and Spatial Architecture in Human Squamous Cell Carcinoma.Cell. 2020; 182: 497-514 e22Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). This observation was also noted in an earlier single cell study of head and neck SCC, where the leading edge of the tumor was highlighted as the most prognostic region (Puram et al., 2017Puram S.V. Tirosh I. Parikh A.S. Patel A.P. Yizhak K. Gillespie S. et al.Single-Cell Transcriptomic Analysis of Primary and Metastatic Tumor Ecosystems in Head and Neck Cancer.Cell. 2017; 171: 1611-1624 e24Abstract Full Text Full Text PDF PubMed Scopus (1006) Google Scholar). These studies support the idea that the dermal compartment may well hold key prognosticating information in the context of AK and cSCC. Collectively, these data suggest that while the epidermal compartment harbors the cell of origin for SCC and AK, the greatest change in transcriptional homeostasis in the progression from sun-protected skin to AK is observed in the dermal compartment and may well be a fruitful avenue of investigation for delineating molecular markers of initiation and progression. All data are available on request. Datasets related to this article can be found at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE221001, hosted by the Gene Expression Omnibus (accession number GSE221001). The authors have no conflicts of interest to declare. This research was funded by the National Cancer Institute, grant numbers P01CA229112 and T32CA078447. This project utilized the Genomics and Flow Cytometry Shared Resources at the Sidney Kimmel Cancer Center, supported by the NCI, grant 5P30CA056036-17. Author Contributions Statement (CRediT-compliant) Conceptualization: all authors; data curation: all authors; formal analysis: all authors; funding acquisition: all authors; investigation: all authors; methodology: all authors; project administration: all authors; resources: all authors; software: all authors; supervision: all authors; validation: all authors; visualization: all authors; writing – original draft: all authors; and writing – review and editing: all authors. Download .xlsx (.07 MB) Help with xlsx files Patient Details SAZT20099, Male, 75 years old, white not Hispanic individual. SAZT20088, Male, 67years old, white not Hispanic individual. SAZT200086, Male, 78 years old, white not Hispanic individual. SAZT200080, Female, 61 years old, white not Hispanic individual. SAZT200015, Female, 75 years old, white not Hispanic individual. SAZT200007, Male, 75 years old, white not Hispanic individual. Supplemental Methods FFPE tissues All human tissue was sourced from the Skin Cancer Prevention Program Biorepository at the University of Arizona Cancer Center. The protocol has been approved by the Institutional Review Board at the University of Arizona (Protocol Title: Skin Cancer Prevention Program Biorepository; Protocol Number: 1200000229). A set of matched sun protected and actinic keratosis from six individuals were used for this study. Tissue blocks were sectioned with a 5 μm thickness and mounted in the center of the slide (Superfrost Plus Micro slides, VWR) within an area of 35.3 mm long by 14.1 mm wide to allow detection by the NanoString GeoMx DSP instrument. NanoString GeoMx Digital Space Profiling Whole Transcriptome Atlas for NGS Sample preparation was performed following Leica Biosystems Bond RX FFPE RNA Slide Preparation Protocol in the GeoMx NGS Slide Preparation User Manual (NanoString, MAN-10115-03 for software version v2.1). This assay relies upon photocleavable RNA probes coupled to oligonucleotide tags that allow the selection of tissue compartments of interest for RNA analysis. Slides were baked at 60°C for 60 minutes. Samples were loaded into Leica Bond Rx for deparaffinization and antigen retrieval with the following settings; Bake and Dewax preparation program, 150μl buffer dispense volume, HIER 20 minutes with ER2 at 100°C, 1μ/mL proteinase K for 15 minutes. Slides were briefly stored in 1x PBS. Excess PBS was removed from slides before the addition of 200μL Whole Transcriptome Assay (WTA) probe sets diluted 1:10 in Buffer R (NanoString), covered with Hybrislips (Grace Biolabs, 714022) and incubated at 37°C overnight in a hybridization chamber lined with Kimwipes moistened with 2x SSC. Unbound probes were removed by washing twice at 37°C for 25 minutes in 50% Formamide (ThermoFisher, AM9324)/2x SSC followed by two washes at room temperature in 2x SSC for 2 minutes. Slides were blocked with 200μL Buffer W (NanoString) for 30 minutes followed by 1 hour incubation at room temperature in a humidity chamber with morphology marker solution comprised 0.66μL Pan-cytokeratin (Novus Biologicals, NBP2-33200AF488) and 10μL Syto83 (ThermoFisher, S11364) in Buffer W (NanoString) to 200μL per slide. Slides were subsequently washed twice with 2x SSC before loading onto NanoString GeoMx DSP instrument. Slides were loaded and scanned on GeoMx instrument according to GeoMx DSP Instrument User Manual (NanoString, MAN-10116-03 for software version v2.1) with the following scan exposure times; 200ms FITC/525nm, 60ms Cy3/568nm, 250ms Texas Red/615nm, 300ms Cy5/666nm. 12 polygonal regions of interest (ROI) were selected per slide with segmentation masks applied to each ROI selecting Pan-cytokeratin positive and Pan-cytokeratin negative areas within each ROI. Channel thresholds were adjusted to maximize inclusion of signal areas and minimize inclusion of non-signal areas with default settings for erode, N-dilate, hole size and particle size. Nuclei number were calculated for each region and are presented in Figure S1. WTA probes were aspirated from each region by way of UV cleavage and deposited into individual wells of 96-well plate. Sequencing NGS libraries were prepared according to GeoMx -NGS Readout Library Prep User Manual (NanoString, MAN-10117-03 for software version V2.1). DSP aspirates were dried down at room temperature overnight followed by resuspension in 10μL DEPC-treated water for 10 minutes. 96-well PCR plates were prepared by addition of 2μL PCR mix (NanoString), 4μL of index primer mix (NanoString) and 4μL of resuspended DSP aspirate. PCR products were generated with the following program; 37°C for 30 minutes, 50°C for 10 minutes, 95°C for 3 minutes followed by 18 cycles of (95°C for 15 seconds, 65°C for 1 minute, 68°C for 30 seconds), final extension at 68°C for 5 minutes and hold at 4°C. Indexed libraries were pooled into small and large pools based on ROI area with small pool ROI being approximately 4-fold smaller than that of large pool ROI. Pools were incubated with AMPure XP beads (Beckman Coulter, A63880) at 1.2x bead to sample ratio for 5 minutes. Beads were collected on a magnetic stand for 5 minutes, the supernatant removed and the beads washed twice with 200μL freshly prepared 80% ethanol in DEPC-treated water for 30sec each. Excess ethanol was allowed to evaporate and the beads were resuspended in elution buffer (10nM Tris-HCl pH 8, 0.05% Tween-20)(15μL for large pool, 10μL small pool). Libraries were rebound to AMPure beads by addition of Ampure Buffer at 1.2x elution buffer volume and the above process repeated and the libraries finally eluted in 12μL for small pool and 16μL for the large pool (with elution buffer). Total target counts per DSP collection plate for sequencing were calculated from the total sampled areas (μm2) reported in the DSP generated Lab Worksheet with the target sequencing depth being 100 counts/μm2. Each library was diluted to 4-10nM and combined to reach the estimated counts/μm2per library in the final pool. All sequencing libraries were generated with unique indexes allowing pooled sequencing. We combined the WTA libraries for sequencing with an Illumina NovaSeq S4 platform at a loading concentration of 250pM with 5% PhiX. The sequencing parameters used were: read 1, 27 cycles; read 2, 27 cycles; index 1, 8 cycles; index 2, 8 cycles. Q3 Normalized data for all 18,000+ genes for each AOI were generated using the DSP workflow according to the manufacturer's specifications. Analysis of samples SAZT20099 and SAZT20088 showed clear separation of cytokeratin gene expression within the epidermal and dermal compartments (Figure S2). Differentially Expressed Gene Identification and Pathways Analysis Beginning with Log2 transformation of Q3 normalized expression (generated from the DSP) reads were parametric batch adjusted with non-informative prior (Combat function in SVA Bioconductor package version 3.38.0, R version 4.2.0, 64-bit Windows build 22000). After random intercept mixed model analysis of variance (by ID) using the R package LMER (version 3.1-3) a significance level of 0.05 for log ratio χ2 test statistic for overall difference in ROIs (sun protected and AK edge and center) were used for pairwise comparisons with FDR adjusted p-value ≤ 0.05. Generated gene lists were used to query Gene Analytics Gene Set Analysis available at https://geneanalytics.genecards.org/ (Ben-Ari Fuchs et al., 2016) and a priori pathways of interest were CLR transformed for biplots. Biplot and centered log-ratio transformed read analysis The centered log-ratio (CLR) transformation is a within sample standardization that centers reads for each gene by the geometric mean over reads for the all the genes, this is a transformation that is similar to CPM and TKPM transformations, and justification for this transformation under the in the context of compositional data analysis and biplots can be found in Greenacre (Greenacre, 2021). Biplots are a combination of Principle Components Analysis and the relationship, both correlation and variance, between the elements making up the PCA plot (Gabriel, 1971). Biplots can be used to evaluate the quality of dimension reduction using the first two principal components (32.7%, 60.3%, 26.4% and 49.5% of the variability in the TP53, P38, DNA damage, and ECM pathways, respectively). We provide additional evidence about the relative importance of the pathways shown in Figure 2 by using the Fisher combined probabilty test (see below for detail), which combines the p-values from individual genes within the each pathway into a single test statistic. Combined Fisher probability analysis Combing many tests for significance within a population, like pathways, is a possible approach for filtering pathways of interest. This method is based on summation of individual gene-level p-values into a single one-sided χ2 test statistic (Fisher, 1992; Mosteller and Fisher, 1948). The p-values for our example in Figure S3 are the overall-test statistic based on mixed effects analysis of variance that includes an adjustment for correlation that is induced by sampling (both ROIs and AOIs) within a tissue sample. Supplemental References Fisher RA. Statistical Methods for Research Workers. In: Kotz S, Johnson NL (eds) Breakthroughs in Statistics. Springer Series in Statistics. Springer, New York, NY. 1992. https://doi.org/10.1007/978-1-4612-4380-9_6 Gabriel KR. The Biplot Graphic Display of Matrices with Application to Principal Component Analysis. Biometrika, 1971 58, 3: 453–467. https://doi.org/10.1093/biomet/58.3.453 Greenacre M. Compositional Data Analysis. Annu. Rev. Stat. Appl. 2021 8: 271-299. https://www.annualreviews.org/doi/pdf/10.1146/annurev-statistics-042720-124436 Mosteller F and Fisher RA. "Questions and Answers." The American Statistician 1948 2: 30–31. https://doi.org/10.2307/2681650Figure S2: Keratin and collagen gene expression differentiates areas of interest. Q3 normalized read counts (y-axis) for the genes encoding keratin 1 (KRT1) and keratin 10 (KRT10) as well as genes encoding collagen 1 (COL1A1) and collagen 3 (COL3A1) components clearly differentiate cytokeratin positive (Pos_) from cytokeratin negative (Neg_) areas of interest. Data from samples SAZT20099 and SAZT20088 are presented.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure S3: Pathway Analysis Comparisons. Combined Fisher probability analysis confirms significant change in p38, toll-like receptor signaling, p53 and DNA damage pathways in cytokeratin positive AOIs (CK+) and significant change in ECM and DNA damage pathways in cytokeratin negative AOIs (CK-). P-values (P-val) are given for each compartment as indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

<p>Relationship between baseline percent breast density by MRI and descriptive breast density by mammogram in the study population.</p>

Background Selenium (Se) is a trace element that has been investigated as a potential chemopreventive agent for colorectal cancer. Dietary intake of other antioxidant nutrients may modify the effect of Se. Objective We examined the association between intake and serum concentrations of retinol, beta-carotene, beta-cryptoxanthin, lycopene, lutein/zeaxanthin, and alpha- and gamma-tocopherol and the development of metachronous colorectal adenoma, and if these nutrients modified the effect of Se. Methods We conducted a prospective study of 1874 participants from the Se Trial with data for antioxidant intake, as well as a subcohort of 508 participants with serum biomarker concentrations. Results Statistically significantly lower odds for the development of metachronous adenoma were observed for those participants in the highest tertile of intake for lutein/zeaxanthin compared to the lowest, with an OR (95% CI) of 0.72 (0.56-0.94). No effect modification for intake of any nutrient was observed. However, circulating concentrations of lycopene exhibited statistically significant effect modification of selenium supplementation (p < 0.06), Conclusion These findings show that intake and circulating concentrations of antioxidant nutrients were not consistently associated with reduced odds for the development of metachronous lesions, although blood concentrations of lycopene may modify the effect of selenium supplementation.

<p>Relationship between baseline percent breast density by MRI and descriptive breast density by mammogram in the study population.</p>

Table showing within subject variability in collagen fiber features.

OBJECTIVESA single dose of human papillomavirus (HPV) vaccine would simplify logistics and reduce costs of vaccination programs worldwide. We conducted a phase IIa trial to determine the stability of HPV type-specific antibody responses after a single dose of the nonavalent HPV vaccine, Gardasil9. METHODSTwo hundred-and-one healthy 9 to 11-year-old girls and boys were enrolled at 2 centers in the United States to receive a prime dose of the nonavalent vaccine at baseline, a delayed dose at month 24, and an optional third dose at month 30. Blood samples were collected to measure HPV type-specific antibodies at baseline and at 6, 12, 18, 24, and 30 months after the prime dose. The primary outcomes were serum HPV16 and HPV18 antibody responses. RESULTSIn both girls and boys, geometric mean concentrations of HPV16 and HPV18 antibodies increased at 6 months, declined between months 6 to 12, and then remained stable and high (at 20- and 10-times those at baseline for HPV16 and HPV18, respectively) throughout months 12, 18, and 24 (prebooster) visits. Both HPV16 and HPV18 antibody responses demonstrated anamnestic boosting effect at 30-months after the delayed (24-month) booster dose. CONCLUSIONSA single dose of the nonavalent HPV vaccine induced persistent and stable HPV16 and HPV18 antibody responses up to 24 months. This study contributes important immunogenicity data to inform feasibility of the single dose HPV vaccination paradigm. Further research is needed to assess the long-term antibody stability and individual clinical and public health benefit of the single dose schedule.

Birthplace, as a proxy for environmental exposures (e.g., diet), may influence metabolomic profiles and influence risk of cancer. This secondary analysis investigated metabolomic profile differences between foreign and U.S.-born Mexican-origin (MO) Hispanic men to shed light on potential mechanisms through which foreign- and U.S.-born individuals experience differences in cancer risk and risk factors. Plasma samples from MO Hispanic men ( N = 42) who participated in a previous lifestyle intervention were collected pre-and post-intervention. Metabolomic profiles were characterized from samples using ultra performance liquid chromatography–quadrupole time of flight mass spectrometry (UPLC-QTOF). Models were visualized using supervised orthogonal projections to latent structures–discriminant analysis (OPLS-DA). Progenesis QI was used for peak integration and metabolite identification. Plasma metabolomic profiles differed between foreign- and U.S.-born pre-intervention (R2 = .65) and post-intervention (R2 = .62). Metabolomic profiles differed pre- versus post-intervention (R2 = .35 and R2 = .65) for the foreign- and U.S.-born group, respectively. Both endogenous metabolites and dietary components characterized differences between foreign- and U.S.-born participants pre- and post-intervention. Plasma metabolomic profiles from MO Hispanic men differed by birthplace. These results advance our understanding of relevant exposures that may affect cancer risk among MO Hispanic men born abroad or in the United States.

Background: There is an unmet clinical need for interventions to prevent disease progression in patients with localized prostate cancer on active surveillance (AS).Objective: To determine the immunologic response to the PROSTVAC vaccine and the clinical indicators of disease progression in patients with localized prostate cancer on AS.Design, setting, and participants: This was a phase 2, double-blind, randomized controlled trial in 154 men with low-or intermediate-risk prostate cancer on AS.Intervention: Participants were randomized (2:1) to receive seven doses of subcutaneous PROSTVAC, a vaccinia/fowlpox viral vector-based immunotherapy containing a prostate-specific antigen (PSA) transgene and three T-cell co-stimulatory molecules, or an empty fowlpox vector (EV) over 140 d.Outcome measurements and statistical analysis: The primary outcome was the change from baseline in CD4 and CD8 T-cell infiltration in biopsy tumor tissue. Key secondary outcomes were safety and changes in prostate biopsy tumor pathology, peripheral antigen-specific T cells, and serum PSA. Continuous variables were compared using non parametric tests. Categorical variables were compared using Fisher's exact test.Results and limitations: The PROSTVAC/EV vaccination was well tolerated. All except one participant completed the vaccination series. Changes in CD4 or CD8 density in biopsy tumor tissue did not differ between the PROSTVAC and EV arms. The proportions of patients with Gleason upgrading to grade group 3 after treatment was similar between the arms. There were no differences in postvaccination peripheral T-cell responses or the PSA change from baseline to 6-mo post-treatment follow-up between the groups.Conclusions: In this first-of-kind trial of immunotherapy in patients on AS for prostate cancer, PROSTVAC did not elicit more favorable prostate tissue or peripheral T-cell responses than the EV. There was no difference between the arms in clinicopathologic effects. Despite the null findings, this is the first study reporting the feasibility and acceptability of an immunotherapy intervention in the AS setting.Patient summary: We looked at responses after an experimental prostate cancer vaccine in patients with prostate cancer on active surveillance (AS). Participants who received the vaccine did not show more favorable outcomes than those receiving the control. Despite these findings, this is the first report showing the feasibility and acceptability of immunotherapy for prostate cancer in patients on AS. & COPY; 2022 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Spaghetti plots of mean arterial pressure (MAP) from clinical blood pressure measurements at three time points by study group.

Objectives: To determine the feasibility (as measured by tolerability and safety) and efficacy of topical 5-fluorouracil (5-FU) and imiquimod for the treatment of cervical intraepithelial neoplasia (CIN) 2/3. Methods: This pilot prospective study was conducted in women aged 18-45years with p16+ CIN 2/3. Participants underwent an 8-week alternating regimen of self-applied 5% 5-FU on weeks 1, 3, 5, and 7 and physician-applied imiquimod on weeks 2, 4, 6, and 8. Adverse events (AEs) were collected by symptom diary and clinical exam. Feasibility was measured by tolerability and safety (AEs) of the study intervention. Tolerability was assessed as the number of participants able to apply 50% or more of the treatment doses. The safety outcome was calculated as the number of participants who experienced "specified AEs" defined as possibly, probably, or definitely related grade 2 or worse AE or grade 1 genital AEs (blisters, ulcerations, or pustules) lasting more than 5days. The efficacy of the intervention was determined by histology and high-risk human papillomavirus (hrHPV) testing was done after treatment. Results: The median age of the 13 participants was 27 +/- 2.9years. Eleven (84.61%) participants applied 50% or more of the treatment. All participants reported grade 1 AEs; 6 (46.15%) reported grade 2 AEs; and 0 reported grade 3/4 AEs. Three (23.08%) participants had specified AEs. Histologic regression to normal or CIN 1 among those completing 50% or more of the treatment doses was observed in 10 (90.91%) participants, and 7 (63.63%) tested negative for hr-HPV at the end of the study. Conclusions: Topical treatment for CIN 2/3 with 5-FU/imiquimod is feasible, with preliminary evidence of efficacy. Topical therapies need further investigation as adjuncts or alternatives to surgical therapy for CIN 2/3.

Nonmelanoma skin cancers (NMSCs) are the most common malignancies worldwide and affect more than 5 million people in the United States every year. NMSC is directly linked to the excessive exposure of the skin to solar ultraviolet (UV) rays. The toll-like receptor 4 (TLR4) antagonist, resatorvid (TAK-242), is a novel prototype chemo preventive agent that suppresses the production of inflammation mediators induced by UV exposure. This study aimed to design and develop TAK-242 into topical formulations using FDA-approved excipients, including DermaBaseTM, PENcreamTM, polyethylene glycol (PEG)-400, propylene glycol (PG), carbomer gel, hyaluronic acid (HA) gel, and Pluronic® F-127 poloxamer triblock copolymer gel for the prevention of skin cancer. The physicochemical properties of raw TAK-242, which influence the compatibility and solubility in the selected base materials, were confirmed using X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), hot-stage microscopy (HSM), Raman spectroscopy, and attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopic analysis. The permeation behavior of TAK-242 from the prepared formulations was determined using Strat-M® transdermal diffusion membranes, and 3D cultured primary human-derived epidermal keratinocytes (EpiDermTM). Despite TAK-242′s high molecular weight and hydrophobicity, it can permeate through reconstructed human epidermis from all formulations. The findings, reported for the first time in this study, emphasize the capabilities of the topical application of TAK-242 via these multiple innovative topical drug delivery formulation platforms.

Background: The increasing rate of obesity in the United States is accompanied by serious health concerns. Obesity increases breast cancer burden and is associated with increased risk of triple-negative breast cancer in premenopausal women and overall poor prognosis in breast cancer patients. There is a need for intervention strategies aiming to reduce obesity-associated dysregulation to attenuate breast cancer risk.Methods: We conducted a Phase II, double-blind, randomized, placebo-controlled clinical trial in overweight/obese premenopausal women with elements of metabolic syndrome to assess the potential of metformin to reduce obesity-associated breast cancer risk. Study participants received metformin (850 mg BID, n = 76) or placebo (n = 75) for 12 months. Fasting blood samples were collected at baseline, 6-months and 12-months from each participant. We analyzed the effects of metformin on circulating levels of insulin/IGF axis, adipokines, and neutrophil-to-lymphocyte ratio in samples collected from this trial. Serum concentrations of insulin, IGF-1, IGFBP-3, leptin and high-molecular weight adiponectin were measured using ELISA immunoassays. Results: The study population included 151 women and had a mean age of 39.5 years, mean body mass index (BMI) was 37.8 and study participants had a large waist and at least one other component of metabolic syndrome. Metformin treatment did not result in significant changes in members of the insulin/IGF axis compared to the placebo group, however, limiting the analysis to participants with detectable metformin in the blood serum resulted in favorable changes in insulin (p=0.0215), HOMA-IR (p<0.001) and a significant increase in IGFBP-3 (p=0.0176) in the metformin group after the intervention. We observed significant decreases in leptin (p=0.0018) and the leptin-to-adiponectin ratio (p=0.0036) in the metformin arm longitudinally. Additionally, we observed a significant reduction in the neutrophil-to-lymphocyte ratio (NLR), a systemic inflammation marker, in the metformin group compared to the placebo group (p=0.0170). Conclusions: We conclude that metformin led to favorable changes in metabolic markers associated with breast cancer risk in the metformin treated participants, however, the changes were not significantly different from the placebo group. The NLR was significantly reduced after metformin intervention compared to the placebo group. More research is needed to understand the effects of metformin on the insulin/IGF axis and adipokines in overweight/obese premenopausal women. Citation Format: Edgar Tapia, Diana Villa-Guilen, Pavani Chalasani, Sara Centuori, Denise J. Roe, Jose Guillen, Catherine Cordova, Liane Pinto, Sherry Chow. Effect of metformin on metabolic markers associated with breast cancer risk in a phase II clinical trial in overweight/obese premenopausal women [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 P1-10-02.