bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from Alternate Metabolic Programs Define Regional Variation of Relevant Biological Features in Renal Cell Carcinoma Progression
Authors: Brooks Samira A. , Khandani Amir H. , Fielding Julia R. , Lin Weili , Sills Tiffany , Lee Yueh , Arreola Alexandra , Milowsky Mathew I. , Wallen Eric M. , Woods Michael E. , Smith Angie B. , Nielsen Mathew E. , Parker Joel S. , Lalush David S. , Rathmell W. Kimryn
Published: April 2023

Abstract
Purpose: Clear cell renal cell carcinoma (ccRCC) has recently been redefined as a highly heterogeneous disease. In addition to genetic heterogeneity, the tumor displays risk variability for developing metastatic disease, therefore underscoring the urgent need for tissue-based prognostic strategies applicable to the clinical setting. We have recently employed the novel PET/magnetic resonance (MR) image modality to enrich our understanding of how tumor heterogeneity can relate to gene expression and tumor biology to assist in defining individualized treatment plans.

Experimental Design: ccRCC patients underwent PET/MR imaging, and these images subsequently used to identify areas of varied intensity for sampling. Samples from 8 patients were subjected to histologic, immunohistochemical, and microarray analysis.

Results: Tumor subsamples displayed a range of heterogeneity for common features of hypoxia-inducible factor expression and microvessel density, as well as for features closely linked to metabolic processes, such as GLUT1 and FBP1. In addition, gene signatures linked with disease risk (ccA and ccB) also demonstrated variable heterogeneity, with most tumors displaying a dominant panel of features across the sampled regions. Intriguingly, the ccA- and ccB-classified samples corresponded with metabolic features and functional imaging levels. These correlations further linked a variety of metabolic pathways (i.e., the pentose phosphate and mTOR pathways) with the more aggressive, and glucose avid ccB subtype.

Conclusions: Higher tumor dependency on exogenous glucose accompanies the development of features associated with the poor risk ccB subgroup. Linking these panels of features may provide the opportunity to create functional maps to enable enhanced visualization of the heterogeneous biologic processes of an individual's disease. Clin Cancer Res; 22(12); 2950–9. ©2016 AACR.
Data from Hypoxia-Activated Prodrug TH-302 Targets Hypoxic Bone Marrow Niches in Preclinical Leukemia Models
Authors: Benito Juliana , Ramirez Marc S. , Millward Niki Zacharias , Velez Juliana , Harutyunyan Karine G. , Lu Hongbo , Shi Yue-xi , Matre Polina , Jacamo Rodrigo , Ma Helen , Konoplev Sergej , McQueen Teresa , Volgin Andrei , Protopopova Marina , Mu Hong , Lee Jaehyuk , Bhattacharya Pratip K. , Marszalek Joseph R. , Davis R. Eric , Bankson James A. , Cortes Jorge E. , Hart Charles P. , Andreeff Michael , Konopleva Marina
Published: April 2023

Abstract
Purpose: To characterize the prevalence of hypoxia in the leukemic bone marrow, its association with metabolic and transcriptional changes in the leukemic blasts and the utility of hypoxia-activated prodrug TH-302 in leukemia models.

Experimental Design: Hyperpolarized magnetic resonance spectroscopy was utilized to interrogate the pyruvate metabolism of the bone marrow in the murine acute myeloid leukemia (AML) model. Nanostring technology was used to evaluate a gene set defining a hypoxia signature in leukemic blasts and normal donors. The efficacy of the hypoxia-activated prodrug TH-302 was examined in the in vitro and in vivo leukemia models.

Results: Metabolic imaging has demonstrated increased glycolysis in the femur of leukemic mice compared with healthy control mice, suggesting metabolic reprogramming of hypoxic bone marrow niches. Primary leukemic blasts in samples from AML patients overexpressed genes defining a “hypoxia index” compared with samples from normal donors. TH-302 depleted hypoxic cells, prolonged survival of xenograft leukemia models, and reduced the leukemia stem cell pool in vivo. In the aggressive FLT3/ITD MOLM-13 model, combination of TH-302 with tyrosine kinase inhibitor sorafenib had greater antileukemia effects than either drug alone. Importantly, residual leukemic bone marrow cells in a syngeneic AML model remain hypoxic after chemotherapy. In turn, administration of TH-302 following chemotherapy treatment to mice with residual disease prolonged survival, suggesting that this approach may be suitable for eliminating chemotherapy-resistant leukemia cells.

Conclusions: These findings implicate a pathogenic role of hypoxia in leukemia maintenance and chemoresistance and demonstrate the feasibility of targeting hypoxic cells by hypoxia cytotoxins. Clin Cancer Res; 22(7); 1687–98. ©2015 AACR.
Data from PTEN Loss Is Associated with Worse Outcome in HER2-Amplified Breast Cancer Patients but Is Not Associated with Trastuzumab Resistance
Authors: Stern Howard M. , Gardner Humphrey , Burzykowski Tomasz , Elatre Wafaa , O'Brien Carol , Lackner Mark R. , Pestano Gary A. , Santiago Angela , Villalobos Ivonne , Eiermann Wolfgang , Pienkowski Tadeusz , Martin Miguel , Robert Nicholas , Crown John , Nuciforo Paolo , Bee Valerie , Mackey John , Slamon Dennis J. , Press Michael F.
Published: April 2023

Abstract
Purpose: To investigate the clinical relevance of PTEN in HER2-amplified and HER2-nonamplified disease.

Experimental Design: We assessed PTEN status in two large adjuvant breast cancer trials (BCIRG-006 and BCIRG-005) using a PTEN immunohistochemical (IHC) assay that was previously validated in a panel of 33 breast cancer cell lines and prostate cancer tissues with known PTEN gene deletion.

Results: In the HER2-positive patient population, absence of tumor cell PTEN staining occurred at a rate of 5.4% and was independent of ER/PR status. In contrast, 15.9% of HER2-negative patients exhibited absence of PTEN staining with the highest frequency seen in triple-negative breast cancer (TNBC) subgroup versus ER/PR-positive patients (35.1% vs. 10.9%). Complete absence of PTEN staining in tumor cells was associated with poor clinical outcome in HER2-positive disease. Those patients whose cancers demonstrated absent PTEN staining had a significant decrease in disease-free survival (DFS) and overall survival (OS) compared with patients with tumors exhibiting any PTEN staining patterns (low, moderate, or high). Trastuzumab appeared to provide clinical benefit even for patients lacking PTEN staining. In the HER2-negative population, there were no statistically significant differences in clinical outcome based on PTEN status.

Conclusions: This study is the largest to date examining PTEN status in breast cancer and the data suggest that the rate and significance of PTEN status differ between HER2-positive and HER2-negative disease. Furthermore, the data clearly suggest that HER2-positive patients with PTEN loss still benefit from trastuzumab. Clin Cancer Res; 21(9); 2065–74. ©2015 AACR.
Data from Vaccination against Oncoproteins of HPV16 for Noninvasive Vulvar/Vaginal Lesions: Lesion Clearance Is Related to the Strength of the T-Cell Response
Authors: van Poelgeest Mariëtte I.E. , Welters Marij J.P. , Vermeij Renee , Stynenbosch Linda F.M. , Loof Nikki M. , Berends-van der Meer Dorien M.A. , Löwik Margriet J.G. , Hamming Ineke L.E. , van Esch Edith M.G. , Hellebrekers Bart W.J. , van Beurden Marc , Schreuder Henk W. , Kagie Marjolein J. , Trimbos J. Baptist M.Z. , Fathers Lorraine M. , Daemen Toos , Hollema Harry , Valentijn A. Rob P.M. , Oostendorp Jaap , Oude Elberink J. Hanneke N.G. , Fleuren Gertjan J. , Bosse Tjalling , Kenter Gemma G. , Stijnen Theo , Nijman Hans W. , Melief Cornelis J.M. , van der Burg Sjoerd H.
Published: April 2023

Abstract
Purpose: Therapeutic vaccination with human papillomavirus type 16 (HPV16) E6 and E7 synthetic long peptides (SLP) is effective against HPV16-induced high-grade vulvar and vaginal intraepithelial neoplasia (VIN/VaIN). However, clinical nonresponders displayed weak CD8+ T-cell reactivity. Here, we studied if imiquimod applied at the vaccine site could improve CD8+ T-cell reactivity, clinical efficacy, and safety of HPV16-SLP (ISA101).

Experimental Design: A multicenter open-label, randomized controlled trial was conducted in patients with HPV16+ high-grade VIN/VaIN. Patients received ISA101 vaccination with or without application of 5% imiquimod at the vaccine site. The primary objective was the induction of a directly ex vivo detectable HPV16-specific CD8+ T-cell response. The secondary objectives were clinical responses (lesion size, histology, and virology) and their relation with the strength of vaccination-induced immune responses.

Results: Forty-three patients were assigned to either ISA101 with imiquimod (n = 21) or ISA101 only (n = 22). Imiquimod did not improve the outcomes of vaccination. However, vaccine-induced clinical responses were observed in 18 of 34 (53%; 95% CI, 35.1–70.2) patients at 3 months and in 15 of 29 (52%; 95% CI, 32.5–70.6) patients, 8 of whom displayed a complete histologic response, at 12 months after the last vaccination. All patients displayed vaccine-induced T-cell responses, which were significantly stronger in patients with complete responses. Importantly, viral clearance occurred in all but one of the patients with complete histologic clearance.

Conclusions: This new study confirms that clinical efficacy of ISA101 vaccination is related to the strength of vaccine-induced HPV16-specific T-cell immunity and is an effective therapy for HPV16-induced high-grade VIN/VaIN. Clin Cancer Res; 22(10); 2342–50. ©2016 AACR.

See related commentary by Karaki et al., p. 2317
Data from Treatment Response Assessment in IDH-Mutant Glioma Patients by Noninvasive 3D Functional Spectroscopic Mapping of 2-Hydroxyglutarate
Authors: Andronesi Ovidiu C. , Loebel Franziska , Bogner Wolfgang , Marjańska Małgorzata , Vander Heiden Matthew G. , Iafrate A. John , Dietrich Jorg , Batchelor Tracy T. , Gerstner Elizabeth R. , Kaelin William G. , Chi Andrew S. , Rosen Bruce R. , Cahill Daniel P.
Published: April 2023

Abstract
Purpose: Measurements of objective response rates are critical to evaluate new glioma therapies. The hallmark metabolic alteration in gliomas with mutant isocitrate dehydrogenase (IDH) is the overproduction of oncometabolite 2-hydroxyglutarate (2HG), which plays a key role in malignant transformation. 2HG represents an ideal biomarker to probe treatment response in IDH-mutant glioma patients, and we hypothesized a decrease in 2HG levels would be measureable by in vivo magnetic resonance spectroscopy (MRS) as a result of antitumor therapy.

Experimental Design: We report a prospective longitudinal imaging study performed in 25 IDH-mutant glioma patients receiving adjuvant radiation and chemotherapy. A newly developed 3D MRS imaging was used to noninvasively image 2HG. Paired Student t test was used to compare pre- and posttreatment tumor 2HG values. Test–retest measurements were performed to determine the threshold for 2HG functional spectroscopic maps (fSM). Univariate and multivariate regression were performed to correlate 2HG changes with Karnofsky performance score (KPS).

Results: We found that mean 2HG (2HG/Cre) levels decreased significantly (median = 48.1%; 95% confidence interval = 27.3%–56.5%; P = 0.007) in the posttreatment scan. The volume of decreased 2HG correlates (R2 = 0.88, P = 0.002) with clinical status evaluated by KPS.

Conclusions: We demonstrate that dynamic measurements of 2HG are feasible by 3D fSM, and the decrease of 2HG levels can monitor treatment response in patients with IDH-mutant gliomas. Our results indicate that quantitative in vivo 2HG imaging may be used for precision medicine and early response assessment in clinical trials of therapies targeting IDH-mutant gliomas. Clin Cancer Res; 22(7); 1632–41. ©2015 AACR.
Data from Spatiotemporal Heterogeneity Characterizes the Genetic Landscape of Pheochromocytoma and Defines Early Events in Tumorigenesis
Authors: Crona Joakim , Backman Samuel , Maharjan Rajani , Mayrhofer Markus , Stålberg Peter , Isaksson Anders , Hellman Per , Björklund Peyman
Published: April 2023

Abstract
Purpose: Pheochromocytoma and paraganglioma (PPGL) patients display heterogeneity in the clinical presentation and underlying genetic cause. The degree of inter- and intratumor genetic heterogeneity has not yet been defined.

Experimental Design: In PPGLs from 94 patients, we analyzed LOH, copy-number variations, and mutation status of SDHA, SDHB, SDHC, SDHD, SDHAF2, VHL, EPAS1, NF1, RET, TMEM127, MAX, and HRAS using high-density SNP array and targeted deep sequencing, respectively. Genetic heterogeneity was determined through (i) bioinformatics analysis of individual samples that estimated absolute purity and ploidy from SNP array data and (ii) comparison of paired tumor samples that allowed reconstruction of phylogenetic trees.

Results: Mutations were found in 61% of the tumors and correlated with specific patterns of somatic copy-number aberrations (SCNA) and degree of nontumoral cell admixture. Intratumor genetic heterogeneity was observed in 74 of 136 samples using absolute bioinformatics estimations and in 22 of 24 patients by comparison of paired samples. In addition, a low genetic concordance was observed between paired primary tumors and distant metastases. This allowed for reconstructing the life history of individual tumors, identifying somatic mutations as well as copy-number loss of 3p and 11p (VHL subgroup), 1p (Cluster 2), and 17q (NF1 subgroup) as early events in PPGL tumorigenesis.

Conclusions: Genomic landscapes of PPGL are specific to mutation subtype and characterized by genetic heterogeneity both within and between tumor lesions of the same patient. Clin Cancer Res; 21(19); 4451–60. ©2015 AACR.
Data from Primary and Acquired Resistance to Immune Checkpoint Inhibitors in Metastatic Melanoma
Authors: Gide Tuba N. , Wilmott James S. , Scolyer Richard A. , Long Georgina V.
Published: April 2023

Abstract
Immune checkpoint inhibitors have revolutionized the treatment of patients with advanced-stage metastatic melanoma, as well as patients with many other solid cancers, yielding long-lasting responses and improved survival. However, a subset of patients who initially respond to immunotherapy, later relapse and develop therapy resistance (termed “acquired resistance”), whereas others do not respond at all (termed “primary resistance”). Primary and acquired resistance are key clinical barriers to further improving outcomes of patients with metastatic melanoma, and the known mechanisms underlying each involves various components of the cancer immune cycle, and interactions between multiple signaling molecules and pathways. Due to this complexity, current knowledge on resistance mechanisms is still incomplete. Overcoming therapy resistance requires a thorough understanding of the mechanisms underlying immune evasion by tumors. In this review, we explore the mechanisms of primary and acquired resistance to immunotherapy in melanoma and detail potential therapeutic strategies to prevent and overcome them. Clin Cancer Res; 24(6); 1260–70. ©2017 AACR.
Data from Evaluating Acetate Metabolism for Imaging and Targeting in Multiple Myeloma
Authors: Fontana Francesca , Ge Xia , Su Xinming , Hathi Deep , Xiang Jingyu , Cenci Simone , Civitelli Roberto , Shoghi Kooresh I. , Akers Walter J. , D'avignon Andre , Weilbaecher Katherine N. , Shokeen Monica
Published: April 2023

Abstract
Purpose: We hypothesized that in multiple myeloma cells (MMC), high membrane biosynthesis will induce acetate uptake in vitro and in vivo. Here, we studied acetate metabolism and targeting in MMC in vitro and tested the efficacy of 11C-acetate–positron emission tomography (PET) to detect and quantitatively image myeloma treatment response in vivo.

Experimental design: Acetate fate tracking using 13C-edited-1H NMR (nuclear magnetic resonance) was performed to study in vitro acetate uptake and metabolism in MMC. Effects of pharmacological modulation of acetate transport or acetate incorporation into lipids on MMC cell survival and viability were assessed. Preclinical mouse MM models of subcutaneous and bone tumors were evaluated using 11C-acetate-PET/CT imaging and tissue biodistribution.

Results: In vitro, NMR showed significant uptake of acetate by MMC and acetate incorporation into intracellular metabolites and membrane lipids. Inhibition of lipid synthesis and acetate transport was toxic to MMC, while sparing resident bone cells or normal B cells. In vivo, 11C-acetate uptake by PET imaging was significantly enhanced in subcutaneous and bone MMC tumors compared with unaffected bone or muscle tissue. Likewise, 11C-acetate uptake was significantly reduced in MM tumors after treatment.

Conclusions: Uptake of acetate from the extracellular environment was enhanced in MMC and was critical to cellular viability. 11C-Acetate–PET detected the presence of myeloma cells in vivo, including uptake in intramedullary bone disease. 11C-Acetate–PET also detected response to therapy in vivo. Our data suggested that acetate metabolism and incorporation into lipids was crucial to MM cell biology and that 11C-acetate–PET is a promising imaging modality for MM. Clin Cancer Res; 23(2); 416–29. ©2016 AACR.
Data from Clinical Trial of Oral Nelfinavir before and during Radiation Therapy for Advanced Rectal Cancer
Authors: Hill Esme J. , Roberts Corran , Franklin Jamie M. , Enescu Monica , West Nicholas , MacGregor Thomas P. , Chu Kwun-Ye , Boyle Lucy , Blesing Claire , Wang Lai-Mun , Mukherjee Somnath , Anderson Ewan M. , Brown Gina , Dutton Susan , Love Sharon B. , Schnabel Julia A. , Quirke Phil , Muschel Ruth , McKenna William G. , Partridge Michael , Sharma Ricky A.
Published: April 2023

Abstract
Purpose: Nelfinavir, a PI3K pathway inhibitor, is a radiosensitizer that increases tumor blood flow in preclinical models. We conducted an early-phase study to demonstrate the safety of nelfinavir combined with hypofractionated radiotherapy (RT) and to develop biomarkers of tumor perfusion and radiosensitization for this combinatorial approach.

Experimental Design: Ten patients with T3-4 N0-2 M1 rectal cancer received 7 days of oral nelfinavir (1,250 mg b.i.d.) and a further 7 days of nelfinavir during pelvic RT (25 Gy/5 fractions/7 days). Perfusion CT (p-CT) and DCE-MRI scans were performed pretreatment, after 7 days of nelfinavir and prior to the last fraction of RT. Biopsies taken pretreatment and 7 days after the last fraction of RT were analyzed for tumor cell density (TCD).

Results: There were 3 drug-related grade 3 adverse events: diarrhea, rash, and lymphopenia. On DCE-MRI, there was a mean 42% increase in median Ktrans, and a corresponding median 30% increase in mean blood flow on p-CT during RT in combination with nelfinavir. Median TCD decreased from 24.3% at baseline to 9.2% in biopsies taken 7 days after RT (P = 0.01). Overall, 5 of 9 evaluable patients exhibited good tumor regression on MRI assessed by tumor regression grade (mrTRG).

Conclusions: This is the first study to evaluate nelfinavir in combination with RT without concurrent chemotherapy. It has shown that nelfinavir-RT is well tolerated and is associated with increased blood flow to rectal tumors. The efficacy of nelfinavir-RT versus RT alone merits clinical evaluation, including measurement of tumor blood flow. Clin Cancer Res; 22(8); 1922–31. ©2016 AACR.

See related commentary by Meyn et al., p. 1834
Data from Radiation Enhancement of Head and Neck Squamous Cell Carcinoma by the Dual PI3K/mTOR Inhibitor PF-05212384
Authors: Leiker Andrew J. , DeGraff William , Choudhuri Rajani , Sowers Anastasia L. , Thetford Angela , Cook John A. , Van Waes Carter , Mitchell James B.
Published: April 2023

Abstract
Purpose: Radiation remains a mainstay for the treatment of nonmetastatic head and neck squamous cell carcinoma (HNSCC), a malignancy characterized by a high rate of PI3K/mTOR signaling axis activation. We investigated the ATP-competitive dual PI3K/mTOR inhibitor, PF-05212384, as a radiosensitizer in preclinical HNSCC models.

Experimental Design: Extent of radiation enhancement of two HNSCC cell lines (UMSCC1-wtP53 and UMSCC46-mtP53) and normal human fibroblast (1522) was assessed by in vitro clonogenic assay with appropriate target inhibition verified by immunoblotting. Radiation-induced DNA damage repair was evaluated by γH2AX Western blots with the mechanism of DNA double-strand break repair abrogation investigated by cell cycle analysis, immunoblotting, and RT-PCR. PF-05212384 efficacy in vivo was assessed by UMSCC1 xenograft tumor regrowth delay, xenograft lysate immunoblotting, and tissue section immunohistochemistry.

Results: PF-05212384 effectively inhibited PI3K and mTOR, resulting in significant radiosensitization of exponentially growing and plateau-phase cells with 24-hour treatment following irradiation, and variable radiation enhancement with 24-hour treatment before irradiation. Tumor cells radiosensitized to a greater extent than normal human fibroblasts. Postirradiation PF-05212384 treatment delays γH2AX foci resolution. PF-05212384 24-hour exposure resulted in an evident G1–S phase block in p53-competent cells. Fractionated radiation plus i.v. PF-05212384 synergistically delayed nude mice bearing UMSCC1 xenograft regrowth, with potential drug efficacy biomarkers identified, including pS6, pAkt, p4EBP1, and Ki67.

Conclusions: Taken together, our results of significant radiosensitization both in vitro and in vivo validate the PI3K/mTOR axis as a radiation modification target and PF-05212384 as a potential clinical radiation modifier of nonmetastatic HNSCC. Clin Cancer Res; 21(12); 2792–801. ©2015 AACR.
Data from Sustained Inhibition of HER3 and EGFR Is Necessary to Induce Regression of HER2-Amplified Gastrointestinal Carcinomas
Authors: Leto Simonetta M. , Sassi Francesco , Catalano Irene , Torri Valter , Migliardi Giorgia , Zanella Eugenia R. , Throsby Mark , Bertotti Andrea , Trusolino Livio
Published: April 2023

Abstract
Purpose: Preclinical studies in HER2-amplified gastrointestinal cancer models have shown that cotargeting HER2 with a monoclonal antibody and a small molecule is superior to monotherapy with either inhibitor, but the underlying cooperative mechanisms remain unexplored. We investigated the molecular underpinnings of this synergy to identify key vulnerabilities susceptible to alternative therapeutic opportunities.

Experimental Design: The phosphorylation/activation of HER2, HER3, EGFR (HER receptors), and downstream transducers was evaluated in HER2-overexpressing colorectal and gastric cancer cell lines by Western blotting and/or multiplex phosphoproteomics. The in vivo outcome of antibody-mediated HER2 blockade by trastuzumab, reversible HER2 inhibition by lapatinib, and irreversible HER2 inhibition by afatinib was assessed in patient-derived tumorgrafts and cell-line xenografts by monitoring tumor growth curves and by using antibody-based proximity assays.

Results: Trastuzumab monotherapy reduced HER3 phosphorylation, with minor consequences on downstream transducers. Lapatinib alone acutely inhibited all HER receptors and effectors but led to delayed rephosphorylation of HER3 and EGFR and partial restoration of ERK and AKT activity. When combined with lapatinib, trastuzumab prevented HER3/EGFR reactivation and caused prolonged inhibition of ERK/AKT. Afatinib alone was also very effective in counteracting the reinstatement of HER3, EGFR, and downstream signaling activation. In vivo, the combination of trastuzumab and lapatinib—or, importantly, monotherapy with afatinib—resulted in overt tumor shrinkage.

Conclusions: Only prolonged inhibition of HER3 and EGFR, achievable by dual blockade with trastuzumab and lapatinib or irreversible HER2 inhibition by single-agent afatinib, led to regression of HER2-amplified gastrointestinal carcinomas. Clin Cancer Res; 21(24); 5519–31. ©2015 AACR.
Data from A Novel MAPK–microRNA Signature Is Predictive of Hormone-Therapy Resistance and Poor Outcome in ER-Positive Breast Cancer
Authors: Miller Philip C. , Clarke Jennifer , Koru-Sengul Tulay , Brinkman Joeli , El-Ashry Dorraya
Published: April 2023

Abstract
Purpose: Hyperactivation of ERK1/2 MAPK (hMAPK) leads to loss of estrogen receptor (ER) expression and poor outcome in breast cancer. microRNAs (miRNA) play important regulatory roles and serve as biomarkers of disease. Here, we describe molecular, pathologic, and clinical outcome associations of an hMAPK–miRNA expression signature in breast cancer.

Experimental Design: An hMAPK–miRNA signature was identified, and associations of this signature with molecular and genetic alterations, gene expression, pathologic features, and clinical outcomes were determined in primary breast cancers from training data and validated using independent datasets. Univariate and multivariate analyses identified subsignatures associated with increased disease recurrence and poorer disease survival among ER-positive (ER+) patients, respectively.

Results: High-hMAPK–miRNA status significantly correlated with ER-negativity, enrichment for basal and HER2-subtypes, and reduced recurrence-free and disease-specific survival in publicly available datasets. A robust determination of a recurrence signature and a survival signature identified hMAPK–miRNAs commonly associated with poor clinical outcome, and specific subsets associated more closely with either disease recurrence or disease survival, especially among ER+ cancers of both luminal A and luminal B subtypes. Multivariate analyses indicated that these recurrence and survival signatures significantly associated with increased risk of disease-specific death and disease recurrence in ER+ cancer and ER+ cancers treated with hormone therapy.

Conclusions: We report an hMAPK–miRNA signature and two subsignatures derived from it that associate significantly with adverse clinical features, poor clinical outcome, and poor response to hormone therapy in breast cancer, thus identifying potential effectors of MAPK signaling, and novel predictive and prognostic biomarkers or therapeutic targets in breast cancer. Clin Cancer Res; 21(2); 373–85. ©2014 AACR.
Data from Estrogen Receptor Expression in 21-Gene Recurrence Score Predicts Increased Late Recurrence for Estrogen-Positive/HER2-Negative Breast Cancer
Authors: Dowsett Mitch , Sestak Ivana , Buus Richard , Lopez-Knowles Elena , Mallon Elizabeth , Howell Anthony , Forbes John F. , Buzdar Aman , Cuzick Jack
Published: April 2023

Abstract
Purpose: To identify the individual genes or gene modules that lead to the OncoptypeDx 21-gene recurrence score's reduced performance after 5 years and thereby identify indices of residual risk that may guide selection of patients for extended adjuvant therapy.

Experimental Design: We conducted a retrospective assessment of the relationship between (i) the individual genes and gene modules of the Recurrence Score and (ii) early (0–5 years) and late (5–10 years) recurrence rates in 1,125 postmenopausal patients with primary estrogen receptor–positive breast cancer treated with anastrozole or tamoxifen in the Arimidex, Tamoxifen, Alone or Combined (ATAC) randomized clinical trial.

Results: In the HER2-negative population (n = 1,009), estimates of recurrence risk were similar between years 0–5 and 5–10 for proliferation and invasion modules but markedly different for the estrogen module and genes within it (all split at the median): for low estrogen module, annual recurrence rates were similar across the two time windows (2.06% vs. 2.46%, respectively); for high estrogen module, annual rates were 1.14% versus 2.72%, respectively (Pinteraction = 0.004). Estrogen receptor transcript levels showed inverse prediction across the time windows: HR, 0.88 (0.73–1.07) and 1.19 (0.99–1.43), respectively (Pinteraction = 0.03). Similar time-, module-, and estrogen-dependent relationships were seen for distant recurrence.

Conclusions: Patients with tumors with high estrogen receptor transcript levels benefit most from 5 years' endocrine therapy but show increased recurrence rates after 5 years and may benefit from extended therapy. Improved prognostic profiles may be created by considering period of treatment and follow-up time. Clin Cancer Res; 21(12); 2763–70. ©2015 AACR.
Data from ESR1 Mutations in Circulating Plasma Tumor DNA from Metastatic Breast Cancer Patients
Authors: Chu David , Paoletti Costanza , Gersch Christina , VanDenBerg Dustin A. , Zabransky Daniel J. , Cochran Rory L. , Wong Hong Yuen , Toro Patricia Valda , Cidado Justin , Croessmann Sarah , Erlanger Bracha , Cravero Karen , Kyker-Snowman Kelly , Button Berry , Parsons Heather A. , Dalton W. Brian , Gillani Riaz , Medford Arielle , Aung Kimberly , Tokudome Nahomi , Chinnaiyan Arul M. , Schott Anne , Robinson Dan , Jacks Karen S. , Lauring Josh , Hurley Paula J. , Hayes Daniel F. , Rae James M. , Park Ben Ho
Published: April 2023

Abstract
Purpose: Mutations in the estrogen receptor (ER)α gene, ESR1, have been identified in breast cancer metastases after progression on endocrine therapies. Because of limitations of metastatic biopsies, the reported frequency of ESR1 mutations may be underestimated. Here, we show a high frequency of ESR1 mutations using circulating plasma tumor DNA (ptDNA) from patients with metastatic breast cancer.

Experimental Design: We retrospectively obtained plasma samples from eight patients with known ESR1 mutations and three patients with wild-type ESR1 identified by next-generation sequencing (NGS) of biopsied metastatic tissues. Three common ESR1 mutations were queried for using droplet digital PCR (ddPCR). In a prospective cohort, metastatic tissue and plasma were collected contemporaneously from eight ER-positive and four ER-negative patients. Tissue biopsies were sequenced by NGS, and ptDNA ESR1 mutations were analyzed by ddPCR.

Results: In the retrospective cohort, all corresponding mutations were detected in ptDNA, with two patients harboring additional ESR1 mutations not present in their metastatic tissues. In the prospective cohort, three ER-positive patients did not have adequate tissue for NGS, and no ESR1 mutations were identified in tissue biopsies from the other nine patients. In contrast, ddPCR detected seven ptDNA ESR1 mutations in 6 of 12 patients (50%).

Conclusions: We show that ESR1 mutations can occur at a high frequency and suggest that blood can be used to identify additional mutations not found by sequencing of a single metastatic lesion. Clin Cancer Res; 22(4); 993–9. ©2015 AACR.
Data from Genomic DNA Hypomethylation and Risk of Renal Cell Carcinoma: A Case–Control Study
Authors: Mendoza- Pérez Julia , Gu Jian , Herrera Luis A. , Tannir Nizar M. , Matin Surena F. , Karam Jose A. , Huang Maosheng , Chang David W. , Wood Christopher G. , Wu Xifeng
Published: April 2023

Abstract
Purpose: Genomic DNA hypomethylation is a hallmark of most cancer genomes, promoting genomic instability and cell transformation. In the present study, we sought to determine whether global DNA methylation in peripheral blood is associated with risk of renal cell carcinoma (RCC).

Experimental Design: A retrospective case–control study consisting of 889 RCC cases and an equal number of age, gender, and ethnicity-matched controls was applied. Global DNA methylation was measured as 5-mC% content. Logistic regression was used to estimate odds ratio (OR) and 95% confidence interval (CI) for the association between DNA methylation level and the risk of RCC.

Results: The median 5-mC% was significantly lower in cases than in healthy controls (P < 0.001). In multivariate logistic regression analysis, individuals in the lowest tertile (T1) of 5-mC% had higher risk of RCC with OR of 1.40 (95% CI, 1.06–1.84), compared with individuals in the highest tertile (T3; Pfor trend = 0.02). When stratified by RCC risk factors, associations between hypomethylation and increased RCC risk appeared to be stronger among males (OR, 1.61; Pfor trend = 0.01), younger age (OR, 1.47; Pfor trend = 0.03), never smokers (OR, 1.55; Pfor trend = 0.02), family history of other cancer (OR, 1.64; Pfor trend = 1.22E−03), and late stage (OR, 2.06, Pfor trend = 4.98E−04). Additionally, we observed significant interaction between gender and 5-mC% in elevating RCC risk (Pfor interaction = 0.03).

Conclusions: Our findings suggest an association between global DNA hypomethylation and RCC risk. To establish global DNA hypomethylation as a risk factor for RCC, future prospective studies are warranted. This study may provide further understanding of the etiology of RCC tumorigenesis. Clin Cancer Res; 22(8); 2074–82. ©2015 AACR.
Data from Prognostic and Chemotherapy Predictive Value of Gene-Expression Phenotypes in Primary Lung Adenocarcinoma
Authors: Ringnér Markus , Jönsson Göran , Staaf Johan
Published: April 2023

Abstract
Purpose: Primary lung adenocarcinoma remains a deadly disease. Gene-expression phenotypes (GEPs) in adenocarcinoma have potential to provide clinically relevant disease stratification for improved prognosis and treatment prediction, given appropriate clinical and methodologic validation.

Experimental Design: 2,395 transcriptional adenocarcinoma profiles were assembled from 17 public cohorts and classified by a nearest centroid GEP classifier into three subtypes: terminal respiratory unit (TRU), proximal-proliferative, and proximal-inflammatory, and additionally scored by five transcriptional metagenes representing different biologic processes, including proliferation. Prognostic- and chemotherapy-predictive associations of the subtypes were analyzed by univariate and multivariate analysis using overall survival or distant metastasis-free survival as endpoints.

Results: Overall, GEPs were associated with patient outcome in both univariate and multivariate analyses, although not in all individual cohorts. The prognostically relevant division was between TRU- and non-TRU–classified cases, with expression of proliferation-associated genes as a key prognostic component. In contrast, GEP classification was not predictive of adjuvant chemotherapy response. GEP classification showed stability to random perturbations of genes or samples and alterations to classification procedures (typically <10% of cases/cohort switching subtype). High classification variability (>20% of cases switching subtype) was observed when removing larger or entire fractions of a single subtype, due to gene-centering shifts not addressable by the classifier.

Conclusions: In a large-scale evaluation, we show that GEPs add prognostic value to standard clinicopathologic variables in lung adenocarcinoma. Subject to classifier refinement and confirmation in prospective cohorts, GEPs have potential to affect the prognostication of adenocarcinoma patients through a molecularly driven disease stratification. Clin Cancer Res; 22(1); 218–29. ©2015 AACR.
Data from Stem Cells Increase in Numbers in Perinecrotic Areas in Human Renal Cancer
Authors: Varna Mariana , Gapihan Guillaume , Feugeas Jean-Paul , Ratajczak Philippe , Tan Sophie , Ferreira Irmine , Leboeuf Christophe , Setterblad Niclas , Duval Arnaud , Verine Jérôme , Germain Stéphane , Mongiat-Artus Pierre , Janin Anne , Bousquet Guilhem
Published: April 2023

Abstract
Purpose: Developing strategies to overcome resistance to sunitinib is a major challenge in human renal cell carcinoma (RCC). We hypothesized that sunitinib-induced tumor necrosis–associated hypoxia could interact with renal cancer stem cells in patients with metastatic RCC.

Experimental Design: We studied tissue samples from 7 patients with primary metastatic RCC, before and after sunitinib treatment, and from six xenograft models derived from human RCC. Two xenograft models were responders to sunitinib, the four others were nonresponders. CD133/CXCR4–coexpressing cells derived from the two responder xenograft models were used for in vitro studies.

Results: In the seven primary RCCs, we identified a significantly larger number of CD133/CXCR4–coexpressing cells in perinecrotic versus perivascular areas. Their numbers also significantly increased after treatment, in perinecrotic areas. We reproduced these clinical and pathologic results in all six RCC xenograft models with again a preferential perinecrotic distribution of CD133-expressing cells. Necrosis occurred at day 7 in the two responder models treated with sunitinib, whereas it occurred at day 21 in the untreated controls and in the four nonresponder models. Strikingly, when we studied the six RCC xenograft models at the time necrosis, whether spontaneous or sunitinib-induced, occurred, necrosis area correlated with stem-cell number in all 120 xenografted RCCs. When studied under experimental hypoxia, the number of CD133/CXCR4–coexpressing cells and their tumorigenic potency increased whereas their sensitivity to sunitinib decreased.

Conclusions: In human RCC, sunitinib was able to generate resistance to its own therapeutic effect via induced hypoxia in perinecrotic areas where cancer stem cells were found in increased numbers. Clin Cancer Res; 21(4); 916–24. ©2014 AACR.
Data from Cabozantinib (XL184) Inhibits Growth and Invasion of Preclinical TNBC Models
Authors: Sameni Mansoureh , Tovar Elizabeth A. , Essenburg Curt J. , Chalasani Anita , Linklater Erik S. , Borgman Andrew , Cherba David M. , Anbalagan Arulselvi , Winn Mary E. , Graveel Carrie R. , Sloane Bonnie F.
Published: April 2023

Abstract
Purpose: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype that is associated with poor clinical outcome. There is a vital need for effective targeted therapeutics for TNBC patients, yet treatment strategies are challenged by the significant intertumoral heterogeneity within the TNBC subtype and its surrounding microenvironment. Receptor tyrosine kinases (RTK) are highly expressed in several TNBC subtypes and are promising therapeutic targets. In this study, we targeted the MET receptor, which is highly expressed across several TNBC subtypes.

Experimental Design: Using the small-molecule inhibitor cabozantinib (XL184), we examined the efficacy of MET inhibition in preclinical models that recapitulate human TNBC and its microenvironment. To analyze the dynamic interactions between TNBC cells and fibroblasts over time, we utilized a 3D model referred to as MAME (Mammary Architecture and Microenvironment Engineering) with quantitative image analysis. To investigate cabozantinib inhibition in vivo, we used a novel xenograft model that expresses human HGF and supports paracrine MET signaling.

Results: XL184 treatment of MAME cultures of MDA-MB-231 and HCC70 cells (± HGF-expressing fibroblasts) was cytotoxic and significantly reduced multicellular invasive outgrowths, even in cultures with HGF-expressing fibroblasts. Treatment with XL184 had no significant effects on METneg breast cancer cell growth. In vivo assays demonstrated that cabozantinib treatment significantly inhibited TNBC growth and metastasis.

Conclusions: Using preclinical TNBC models that recapitulate the breast tumor microenvironment, we demonstrate that cabozantinib inhibition is an effective therapeutic strategy in several TNBC subtypes. Clin Cancer Res; 22(4); 923–34. ©2015 AACR.
Data from CD44 Isoform Status Predicts Response to Treatment with Anti-CD44 Antibody in Cancer Patients
Authors: Birzele Fabian , Voss Edgar , Nopora Adam , Honold Konrad , Heil Florian , Lohmann Sabine , Verheul Henk , Le Tourneau Christophe , Delord Jean-Pierre , van Herpen Carla , Mahalingam Devalingam , Coveler Andrew L. , Meresse Valerie , Weigand Stefan , Runza Valeria , Cannarile Michael
Published: April 2023

Abstract
Purpose: CD44, a cell surface glycoprotein, plays important roles in the development, progression, and metastasis of various tumor types. The aim of this study was to investigate how the expression of CD44 isoforms influences the interaction with hyaluronic acid (HA) and how differential isoform expression impacts antitumoral responses in vivo to treatment with RG7356, a humanized anti-CD44 antibody inhibiting CD44–HA interaction.

Experimental Design: CD44 isoform expression on various tumor cell lines was analyzed by RNASeq while data on patients with different tumor types were obtained from the publicly available TCGA RNASeq dataset as well as a phase I clinical study (NCT01358903). We analyzed the link between HA production and CD44 isoform expression as well as the consequences of blocking the CD44-mediated cell adhesion to HA using RG7356. The correlation between CD44 isoform expression and antitumor response to RG7356 treatment was investigated in the corresponding murine xenograft in vivo models as well as in a subset of patients treated with RG7356 from a recently completed phase I clinical trial.

Results: CD44 isoform expression, in particular expression of CD44s, is associated with HA production and predicts response to treatment with RG7356 in tumor xenograft models. Furthermore, patient data suggest that CD44 isoform status is a potential predictive biomarker for clinical response to treatment with RG7356.

Conclusions: We provide new insights into the close interplay between CD44 and HA and a potential biomarker to enrich patient responses to RG7356 in the clinic. Clin Cancer Res; 21(12); 2753–62. ©2015 AACR.
Data from Inhibition of Tumor-Derived Prostaglandin-E2 Blocks the Induction of Myeloid-Derived Suppressor Cells and Recovers Natural Killer Cell Activity
Authors: Mao Yumeng , Sarhan Dhifaf , Steven André , Seliger Barbara , Kiessling Rolf , Lundqvist Andreas
Published: April 2023

Abstract
Purpose: Increased frequencies of myeloid-derived suppressor cells (MDSC) correlate with poor prognosis in patients with cancers. Tumor-derived prostaglandin-E2 (PGE2) plays an important role in inducing MDSCs. However, the detailed mechanisms of this induction remain unknown. To develop targeted therapies for MDSCs, we sought to investigate the molecular basis of PGE2-regulated accumulation of MDSCs and their functional consequence on natural killer (NK) cell activity.

Experimental Design: The effects of PGE2 in inducing phenotypic, signaling, and functional alternations on monocytes were analyzed in vitro. Suppression of NK-cell activity by PGE2-treated monocytes was compared with that of freshly isolated CD14+HLA-DRlow/− monocytic MDSCs (moMDSC) from patients with melanoma. In addition, to explore the in vivo relevance of targeting PGE2 to reduce MDSC-mediated suppression of NK cells, we established a murine model, where tumor cells were disabled from cyclooxygenase-2 (COX-2) production.

Results: Patient-derived moMDSCs inhibited NK-cell activity through the production of TGFβ. In vitro, binding of PGE2 to EP2 and EP4 receptors on monocytes activated the p38MAPK/ERK pathway and resulted in elevated secretion of TGFβ. Similar to moMDSCs, PGE2-treated monocytes potently suppressed NK-cell activity through production of TGFβ. Furthermore, silencing COX-2 in murine 4T1 tumor cells reduced the accumulation of CD11b+Gr1+ MDSCs in the spleen, resulting in concomitant improved in vivo clearance of NK-cell sensitive YAC-1 cells.

Conclusions: Our results reveal an indispensable role of tumor-derived PGE2 in inducing MDSCs and suggest a favorable outcome of combining COX-2–targeted therapy and adoptive NK-cell transfer in patients with cancer. Clin Cancer Res; 20(15); 4096–106. ©2014 AACR.
Data from Deoxycytidine Kinase Expression Underpins Response to Gemcitabine in Bladder Cancer
Authors: Kerr Martin , Scott Helen E. , Groselj Blaz , Stratford Michael R.L. , Karaszi Katalin , Sharma Naomi L. , Kiltie Anne E.
Published: April 2023

Abstract
Purpose: In a recent phase II clinical trial, low-dose (100 mg/m2) gemcitabine showed promise as a radiosensitizer in bladder cancer, but underlying mechanisms lack elucidation. Here, we investigated the mechanism of radiosensitization by low-dose gemcitabine in bladder cancer cell lines.

Experimental Design: Four bladder cancer cell lines were screened for radiosensitization by low-dose gemcitabine using clonogenic assay, and gemcitabine-resistant RT112gem and CALgem cells created by exposure to increasing gemcitabine doses. Four key gemcitabine-regulatory genes were knocked down by transient siRNA. Nude mice carrying CALgem subcutaneous xenografts were exposed to 100 mg/kg gemcitabine ± ionizing radiation (IR) and response assessed by tumor growth delay.

Results: Gemcitabine was cytotoxic in the low nanomolar range (10–40 nmol/L) in four bladder cancer cell lines and radiosensitized all four lines. Sensitizer enhancement ratios at 10% survival were: RT112 1.42, CAL29 1.55, T24 1.63, and VMCUB1 1.47. Transient siRNA knockdown of deoxycytidine kinase (dCK) significantly reduced radiosensitization by gemcitabine (P = 0.02). RT112gem and CALgem cells displayed robust decreases of dCK mRNA and protein levels; reexpression of dCK restored gemcitabine sensitivity. However, CALgem xenografts responded better to combination gemcitabine/IR than either treatment alone (P < 0.001) with dCK strongly expressed in the tumor vasculature and stroma.

Conclusions: Gemcitabine resistance in bladder cancer cell lines was associated with decreased dCK expression, but gemcitabine-resistant xenografts were responsive to combination low-dose gemcitabine/IR. We propose that dCK activity in tumor vasculature renders it gemcitabine sensitive, which is sufficient to invoke a tumor response and permit tumor cell kill in gemcitabine-resistant tumors. Clin Cancer Res; 20(21); 5435–45. ©2014 AACR.
Data from Treatment Regimen, Surgical Outcome, and T-cell Differentiation Influence Prognostic Benefit of Tumor-Infiltrating Lymphocytes in High-Grade Serous Ovarian Cancer
Authors: Wouters Maartje C.A. , Komdeur Fenne L. , Workel Hagma H. , Klip Harry G. , Plat Annechien , Kooi Neeltje M. , Wisman G. Bea A. , Mourits Marian J.E. , Arts Henriette J.G. , Oonk Maaike H.M. , Yigit Refika , de Jong Steven , Melief Cornelis J.M. , Hollema Harry , Duiker Evelien W. , Daemen Toos , de Bruyn Marco , Nijman Hans W.
Published: April 2023

Abstract
Purpose: Tumor-infiltrating lymphocytes (TIL) are associated with a better prognosis in high-grade serous ovarian cancer (HGSC). However, it is largely unknown how this prognostic benefit of TIL relates to current standard treatment of surgical resection and (neo-)adjuvant chemotherapy. To address this outstanding issue, we compared TIL infiltration in a unique cohort of patients with advanced-stage HGSC primarily treated with either surgery or neoadjuvant chemotherapy.

Experimental Design: Tissue microarray slides containing samples of 171 patients were analyzed for CD8+ TIL by IHC. Freshly isolated CD8+ TIL subsets were characterized by flow cytometry based on differentiation, activation, and exhaustion markers. Relevant T-cell subsets (CD27+) were validated using IHC and immunofluorescence.

Results: A prognostic benefit for patients with high intratumoral CD8+ TIL was observed if primary surgery had resulted in a complete cytoreduction (no residual tissue). By contrast, optimal (<1 cm of residual tumor) or incomplete cytoreduction fully abrogated the prognostic effect of CD8+ TIL. Subsequent analysis of primary TIL by flow cytometry and immunofluorescence identified CD27 as a key marker for a less-differentiated, yet antigen-experienced and potentially tumor-reactive CD8+ TIL subset. In line with this, CD27+ TIL were associated with an improved prognosis even in incompletely cytoreduced patients. Neither CD8+ nor CD27+ cell infiltration was of prognostic benefit in patients treated with neoadjuvant chemotherapy.

Conclusions: Our findings indicate that treatment regimen, surgical result, and the differentiation of TIL should all be taken into account when studying immune factors in HGSC or, by extension, selecting patients for immunotherapy trials. Clin Cancer Res; 22(3); 714–24. ©2015 AACR.