bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from Low PD-1 Expression in Cytotoxic CD8+ Tumor-Infiltrating Lymphocytes Confers an Immune-Privileged Tissue Microenvironment in NSCLC with a Prognostic and Predictive Value
Authors: Mazzaschi Giulia , Madeddu Denise , Falco Angela , Bocchialini Giovanni , Goldoni Matteo , Sogni Francesco , Armani Giovanna , Lagrasta Costanza Annamaria , Lorusso Bruno , Mangiaracina Chiara , Vilella Rocchina , Frati Caterina , Alfieri Roberta , Ampollini Luca , Veneziani Michele , Silini Enrico Maria , Ardizzoni Andrea , Urbanek Konrad , Aversa Franco , Quaini Federico , Tiseo Marcello
Published: April 2023

Abstract
Purpose: The success of immune checkpoint inhibitors strengthens the notion that tumor growth and regression are immune regulated. To determine whether distinct tissue immune microenvironments differentially affect clinical outcome in non–small cell lung cancer (NSCLC), an extended analysis of PD-L1 and tumor-infiltrating lymphocytes (TIL) was performed.

Experimental Design: Samples from resected adenocarcinoma (ADC 42), squamous cell carcinoma (SCC 58), and 26 advanced diseases (13 ADC and 13 SCC) treated with nivolumab were analyzed. PD-L1 expression and the incidence of CD3, CD8, CD4, PD-1, CD57, FOXP3, CD25, and Granzyme B TILs were immunohistochemically assessed.

Results: PD-L1 levels inversely correlated with N involvement, although they did not show a statistically significant prognostic value in resected patients. The incidence and phenotype of TILs differed in SCC versus ADC, in which EGFR and KRAS mutations conditioned a different frequency and tissue localization of lymphocytes. NSCLC resected patients with high CD8pos lymphocytes lacking PD-1 inhibitory receptor had a longer overall survival (OS: HR = 2.268; 95% CI, 1.056–4.871, P = 0.03). PD-1-to-CD8 ratio resulted in a prognostic factor both on univariate (HR = 1.952; 95% CI, 1.34–3.12, P = 0.001) and multivariate (HR = 1.943; 95% CI, 1.38–2.86, P = 0.009) analysis. Moreover, low PD-1 incidence among CD8pos cells was a distinctive feature of nivolumab-treated patients, showing clinical benefit with a prolonged progression-free survival (PFS: HR = 4.51; 95% CI, 1.45–13.94, P = 0.004).

Conclusions: In the presence of intrinsic variability in PD-L1 expression, the reservoir of PD-1–negative effector T lymphocytes provides an immune-privileged microenvironment with a positive impact on survival of patients with resected disease and response to immunotherapy in advanced NSCLC. Clin Cancer Res; 24(2); 407–19. ©2017 AACR.
Data from Evaluation of Tumor-Derived Exosomal miRNA as Potential Diagnostic Biomarkers for Early-Stage Non–Small Cell Lung Cancer Using Next-Generation Sequencing
Authors: Jin Xiance , Chen Yanfan , Chen Hanbin , Fei Shaoran , Chen Didi , Cai Xiaona , Liu Linger , Lin Baochai , Su Huafang , Zhao Lihao , Su Meng , Pan Huanle , Shen Lanxiao , Xie Deyao , Xie Congying
Published: April 2023

Abstract
Purpose: To identify tumor-derived exosomal biomarkers that are able to discriminate between adenocarcinoma and squamous cell carcinoma (SCC) as a noninvasive method in the early diagnosis of non–small cell lung cancer (NSCLC).

Experimental Design: Tumor-derived exosomes from the plasma of early-stage NSCLC patients were isolated. Exosomal miRNA profiling of 46 stage I NSCLC patients and 42 healthy individuals was performed using miRNA-seq to identify and validate adenocarcinoma- and SCC-specific miRNAs. The diagnostic accuracy of select miRNAs was tested further with an additional 60 individuals.

Results: There were 11 and 6 miRNAs expressed at remarkably higher levels, 13 and 8 miRNAs expressed at lower levels in adenocarcinoma and SCC patients, respectively, compared with healthy volunteers. Distinct adenocarcinoma- and SCC-specific exosomal miRNAs were validated. The reliability of miRNA-seq data was verified with several demonstrated diagnostic potential miRNAs for NSCLC and other carcinomas, as reported in previous studies, such as let-7, miR-21, miR-24, and miR-486. The results indicated that miR-181-5p, miR-30a-3p, miR-30e-3p, and miR-361-5p were adenocarcinoma-specific, and miR-10b-5p, miR-15b-5p, and miR-320b were SCC-specific. The diagnostic accuracy of three combination miRNA panels was evaluated using an AUC value of 0.899, 0.936, and 0.911 for detecting NSCLC, adenocarcinoma, and SCC, respectively.

Conclusions: Tumor-derived exosomal miRNAs, adenocarcinoma-specific miR-181-5p, miR-30a-3p, miR-30e-3p and miR-361-5p, and SCC-specific miR-10b-5p, miR-15b-5p, and miR-320b were observed by next-generation sequencing, and their diagnostic accuracy were verified. These miRNAs may be promising and effective candidates in the development of highly sensitive, noninvasive biomarkers for early NSCLC diagnosis. Clin Cancer Res; 23(17); 5311–9. ©2017 AACR.
Data from Blocking Tumor-Educated MSC Paracrine Activity Halts Osteosarcoma Progression
Authors: Baglio S. Rubina , Lagerweij Tonny , Pérez-Lanzón Maria , Ho Xuan Dung , Léveillé Nicolas , Melo Sonia A. , Cleton-Jansen Anne-Marie , Jordanova Ekaterina S. , Roncuzzi Laura , Greco Michelina , van Eijndhoven Monique A.J. , Grisendi Giulia , Dominici Massimo , Bonafede Roberta , Lougheed Sinead M. , de Gruijl Tanja D. , Zini Nicoletta , Cervo Silvia , Steffan Agostino , Canzonieri Vincenzo , Martson Aare , Maasalu Katre , Köks Sulev , Wurdinger Tom , Baldini Nicola , Pegtel D. Michiel
Published: April 2023

Abstract
Purpose: Human osteosarcoma is a genetically heterogeneous bone malignancy with poor prognosis despite the employment of aggressive chemotherapy regimens. Because druggable driver mutations have not been established, dissecting the interactions between osteosarcoma cells and supporting stroma may provide insights into novel therapeutic targets.

Experimental Design: By using a bioluminescent orthotopic xenograft mouse model of osteosarcoma, we evaluated the effect of tumor extracellular vesicle (EV)–educated mesenchymal stem cells (TEMSC) on osteosarcoma progression. Characterization and functional studies were designed to assess the mechanisms underlying MSC education. Independent series of tissue specimens were analyzed to corroborate the preclinical findings, and the composition of patient serum EVs was analyzed after isolation with size-exclusion chromatography.

Results: We show that EVs secreted by highly malignant osteosarcoma cells selectively incorporate a membrane-associated form of TGFβ, which induces proinflammatory IL6 production by MSCs. TEMSCs promote tumor growth, accompanied with intratumor STAT3 activation and lung metastasis formation, which was not observed with control MSCs. Importantly, intravenous administration of the anti-IL6 receptor antibody tocilizumab abrogated the tumor-promoting effects of TEMSCs. RNA-seq analysis of human osteosarcoma tissues revealed a distinct TGFβ-induced prometastatic gene signature. Tissue microarray immunostaining indicated active STAT3 signaling in human osteosarcoma, consistent with the observations in TEMSC-treated mice. Finally, we isolated pure populations of EVs from serum and demonstrated that circulating levels of EV-associated TGFβ are increased in osteosarcoma patients.

Conclusions: Collectively, our findings suggest that TEMSCs promote osteosarcoma progression and provide the basis for testing IL6- and TGFβ-blocking agents as new therapeutic options for osteosarcoma patients. Clin Cancer Res; 23(14); 3721–33. ©2017 AACR.
Data from Antihypoxic Potentiation of Standard Therapy for Experimental Colorectal Liver Metastasis through Myo-Inositol Trispyrophosphate
Authors: Limani Perparim , Linecker Michael , Kachaylo Ekaterina , Tschuor Christoph , Kron Philipp , Schlegel Andrea , Ungethuem Udo , Jang Jae Hwi , Georgiopoulou Stavroula , Nicolau Claude , Lehn Jean-Marie , Graf Rolf , Humar Bostjan , Clavien Pierre-Alain
Published: April 2023

Abstract
Purpose: Tumor hypoxia activates hypoxia-inducible factors (Hifs), which induce a range of malignant changes including vascular abnormalities. Here, we determine whether inhibition of the hypoxic tumor response through myo-inositol trispyrophosphate (ITPP), a compound with antihypoxic properties, is able to cause prolonged vascular normalization that can be exploited to improve standard-of-care treatment.

Experimental Design: We tested ITPP on two syngeneic orthotopic mouse models of lethal colorectal cancer liver metastasis. Tumors were monitored by MRI and analyzed for the hypoxic response and their malignant potential. A Hif activator and in vitro assays were used to define the working mode of ITPP. Hypoxic response and vasculature were re-evaluated 4 weeks after treatment. Finally, we determined survival following ITPP monotherapy, FOLFOX monotherapy, FOLFOX plus Vegf antibody, and FOLFOX plus ITPP, both overlapping and sequential.

Results: ITPP reduced tumor load, efficiently inhibited the hypoxic response, and improved survival. These effects were lost when mice were pretreated with a Hif activator. Its immediate effects on the hypoxic response, including an apparent normalization of tumor vasculature, persisted for at least 4 weeks after treatment cessation. Compared with FOLFOX alone, Vegf antibody combined with FOLFOX prolonged survival by <30%, whereas ITPP combined with FOLFOX extended survival by >140%, regardless of whether FOLFOX was given in overlap or after ITPP exposure.

Conclusions: Our findings reveal a truly antihypoxic mechanism for ITPP and demonstrate the capacity of this nontoxic compound to potentiate the efficacy of existing anticancer treatment in a way amenable to clinical translation. Clin Cancer Res; 22(23); 5887–97. ©2016 AACR.
Data from Whole-Exome Sequencing of Salivary Gland Mucoepidermoid Carcinoma
Authors: Kang Hyunseok , Tan Marietta , Bishop Justin A. , Jones Siân , Sausen Mark , Ha Patrick K. , Agrawal Nishant
Published: April 2023

Abstract
Purpose: Mucoepidermoid carcinoma (MEC) is the most common salivary gland malignancy. To explore the genetic origins of MEC, we performed systematic genomic analyses of these tumors.

Experimental Design: Whole-exome sequencing and gene copy-number analyses were performed for 18 primary cancers with matched normal tissue. FISH was used to determine the presence or absence of the MECT1-MAML2 translocation in 17 tumors.

Results: TP53 was the most commonly mutated gene in MEC (28%), and mutations were found only in intermediate- and high-grade tumors. Tumors with TP53 mutations had more mutations overall than tumors without TP53 mutations (P = 0.006). POU6F2 was the second most frequently mutated gene, found in three low-grade MECs with the same in-frame deletion. Somatic alterations in IRAK1, MAP3K9, ITGAL, ERBB4, OTOGL, KMT2C, and OBSCN were identified in at least two of the 18 tumors sequenced. FISH analysis confirmed the presence of the MECT1-MAML2 translocation in 15 of 17 tumors (88%).

Conclusions: Through these integrated genomic analyses, MECT1-MAML2 translocation and somatic TP53 and POU6F2 mutations appear to be the main drivers of MEC. Clin Cancer Res; 23(1); 283–8. ©2016 AACR.
Data from Clinical and Immunologic Biomarkers for Histologic Regression of High-Grade Cervical Dysplasia and Clearance of HPV16 and HPV18 after Immunotherapy
Authors: Morrow Matthew P. , Kraynyak Kimberly A. , Sylvester Albert J. , Dallas Michael , Knoblock Dawson , Boyer Jean D. , Yan Jian , Vang Russell , Khan Amir S. , Humeau Laurent , Sardesai Niranjan Y. , Kim J. Joseph , Plotkin Stanley , Weiner David B. , Trimble Cornelia L. , Bagarazzi Mark L.
Published: April 2023

Abstract
Purpose: As previously reported, treatment of high-grade cervical dysplasia with VGX-3100 resulted in complete histopathologic regression (CR) concomitant with elimination of HPV16/18 infection in 40.0% of VGX-3100–treated patients compared with only 14.3% in placebo recipients in a randomized phase IIb study. Here, we identify clinical and immunologic characteristics that either predicted or correlated with therapeutic benefit from VGX-3100 to identify parameters that might guide clinical decision-making for this disease.

Experimental Design: We analyzed samples taken from cervical swabs, whole blood, and tissue biopsies/resections to determine correlates and predictors of treatment success.

Results: At study entry, the presence of preexisting immunosuppressive factors such as FoxP3 and PD-L1 in cervical lesions showed no association with treatment outcome. The combination of HPV typing and cervical cytology following dosing was predictive for both histologic regression and elimination of detectable virus at the efficacy assessment 22 weeks later (negative predictive value 94%). Patients treated with VGX-3100 who had lesion regression had a statistically significant >2-fold increase in CD137+perforin+CD8+ T cells specific for the HPV genotype causing disease. Increases in cervical mucosal CD137+ and CD103+ infiltrates were observed only in treated patients. Perforin+ cell infiltrates were significantly increased >2-fold in cervical tissue only in treated patients who had histologic CR.

Conclusions: Quantitative measures associated with an effector immune response to VGX-3100 antigens were associated with lesion regression. Consequently, these analyses indicate that certain immunologic responses associate with successful resolution of HPV-induced premalignancy, with particular emphasis on the upregulation of perforin in the immunotherapy-induced immune response. Clin Cancer Res; 24(2); 276–94. ©2017 AACR.
Data from ImmunoPET of Malignant and Normal B Cells with 89Zr- and 124I-Labeled Obinutuzumab Antibody Fragments Reveals Differential CD20 Internalization In Vivo
Authors: Zettlitz Kirstin A. , Tavaré Richard , Knowles Scott M. , Steward Kristopher K. , Timmerman John M. , Wu Anna M.
Published: April 2023

Abstract
Purpose: The B-cell antigen CD20 provides a target for antibody-based positron emission tomography (immunoPET). We engineered antibody fragments targeting human CD20 and studied their potential as immunoPET tracers in transgenic mice (huCD20TM) and in a murine lymphoma model expressing human CD20.

Experimental Design: Anti-CD20 cys-diabody (cDb) and cys-minibody (cMb) based on rituximab and obinutuzumab (GA101) were radioiodinated and used for immunoPET imaging of a murine lymphoma model. Pairwise comparison of obinutuzumab-based antibody fragments labeled with residualizing (89Zr) versus non-residualizing (124I) radionuclides by region of interest analysis of serial PET images was conducted both in the murine lymphoma model and in huCD20TM to assess antigen modulation in vivo.

Results:124I-GAcDb and 124I-GAcMb produced high-contrast immunoPET images of B-cell lymphoma and outperformed the respective rituximab-based tracers. ImmunoPET imaging of huCD20TM showed specific uptake in lymphoid tissues. The use of the radiometal 89Zr as alternative label for GAcDb and GAcMb yielded greater target-specific uptake and retention compared with 124I-labeled tracers. Pairwise comparison of 89Zr- and 124I-labeled GAcDb and GAcMb allowed assessment of in vivo internalization of CD20/antibody complexes and revealed that CD20 internalization differs between malignant and endogenous B cells.

Conclusions: These obinutuzumab-based PET tracers have the ability to noninvasively and quantitatively monitor CD20-expression and have revealed insights into CD20 internalization upon antibody binding in vivo. Because they are based on a humanized mAb they have the potential for direct clinical translation and could improve patient selection for targeted therapy, dosimetry prior to radioimmunotherapy, and prediction of response to therapy. Clin Cancer Res; 23(23); 7242–52. ©2017 AACR.
Data from Synergistic Drug Combinations with a CDK4/6 Inhibitor in T-cell Acute Lymphoblastic Leukemia
Authors: Pikman Yana , Alexe Gabriela , Roti Giovanni , Conway Amy Saur , Furman Andrew , Lee Emily S. , Place Andrew E. , Kim Sunkyu , Saran Chitra , Modiste Rebecca , Weinstock David M. , Harris Marian , Kung Andrew L. , Silverman Lewis B. , Stegmaier Kimberly
Published: April 2023

Abstract
Purpose: Although significant progress has been made in the treatment of T-cell acute lymphoblastic leukemia (T-ALL), many patients will require additional therapy for relapsed/refractory disease. Cyclin D3 (CCND3) and CDK6 are highly expressed in T-ALL and have been effectively targeted in mutant NOTCH1-driven mouse models of this disease with a CDK4/6 small-molecule inhibitor. Combination therapy, however, will be needed for the successful treatment of human disease.

Experimental Design: We performed preclinical drug testing using a panel of T-ALL cell lines first with LEE011, a CDK4/6 inhibitor, and next with the combination of LEE011 with a panel of drugs relevant to T-ALL treatment. We then tested the combination of LEE011 with dexamethasone or everolimus in three orthotopic mouse models and measured on-target drug activity.

Results: We first determined that both NOTCH1-mutant and wild-type T-ALL are highly sensitive to pharmacologic inhibition of CDK4/6 when wild-type RB is expressed. Next, we determined that CDK4/6 inhibitors are antagonistic when used either concurrently or in sequence with many of the drugs used to treat relapsed T-ALL (methotrexate, mercaptopurine, asparaginase, and doxorubicin) but are synergistic with glucocorticoids, an mTOR inhibitor, and gamma secretase inhibitor. The combinations of LEE011 with the glucocorticoid dexamethasone or the mTOR inhibitor everolimus were tested in vivo and prolonged survival in three orthotopic mouse models of T-ALL. On-target activity was measured in peripheral blood and tissue of treated mice.

Conclusions: We conclude that LEE011 is active in T-ALL and that combination therapy with corticosteroids and/or mTOR inhibitors warrants further investigation. Clin Cancer Res; 23(4); 1012–24. ©2016 AACR.

See related commentary by Carroll et al., p. 873
Data from Active Estrogen Receptor-alpha Signaling in Ovarian Cancer Models and Clinical Specimens
Authors: Andersen Courtney L. , Sikora Matthew J. , Boisen Michelle M. , Ma Tianzhou , Christie Alec , Tseng George , Park Yongseok , Luthra Soumya , Chandran Uma , Haluska Paul , Mantia-Smaldone Gina M. , Odunsi Kunle , McLean Karen , Lee Adrian V. , Elishaev Esther , Edwards Robert P. , Oesterreich Steffi
Published: April 2023

Abstract
Purpose: High-grade serous ovarian cancer (HGSOC) is an aggressive disease with few available targeted therapies. Despite high expression of estrogen receptor-alpha (ERα) in approximately 80% of HGSOC and some small but promising clinical trials of endocrine therapy, ERα has been understudied as a target in this disease. We sought to identify hormone-responsive, ERα-dependent HGSOC.

Experimental Design: We characterized endocrine response in HGSOC cells across culture conditions [ two-dimensional (2D), three-dimensional (3D), forced suspension] and in patient-derived xenograft (PDX) explants, assessing proliferation and gene expression. Estrogen-regulated transcriptome data were overlapped with public datasets to develop a comprehensive panel of ERα target genes. Expression of this panel and ERα H-score were assessed in HGSOC samples from patients who received endocrine therapy. Time on endocrine therapy was used as a surrogate for clinical response.

Results: Proliferation is ERα-regulated in HGSOC cells in vitro and in vivo, and is partly dependent on 3D context. Transcriptomic studies identified genes shared by cell lines and PDX explants as ERα targets. The selective ERα downregulator (SERD) fulvestrant is more effective than tamoxifen in blocking ERα action. ERα H-score is predictive of efficacy of endocrine therapy, and this prediction is further improved by inclusion of target gene expression, particularly IGFBP3.

Conclusions: Laboratory models corroborate intertumor heterogeneity of endocrine response in HGSOC but identify features associated with functional ERα and endocrine responsiveness. Assessing ERα function (e.g., IGFBP3 expression) in conjunction with H-score may help select patients who would benefit from endocrine therapy. Preclinical data suggest that SERDs might be more effective than tamoxifen. Clin Cancer Res; 23(14); 3802–12. ©2017 AACR.
Data from A Four-Gene Promoter Methylation Marker Panel Consisting of GREM1, NEURL, LAD1, and NEFH Predicts Survival of Clear Cell Renal Cell Cancer Patients
Authors: van Vlodrop Iris J.H. , Joosten Sophie C. , De Meyer Tim , Smits Kim M. , Van Neste Leander , Melotte Veerle , Baldewijns Marcella M.L.L. , Schouten Leo J. , van den Brandt Piet A. , Jeschke Jana , Yi Joo Mi , Schuebel Kornel E. , Ahuja Nita , Herman James G. , Aarts Maureen J. , Bosman Fred T. , Van Criekinge Wim , van Engeland Manon
Published: April 2023

Abstract
Purpose: The currently used prognostic models for patients with nonmetastatic clear cell renal cell carcinoma (ccRCC) are based on clinicopathologic features and might be improved by adding molecular markers. Epigenetic alterations occur frequently in ccRCC and are promising biomarkers. The aim of this study is to identify prognostic promoter methylation markers for ccRCC.

Experimental Design: We integrated data generated by massive parallel sequencing of methyl-binding domain enriched DNA and microarray-based RNA expression profiling of 5-aza-2′-deoxycytidine–treated ccRCC cell lines to comprehensively characterize the ccRCC methylome. A selection of the identified methylation markers was evaluated in two independent series of primary ccRCC (n = 150 and n = 185) by methylation-specific PCR. Kaplan–Meier curves and log-rank tests were used to estimate cause-specific survival. HRs and corresponding 95% confidence intervals (CI) were assessed using Cox proportional hazard models. To assess the predictive capacity and fit of models combining several methylation markers, HarrellC statistic and the Akaike Information Criterion were used.

Results: We identified four methylation markers, that is, GREM1, NEURL, LAD1, and NEFH, that individually predicted prognosis of patients with ccRCC. The four markers combined were associated with poorer survival in two independent patient series (HR, 3.64; 95% CI, 1.02–13.00 and HR, 7.54; 95% CI, 2.68–21.19). These findings were confirmed in a third series of ccRCC cases from The Cancer Genome Atlas (HR, 3.60; 95% CI, 2.02–6.40).

Conclusions: A four-gene promoter methylation marker panel consisting of GREM1, NEURL, LAD1, and NEFH predicts outcome of patients with ccRCC and might be used to improve current prognostic models. Clin Cancer Res; 23(8); 2006–18. ©2016 AACR.
Data from Age and Gender Associations of Virus Positivity in Merkel Cell Carcinoma Characterized Using a Novel RNA In Situ Hybridization Assay
Authors: Wang Lisha , Harms Paul W. , Palanisamy Nallasivam , Carskadon Shannon , Cao Xuhong , Siddiqui Javed , Patel Rajiv M. , Zelenka-Wang Sylvia , Durham Alison B. , Fullen Douglas R. , Harms Kelly L. , Su Fengyun , Shukla Sudhanshu , Mehra Rohit , Chinnaiyan Arul M.
Published: April 2023

Abstract
Purpose: Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine tumor of the skin. Merkel cell polyomavirus (MCPyV) plays an oncogenic role in the majority of MCCs. Detection of MCPyV in MCCs has diagnostic utility and prognostic potential. We investigated whether RNAscope, an RNA in situ hybridization (ISH) assay for detection of RNA transcripts in tissues, is useful for MCPyV detection.

Experimental Design: We applied an RNAscope probe targeting MCPyV T antigen transcripts on tissue microarrays (TMA) and whole-tissue sections encompassing 87 MCCs from 75 patients, 14 carcinomas of other types, and benign tissues. For comparison, qPCR was performed on 57 cases of MCC from 52 patients.

Results: RNA-ISH demonstrated the presence of MCPyV in 37 of 75 cases (49.3%). Notably, tumors from younger patients (<73 years) had a significantly higher virus positivity than those from elderly patients (≥73 years; 64.9% vs. 34.2%, P = 0.011). Female patients had a higher positive rate of MCPyV than male patients (66.7% vs. 39.6%, P = 0.032). Data from both RNA-ISH and qPCR were available for 57 samples. Considering MCPyV qPCR as the gold standard for determining MCPyV status, RNAscope had 100% sensitivity and 100% specificity. There was a strong correlation between qPCR copy number and RNA-ISH product score (Spearman correlation coefficient R2 = 0.932, P < 0.0001).

Conclusions: RNA-ISH is comparably sensitive to qPCR for detection of MCPyV and allows for correlation with tissue morphology. This study also reveals a significant association between age, gender, and MCPyV positivity. Clin Cancer Res; 23(18); 5622–30. ©2017 AACR.
Data from Analysis of Circulating Tumor DNA and Clinical Correlates in Patients with Esophageal, Gastroesophageal Junction, and Gastric Adenocarcinoma
Authors: Kato Shumei , Okamura Ryosuke , Baumgartner Joel M. , Patel Hitendra , Leichman Lawrence , Kelly Kaitlyn , Sicklick Jason K. , Fanta Paul T. , Lippman Scott M. , Kurzrock Razelle
Published: April 2023

AbstractPurpose:
Esophageal, gastroesophageal junction, and gastric adenocarcinoma (herein gastroesophageal adenocarcinomas) are associated with poor prognosis and limited systemic treatment options. To further understand the genomic landscape of gastroesophageal cancers and its clinical correlations, circulating tumor DNA (ctDNA) from patients’ plasma was evaluated using next-generation sequencing (NGS).
Experimental Design:
We analyzed genomic alterations of 55 patients (mostly advanced disease; 9, surgically resectable) with gastroesophageal adenocarcinomas using clinical-grade NGS performed on plasma-derived ctDNA (54–73 gene panel). The test detects single-nucleotide variants, as well as copy number amplifications, fusions, and indels in selected genes.
Results:
Seventy-six percent of patients (42/55) had ≥1 genomic alteration [including variants of unknown significance (VUS)] and 69.1% (38/55) had ≥1 characterized alteration (excluding VUSs). The median number of alterations per patient was 2 (range, 0–15). TP53 (50.9%, 28/55), PIK3CA (16.4%, 9/55), ERBB2 (14.5%, 8/55), and KRAS (14.5%, 8/55) genes were most frequently affected characterized alterations. Thirty-one patients also had tissue NGS. Concordance between tissue and ctDNA ranged from 61.3% (TP53 alterations) to 87.1% (KRAS alterations). ERBB2 alterations were significantly associated with poor overall survival (HR, 14.06; 95% confidence interval, 2.44–81.03; P = 0.003 multivariate analysis). Among patients with ≥1 alteration, no 2 patients had identical molecular portfolios. All patients with ≥1 characterized alteration had theoretically targetable alterations by an FDA-approved agent (on- or off-label). Illustrative case treated with cognate agent is presented.
Conclusions:
Evaluation of ctDNA by NGS among patients with gastroesophageal adenocarcinoma is feasible. Patients harbored heterogeneous patterns of genomics, with most having alterations that are potentially pharmacologically tractable.
Data from Preclinical Development and First-in-Human Imaging of the Integrin αvβ6 with [18F]αvβ6-Binding Peptide in Metastatic Carcinoma
Authors: Hausner Sven H. , Bold Richard J. , Cheuy Lina Y. , Chew Helen K. , Daly Megan E. , Davis Ryan A. , Foster Cameron C. , Kim Edward J. , Sutcliffe Julie L.
Published: April 2023

AbstractPurpose:
The study was undertaken to develop and evaluate the potential of an integrin αvβ6-binding peptide (αvβ6-BP) for noninvasive imaging of a diverse range of malignancies with PET.
Experimental Design:
The peptide αvβ6-BP was prepared on solid phase and radiolabeled with 4-[18F]fluorobenzoic acid. In vitro testing included ELISA, serum stability, and cell binding studies using paired αvβ6-expressing and αvβ6-null cell lines. In vivo evaluation (PET/CT, biodistribution, and autoradiography) was performed in a mouse model bearing the same paired αvβ6-expressing and αvβ6-null cell xenografts. A first-in-human PET/CT imaging study was performed in patients with metastatic lung, colon, breast, or pancreatic cancer.
Results:
[18F]αvβ6-BP displayed excellent affinity and selectivity for the integrin αvβ6 in vitro [IC50(αvβ6) = 1.2 nmol/L vs IC50(αvβ3) >10 μmol/L] in addition to rapid target-specific cell binding and internalization (72.5% ± 0.9% binding and 52.5% ± 1.8%, respectively). Favorable tumor affinity and selectivity were retained in the mouse model and excretion of unbound [18F]αvβ6-BP was rapid, primarily via the kidneys. In patients, [18F]αvβ6-BP was well tolerated without noticeable adverse side effects. PET images showed significant uptake of [18F]αvβ6-BP in both the primary lesion and metastases, including metastasis to brain, bone, liver, and lung.
Conclusions:
The clinical impact of [18F]αvβ6-BP PET imaging demonstrated in this first-in-human study is immediate for a broad spectrum of malignancies.
Data from Plasma HER2 (ERBB2) Copy Number Predicts Response to HER2-targeted Therapy in Metastatic Colorectal Cancer
Authors: Siravegna Giulia , Sartore-Bianchi Andrea , Nagy Rebecca J. , Raghav Kanwal , Odegaard Justin I. , Lanman Richard B. , Trusolino Livio , Marsoni Silvia , Siena Salvatore , Bardelli Alberto
Published: April 2023

AbstractPurpose:
ERBB2 (HER2) amplification is an emerging biomarker in colon cancer, conferring sensitivity to combination anti-HER2 therapy. Measurement of HER2 copy number is typically performed using surgical specimens, but cell-free circulating tumor DNA (ctDNA) analysis may be a noninvasive alternative. We determined the sensitivity of plasma copy number (pCN) for detecting ERBB2 amplifications and whether pCN correlated with tissue-detected copy number. We also assessed response to HER2-targeted therapy based on pCN and suggest a pCN threshold predictive of response.
Experimental Design:
Forty-eight pretreatment and progression plasma samples from 29 HER2-positive patients in the HERACLES A clinical trial were tested using the Guardant360 cfDNA assay. We correlated ERRB2 pCN with progression-free survival (PFS) and best objective response (BOR) and applied an adjustment method based on tumor DNA shedding using the maximum mutant allele fraction as a surrogate for tumor content to accurately determine the pCN threshold predictive of response.
Results:
Forty-seven of 48 samples had detectable ctDNA, and 46 of 47 samples were ERBB2-amplified on the basis of cfDNA [2.55–122 copies; 97.9% sensitivity (95% confidence interval, 87.2%–99.8%)]. An adjusted ERBB2 pCN of ≥25.82 copies correlated with BOR and PFS (P = 0.0347).
Conclusions:
cfDNA is a viable alternative to tissue-based genotyping in the metastatic setting. The cfDNA platform utilized correctly identified 28 of 29 (96.6%) of pretreatment samples as ERBB2-amplified and predicted benefit from HER2-targeted therapy. In this study, an observed pCN of 2.4 and an adjusted pCN of 25.82 copies of ERBB2 are proposed to select patients who will benefit from HER2-targeted therapy.
Data from Patient-derived Interstitial Fluids and Predisposition to Aggressive Sporadic Breast Cancer through Collagen Remodeling and Inactivation of p53
Authors: Kenny Timothy C. , Schmidt Hank , Adelson Kerin , Hoshida Yujin , Koh Anna P. , Shah Nagma , Mandeli John , Ting Jess , Germain Doris
Published: April 2023

Abstract
Purpose: Despite the fact that interstitial fluid (IF) represents a third of our body fluid, it is the most poorly understood body fluid in medicine. Increased IF pressure is thought to result from the increased deposition of extracellular matrix in the affected tissue preventing its reabsorption. In the cancer field, increased rigidity surrounding a cancerous mass remains the main reason that palpation and radiologic examination, such as mammography, are used for cancer detection. While the pressure produced by IF has been considered, the biochemical composition of IF has not been considered in its effect on tumors.

Experimental Design: We classified 135 IF samples from bilateral mastectomy patients based on their ability to promote the invasion of breast cancer cells.

Results: We observed a wide range of invasion scores. Patients with high-grade primary tumors at diagnosis had higher IF invasion scores. In mice, injections of high-score IF (IFHigh) in a normal mammary gland promotes ductal hyperplasia, increased collagen deposition, and local invasion. In a mouse model of residual disease, IFHigh increased disease progression and promoted aggressive visceral metastases. Mechanistically, we found that IFHigh induces myofibroblast differentiation and collagen production through activation of CLIC4. IFHigh also downregulates RYBP, leading to degradation of p53. Furthermore, in mammary glands of heterozygous p53-mutant knock-in mice, IFHigh promotes spontaneous tumor formation.

Conclusions: Our study indicates that IF can increase the deposition of extracellular matrix and raises the provocative possibility that they play an active role in the predisposition, development, and clinical course of sporadic breast cancers. Clin Cancer Res; 23(18); 5446–59. ©2017 AACR.
Data from Alternative Lengthening of Telomeres and Loss of DAXX/ATRX Expression Predicts Metastatic Disease and Poor Survival in Patients with Pancreatic Neuroendocrine Tumors
Authors: Singhi Aatur D. , Liu Ta-Chiang , Roncaioli Justin L. , Cao Dengfeng , Zeh Herbert J. , Zureikat Amer H. , Tsung Allan , Marsh J. Wallis , Lee Kenneth K. , Hogg Melissa E. , Bahary Nathan , Brand Randall E. , McGrath Kevin M. , Slivka Adam , Cressman Kristi L. , Fuhrer Kimberly , O'Sullivan Roderick J.
Published: April 2023

Abstract
Purpose: Pancreatic neuroendocrine tumors (PanNET) are a heterogeneous group of neoplasms with increasing incidence and unpredictable behavior. Whole-exome sequencing has identified recurrent mutations in the genes DAXX and ATRX, which correlate with loss of protein expression and alternative lengthening of telomeres (ALT). Both ALT and DAXX/ATRX loss were initially reported to be associated with a favorable prognosis; however, recent studies suggest the contrary. Our aims were to assess the prevalence and prognostic significance of ALT and DAXX/ATRX in both primary and metastatic PanNETs.

Experimental Design: Telomere-specific FISH and DAXX/ATRX IHC was performed on a multi-institutional cohort of 321 patients with resected PanNET and 191 distant metastases from 52 patients. These results were correlated with clinicopathologic features, including disease-free survival (DFS) and disease-specific survival (DSS).

Results: The prevalence of ALT and DAXX/ATRX loss in resected PanNETs was 31% and 26%, respectively, and associated with larger tumor size, higher WHO grade, lymph node metastasis, and distant metastasis (P < 0.001). The 5-year DFS and 10-year DSS of patients with ALT-positive and DAXX/ATRX-negative PanNETs were 40% and 50%, respectively, as compared with 96% and 89%, respectively, for wild-type PanNETs. Among distant metastases, ALT and DAXX/ATRX loss was 67% and 52%, respectively, and only occurred in the setting of an ALT-positive and DAXX/ATRX-negative primary PanNET. By multivariate analysis, both ALT and DAXX/ATRX loss were negative, independent prognostic factors for DFS.

Conclusions: ALT and DAXX/ATRX loss in PanNETs was associated with shorter DFS and DSS and likely plays a significant role in driving metastatic disease. Clin Cancer Res; 23(2); 600–9. ©2016 AACR.
Data from Circulating Tumor DNA Reveals Clinically Actionable Somatic Genome of Metastatic Bladder Cancer
Authors: Vandekerkhove Gillian , Todenhöfer Tilman , Annala Matti , Struss Werner J. , Wong Amanda , Beja Kevin , Ritch Elie , Brahmbhatt Sonal , Volik Stanislav V. , Hennenlotter Jörg , Nykter Matti , Chi Kim N. , North Scott , Stenzl Arnulf , Collins Colin C. , Eigl Bernhard J. , Black Peter C. , Wyatt Alexander W.
Published: April 2023

Abstract
Purpose: Targeted agents and immunotherapies promise to transform the treatment of metastatic bladder cancer, but therapy selection will depend on practical tumor molecular stratification. Circulating tumor DNA (ctDNA) is established in several solid malignancies as a minimally invasive tool to profile the tumor genome in real-time, but is critically underexplored in bladder cancer.

Experimental Design: We applied a combination of whole-exome sequencing and targeted sequencing across 50 bladder cancer driver genes to plasma cell-free DNA (cfDNA) from 51 patients with aggressive bladder cancer, including 37 with metastatic disease.

Results: The majority of patients with metastasis, but only 14% of patients with localized disease, had ctDNA proportions above 2% of total cfDNA (median 16.5%, range 3.9%–72.6%). Twelve percent of estimable samples had evidence of genome hypermutation. We reveal an aggressive mutational landscape in metastatic bladder cancer with 95% of patients harboring deleterious alterations to TP53, RB1, or MDM2, and 70% harboring a mutation or disrupting rearrangement affecting chromatin modifiers such as ARID1A. Targetable alterations in MAPK/ERK or PI3K/AKT/mTOR pathways were robustly detected, including amplification of ERBB2 (20% of patients) and activating hotspot mutations in PIK3CA (20%), with the latter mutually exclusive to truncating mutations in TSC1. A novel FGFR3 gene fusion was identified in consecutive samples from one patient.

Conclusions: Our study demonstrates that ctDNA provides a practical and cost-effective snapshot of driver gene status in metastatic bladder cancer. The identification of a wide spectrum of clinically informative somatic alterations nominates ctDNA as a tool to dissect disease pathogenesis and guide therapy selection in patients with metastatic bladder cancer. Clin Cancer Res; 23(21); 6487–97. ©2017 AACR.
Data from Frequent Homologous Recombination Deficiency in High-grade Endometrial Carcinomas
Authors: de Jonge Marthe M. , Auguste Aurélie , van Wijk Lise M. , Schouten Philip C. , Meijers Matty , ter Haar Natalja T. , Smit Vincent T.H.B.M. , Nout Remi A. , Glaire Mark A. , Church David N. , Vrieling Harry , Job Bastien , Boursin Yannick , de Kroon Cor D. , Rouleau Etienne , Leary Alexandra , Vreeswijk Maaike P.G. , Bosse Tjalling
Published: April 2023

AbstractPurpose:
The elevated levels of somatic copy-number alterations (SCNAs) in a subset of high-risk endometrial cancers are suggestive of defects in pathways governing genome integrity. We sought to assess the prevalence of homologous recombination deficiency (HRD) in endometrial cancers and its association with histopathologic and molecular characteristics.
Experimental Design:
Fresh tumor tissue was prospectively collected from 36 endometrial cancers, and functional HRD was examined by the ability of replicating tumor cells to accumulate RAD51 protein at DNA double-strand breaks (RAD51 foci) induced by ionizing radiation. Genomic alterations were determined by next-generation sequencing and array comparative genomic hybridization/SNP array. The prevalence of BRCA-associated genomic scars, a surrogate marker for HRD, was determined in the The Cancer Genome Atlas (TCGA) endometrial cancer cohort.
Results:
Most endometrial cancers included in the final analysis (n = 25) were of non-endometrioid (52%), grade 3 (60%) histology, and FIGO stage I (72%). HRD was observed in 24% (n = 6) of cases and was restricted to non-endometrioid endometrial cancers (NEEC), with 46% of NEECs being HRD compared with none of the endometrioid endometrial cancers (EEC, P = 0.014). All but 1 of the HRD cases harbored either a pathogenic BRCA1 variant or high somatic copy-number (SCN) losses of HR genes. Analysis of TCGA cases supported these results, with BRCA-associated genomic scars present in up to 48% (63/132) of NEEC versus 12% (37/312) of EEC (P < 0.001).
Conclusions:
HRD occurs in endometrial cancers and is largely restricted to non-endometrioid, TP53-mutant endometrial cancers. Evaluation of HRD may help select patients that could benefit from treatments targeting this defect, including platinum compounds and PARP inhibitors.
Data from A Prospective Evaluation of Circulating Tumor Cells and Cell-Free DNA in EGFR-Mutant Non–Small Cell Lung Cancer Patients Treated with Erlotinib on a Phase II Trial
Authors: Yanagita Masahiko , Redig Amanda J. , Paweletz Cloud P. , Dahlberg Suzanne E. , O'Connell Allison , Feeney Nora , Taibi Myriam , Boucher David , Oxnard Geoffrey R. , Johnson Bruce E. , Costa Daniel B. , Jackman David M. , Jänne Pasi A.
Published: April 2023

Abstract
Purpose: Genotype-directed therapy is the standard of care for advanced non–small cell lung cancer (NSCLC), but obtaining tumor tissue for genotyping remains a challenge. Circulating tumor cell (CTC) or cell-free DNA (cfDNA) analysis may allow for noninvasive evaluation. This prospective trial evaluated CTCs and cfDNA in EGFR-mutant NSCLC patients treated with erlotinib until progression.

Experimental Design: EGFR-mutant NSCLC patients were enrolled in a phase II trial of erlotinib. Blood was collected at baseline, every 2 months on study, and at disease progression. Plasma genotyping was performed by droplet digital PCR for EGFR19del, L858R, and T790M. CTCs were isolated by CellSave, enumerated, and analyzed by immunofluorescence for CD45 and pan-cytokeratin and EGFR and MET FISH were also performed. Rebiopsy was performed at disease progression.

Results: Sixty patients were enrolled; 44 patients discontinued therapy for disease progression. Rebiopsy occurred in 35 of 44 patients (80%), with paired CTC/cfDNA analysis in 41 of 44 samples at baseline and 36 of 44 samples at progression. T790M was identified in 23 of 35 (66%) tissue biopsies and 9 of 39 (23%) cfDNA samples. CTC analysis at progression identified MET amplification in 3 samples in which tissue analysis could not be performed. cfDNA analysis identified T790M in 2 samples in which rebiopsy was not possible. At diagnosis, high levels of cfDNA but not high levels of CTCs correlated with progression-free survival.

Conclusions: cfDNA and CTCs are complementary, noninvasive assays for evaluation of acquired resistance to first-line EGFR TKIs and may expand the number of patients in whom actionable genetic information can be obtained at acquired resistance. Serial cfDNA monitoring may offer greater clinical utility than serial monitoring of CTCs. Clin Cancer Res; 22(24); 6010–20. ©2016 AACR.
Data from MONARCH 1, A Phase II Study of Abemaciclib, a CDK4 and CDK6 Inhibitor, as a Single Agent, in Patients with Refractory HR+/HER2− Metastatic Breast Cancer
Authors: Dickler Maura N. , Tolaney Sara M. , Rugo Hope S. , Cortés Javier , Diéras Véronique , Patt Debra , Wildiers Hans , Hudis Clifford A. , O'Shaughnessy Joyce , Zamora Esther , Yardley Denise A. , Frenzel Martin , Koustenis Andrew , Baselga José
Published: April 2023

Abstract
Purpose: The phase II MONARCH 1 study was designed to evaluate the single-agent activity and adverse event (AE) profile of abemaciclib, a selective inhibitor of CDK4 and CDK6, in women with refractory hormone receptor–positive (HR+), HER2− metastatic breast cancer (MBC).

Experimental Design: MONARCH 1 was a phase II single-arm open-label study. Women with HR+/HER2− MBC who had progressed on or after prior endocrine therapy and had 1 or 2 chemotherapy regimens in the metastatic setting were eligible. Abemaciclib 200 mg was administered orally on a continuous schedule every 12 hours until disease progression or unacceptable toxicity. The primary objective of MONARCH 1 was investigator-assessed objective response rate (ORR). Other endpoints included clinical benefit rate, progression-free survival (PFS), and overall survival (OS).

Results: Patients (n = 132) had a median of 3 (range, 1–8) lines of prior systemic therapy in the metastatic setting, 90.2% had visceral disease, and 50.8% had ≥3 metastatic sites. At the 12-month final analysis, the primary objective of confirmed objective response rate was 19.7% (95% CI, 13.3–27.5; 15% not excluded); clinical benefit rate (CR+PR+SD≥6 months) was 42.4%, median progression-free survival was 6.0 months, and median overall survival was 17.7 months. The most common treatment-emergent AEs of any grade were diarrhea, fatigue, and nausea; discontinuations due to AEs were infrequent (7.6%).

Conclusions: In this poor-prognosis, heavily pretreated population with refractory HR+/HER2− metastatic breast cancer, continuous dosing of single-agent abemaciciclib was well tolerated and exhibited promising clinical activity. Clin Cancer Res; 23(17); 5218–24. ©2017 AACR.
Data from Clonotypic Diversity of the T-cell Receptor Corroborates the Immature Precursor Origin of Cutaneous T-cell Lymphoma
Authors: Hamrouni Abdelbasset , Fogh Hanne , Zak Zoulika , Ødum Niels , Gniadecki Robert
Published: April 2023

AbstractPurpose:
Mycosis fungoides is one of the most common types of extranodal T-cell lymphomas, considered to be caused by malignant transformation of the mature T cells residing in the skin. However, some clinical observations such as the multifocal distribution of mycosis fungoides lesions or patterns of relapse after radiotherapy are not readily explainable by the mature T-cell origin theory.
Experimental Design:
We have performed a detailed analysis of T-cell receptor (TCR) rearrangements in single malignant cells and in biopsies from mycosis fungoides tumors composed of >80% of malignant cells using next-generation sequencing (NGS) to pinpoint the relationship between neoplastic cells in mycosis fungoides. We have also aimed to detect malignant, circulating T-cell by whole blood TCR sequencing.
Results:
We found a substantial clonal heterogeneity in the mycosis fungoides samples with regards to TCR, and we demonstrated that lymphoma cells harboring identical TCRγ sequences may harbor different TCRα and β sequences. Lack of absolute TCRα, -β, -γ monoclonality was further confirmed by TCR amplification and sequencing from microdissected lymphoma cells. We have also found the TCR rearrangements characteristic for lymphoma cells in patients' peripheral blood despite the lack of leukemic blood involvement; however, the circulating TCRγ clonotype did not always represent the dominant cutaneous clonotype.
Conclusions:
These findings can be explained by a model where malignant transformation takes place during early T-cell development giving rise to circulating premalignant clones, which home to the skin producing clinically apparent lesions of cutaneous lymphoma. Therapeutic strategies in T-cell lymphoma should therefore target those early lymphoma precursor cells.
Data from Functional Silencing of HSD17B2 in Prostate Cancer Promotes Disease Progression
Authors: Gao Xiaomei , Dai Charles , Huang Shengsong , Tang Jingjie , Chen Guoyuan , Li Jianneng , Zhu Ziqi , Zhu Xuyou , Zhou Shuirong , Gao Yuanyuan , Hou Zemin , Fang Zijun , Xu Chengdang , Wang Jianyang , Wu Denglong , Sharifi Nima , Li Zhenfei
Published: April 2023

AbstractPurpose:
Steroidogenic enzymes are essential for prostate cancer development. Enzymes inactivating potent androgens were not investigated thoroughly, which leads to limited interference strategies for prostate cancer therapy. Here we characterized the clinical relevance, significance, and regulation mechanism of enzyme HSD17B2 in prostate cancer development.
Experimental Design:
HSD17B2 expression was detected with patient specimens and prostate cancer cell lines. Function of HSD17B2 in steroidogenesis, androgen receptor (AR) signaling, and tumor growth was investigated with prostate cancer cell lines and a xenograft model. DNA methylation and mRNA alternative splicing were investigated to unveil the mechanisms of HSD17B2 regulation.
Results:
HSD17B2 expression was reduced as prostate cancer progressed. 17βHSD2 decreased potent androgen production by converting testosterone (T) or dihydrotestosterone (DHT) to each of their upstream precursors. HSD17B2 overexpression suppressed androgen-induced cell proliferation and xenograft growth. Multiple mechanisms were involved in HSD17B2 functional silencing including DNA methylation and mRNA alternative splicing. DNA methylation decreased the HSD17B2 mRNA level. Two new catalytic-deficient isoforms, generated by alternative splicing, bound to wild-type 17βHSD2 and promoted its degradation. Splicing factors SRSF1 and SRSF5 participated in the generation of new isoforms.
Conclusions:
Our findings provide evidence of the clinical relevance, significance, and regulation of HSD17B2 in prostate cancer progression, which might provide new strategies for clinical management by targeting the functional silencing mechanisms of HSD17B2.

See related commentary by Mostaghel, p. 1139