Plasma vesicle miRNAs for therapy response monitoring in Hodgkin lymphoma patients

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Abstract

BACKGROUND. Cell-free circulating nucleic acids, including 22-nt microRNAs (miRNAs), represent noninvasive biomarkers for treatment response monitoring of cancer patients. While the majority of plasma miRNA is bound to proteins, a smaller, less well-characterized pool is associated with extracellular vesicles (EVs). Here, we addressed whether EV-associated miRNAs reflect metabolic disease in classical Hodgkin lymphoma (cHL) patients.

METHODS. With standardized size-exclusion chromatography (SEC), we isolated EV-associated extracellular RNA (exRNA) fractions and protein-bound miRNA from plasma of cHL patients and healthy subjects. We performed a comprehensive small RNA sequencing analysis and validation by TaqMan qRT-PCR for candidate discovery. Fluorodeoxyglucose-PET (FDG-PET) status before treatment, directly after treatment, and during long-term follow-up was compared directly with EV miRNA levels.

RESULTS. The plasma EV miRNA repertoire was more extensive compared with protein-bound miRNA that was heavily dominated by a few abundant miRNA species and was less informative of disease status. Purified EV fractions of untreated cHL patients and tumor EVs had enriched levels of miR24-3p, miR127-3p, miR21-5p, miR155-5p, and let7a-5p compared with EV fractions from healthy subjects and disease controls. Serial monitoring of EV miRNA levels in patients before treatment, directly after treatment, and during long-term follow-up revealed robust, stable decreases in miRNA levels matching a complete metabolic response, as observed with FDG-PET. Importantly, EV miRNA levels rose again in relapse patients.

CONCLUSION. We conclude that cHL-related miRNA levels in circulating EVs reflect the presence of vital tumor tissue and are suitable for therapy response and relapse monitoring in individual cHL patients.

FUNDING. Cancer Center Amsterdam Foundation (CCA-2013), Dutch Cancer Society (KWF-5510), Technology Foundation STW (STW Perspectief CANCER-ID).

Authors

Monique A.J. van Eijndhoven, Josée M. Zijlstra, Nils J. Groenewegen, Esther E.E. Drees, Stuart van Niele, S. Rubina Baglio, Danijela Koppers-Lalic, Hans van der Voorn, Sten F.W.M. Libregts, Marca H.M. Wauben, Renee X. de Menezes, Jan R.T. van Weering, Rienk Nieuwland, Lydia Visser, Anke van den Berg, Daphne de Jong, D. Michiel Pegtel

The airway epithelium undergoes metabolic reprogramming in individuals at high risk for lung cancer

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Abstract

The molecular determinants of lung cancer risk remain largely unknown. Airway epithelial cells are prone to assault by risk factors and are considered to be the primary cell type involved in the field of cancerization. To investigate risk-associated changes in the bronchial epithelium proteome that may offer new insights into the molecular pathogenesis of lung cancer, proteins were identified in the airway epithelial cells of bronchial brushing specimens from risk-stratified individuals by shotgun proteomics. Differential expression of selected proteins was validated by parallel reaction monitoring mass spectrometry in an independent set of individual bronchial brushings. We identified 2,869 proteins, of which 312 proteins demonstrated a trend in expression. Pathway analysis revealed enrichment of carbohydrate metabolic enzymes in high-risk individuals. Glucose consumption and lactate production were increased in human bronchial epithelial BEAS2B cells treated with cigarette smoke condensate for 7 months. Increased lipid biosynthetic capacity and net reductive carboxylation were revealed by metabolic flux analyses of [U-¹³C₅] glutamine in this in vitro model, suggesting profound metabolic reprogramming in the airway epithelium of high-risk individuals. These results provide a rationale for the development of potentially new chemopreventive strategies and selection of patients for surveillance programs.

Authors

S.M. Jamshedur Rahman, Xiangming Ji, Lisa J. Zimmerman, Ming Li, Bradford K. Harris, Megan D. Hoeksema, Irina A. Trenary, Yong Zou, Jun Qian, Robbert J.C. Slebos, Jennifer Beane, Avrum Spira, Yu Shyr, Rosana Eisenberg, Daniel C. Liebler, Jamey D. Young, Pierre P. Massion

Proapoptotic protein Bim attenuates estrogen-enhanced survival in lymphangioleiomyomatosis

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Abstract

Lymphangioleiomyomatosis (LAM) is a progressive lung disease that primarily affects young women. Genetic evidence suggests that LAM cells bearing TSC2 mutations migrate to the lungs, proliferate, and cause cystic remodeling. The female predominance indicates that estrogen plays a critical role in LAM pathogenesis, and we have proposed that estrogen promotes LAM cell metastasis by inhibition of anoikis. We report here that estrogen increased LAM patient–derived cells’ resistance to anoikis in vitro, accompanied by decreased accumulation of the proapoptotic protein Bim, an activator of anoikis. The resistance to anoikis was reversed by the proteasome inhibitor, bortezomib. Treatment of LAM patient–derived cells with estrogen plus bortezomib promoted anoikis compared with estrogen alone. Depletion of Bim by siRNA in TSC2-deficient cells resulted in anoikis resistance. Treatment of mice with bortezomib reduced estrogen-promoted lung colonization of TSC2-deficient cells. Importantly, molecular depletion of Bim by siRNA in Tsc2-deficient cells increased lung colonization in a mouse model. Collectively, these data indicate that Bim plays a key role in estrogen-enhanced survival of LAM patient–derived cells under detached conditions that occur with dissemination. Thus, targeting Bim may be a plausible future treatment strategy in patients with LAM.

Authors

Chenggang Li, Na Li, Xiaolei Liu, Erik Y. Zhang, Yang Sun, Kouhei Masuda, Jing Li, Julia Sun, Tasha Morrison, Xiangke Li, Yuanguang Chen, Jiang Wang, Nagla A. Karim, Yi Zhang, John Blenis, Mauricio J. Reginato, Elizabeth P. Henske, Jane J. Yu

Institutional implementation of clinical tumor profiling on an unselected cancer population

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Abstract

BACKGROUND. Comprehensive genomic profiling of a patient’s cancer can be used to diagnose, monitor, and recommend treatment. Clinical implementation of tumor profiling in an enterprise-wide, unselected cancer patient population has yet to be reported.

METHODS. We deployed a hybrid-capture and massively parallel sequencing assay (OncoPanel) for all adult and pediatric patients at our combined cancer centers. Results were categorized by pathologists based on actionability. We report the results for the first 3,727 patients tested.

RESULTS. Our cohort consists of cancer patients unrestricted by disease site or stage. Across all consented patients, half had sufficient and available (>20% tumor) material for profiling; once specimens were received in the laboratory for pathology review, 73% were scored as adequate for genomic testing. When sufficient DNA was obtained, OncoPanel yielded a result in 96% of cases. 73% of patients harbored an actionable or informative alteration; only 19% of these represented a current standard of care for therapeutic stratification. The findings recapitulate those of previous studies of common cancers but also identify alterations, including in AXL and EGFR, associated with response to targeted therapies. In rare cancers, potentially actionable alterations suggest the utility of a “cancer-agnostic” approach in genomic profiling. Retrospective analyses uncovered contextual genomic features that may inform therapeutic response and examples where diagnoses revised by genomic profiling markedly changed clinical management.

CONCLUSIONS. Broad sequencing-based testing deployed across an unselected cancer cohort is feasible. Genomic results may alter management in diverse scenarios; however, additional barriers must be overcome to enable precision cancer medicine on a large scale.

FUNDING. This work was supported by DFCI, BWH, and the National Cancer Institute (5R33CA155554 and 5K23CA157631).

Authors

Lynette M. Sholl, Khanh Do, Priyanka Shivdasani, Ethan Cerami, Adrian M. Dubuc, Frank C. Kuo, Elizabeth P. Garcia, Yonghui Jia, Phani Davineni, Ryan P. Abo, Trevor J. Pugh, Paul van Hummelen, Aaron R. Thorner, Matthew Ducar, Alice H. Berger, Mizuki Nishino, Katherine A. Janeway, Alanna Church, Marian Harris, Lauren L. Ritterhouse, Joshua D. Campbell, Vanesa Rojas-Rudilla, Azra H. Ligon, Shakti Ramkissoon, James M. Cleary, Ursula Matulonis, Geoffrey R. Oxnard, Richard Chao, Vanessa Tassell, James Christensen, William C. Hahn, Philip W. Kantoff, David J. Kwiatkowski, Bruce E. Johnson, Matthew Meyerson, Levi A. Garraway, Geoffrey I. Shapiro, Barrett J. Rollins, Neal I. Lindeman, Laura E. MacConaill

Akt and SHP-1 are DC-intrinsic checkpoints for tumor immunity

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Abstract

BM-derived DC (BMDC) are powerful antigen-presenting cells. When loaded with immune complexes (IC), consisting of tumor antigens bound to antitumor antibody, BMDC induce powerful antitumor immunity in mice. However, attempts to employ this strategy clinically with either tumor-associated DC (TADC) or monocyte-derived DC (MoDC) have been disappointing. To investigate the basis for this phenomenon, we compared the response of BMDC, TADC, and MoDC to tumor IgG-IC. Our findings revealed, in both mice and humans, that upon exposure to IgG-IC, BMDC internalized the IC, increased costimulatory molecule expression, and stimulated autologous T cells. In contrast, TADC and, surprisingly, MoDC remained inert upon contact with IC due to dysfunctional signaling following engagement of Fcγ receptors. Such dysfunction is associated with elevated levels of the Src homology region 2 domain–containing phosphatase-1 (SHP-1) and phosphatases regulating Akt activation. Indeed, concomitant inhibition of both SHP-1 and phosphatases that regulate Akt activation conferred upon TADC and MoDC the capacity to take up and process IC and induce antitumor immunity in vivo. This work identifies the molecular checkpoints that govern activation of MoDC and TADC and their capacity to elicit T cell immunity.

Authors

Yaron Carmi, Tyler R. Prestwood, Matthew H. Spitzer, Ian L. Linde, Jonathan Chabon, Nathan E. Reticker-Flynn, Nupur Bhattacharya, Hong Zhang, Xiangyue Zhang, Pamela A. Basto, Bryan M. Burt, Michael N. Alonso, Edgar G. Engleman

Acquired platinum resistance involves epithelial to mesenchymal transition through ubiquitin ligase FBXO32 dysregulation

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Abstract

To identify the molecules involved in epithelial to mesenchymal transition (EMT) in urothelial carcinoma (UC) after acquisition of platinum resistance, here we examined the changes in global gene expression before and after platinum treatment. Four invasive UC cell lines, T24, 5637, and their corresponding sublines T24PR and 5637PR with acquired platinum resistance, were assessed by microarray, and the ubiquitin E3 ligase FBXO32 was newly identified as a negative regulator of EMT in UC tumors after acquisition of platinum resistance. In vitro and in vivo studies showed an intimate relationship between FBXO32 expression and EMT, demonstrating that FBXO32 dysregulation in T24PR cells results in elevated expression of the mesenchymal molecules SNAIL and vimentin and decreased expression of the epithelial molecule E-cadherin. The association between FBXO32 expression and EMT was further validated using clinical samples. Knockdown of MyoD expression, a specific target of FBXO32 polyubiquitination, revealed upregulation of E-cadherin expression and downregulation of SNAIL and vimentin expression in T24PR cells. Comparative genomic hybridization array analysis demonstrated loss of heterozygosity at 8q24.13 in T24PR cells, which harbors FBXO32. Our findings suggest the importance of the association between EMT and ubiquitin-proteasome regulation when tumors develop acquired platinum resistance.

Authors

Nobuyuki Tanaka, Takeo Kosaka, Yasumasa Miyazaki, Shuji Mikami, Naoya Niwa, Yutaro Otsuka, Yoji Andrew Minamishima, Ryuichi Mizuno, Eiji Kikuchi, Akira Miyajima, Hisataka Sabe, Yasunori Okada, Per Uhlén, Makoto Suematsu, Mototsugu Oya

Improving vascular maturation using noncoding RNAs increases antitumor effect of chemotherapy

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Abstract

Current antiangiogenesis therapy relies on inhibiting newly developed immature tumor blood vessels and starving tumor cells. This strategy has shown transient and modest efficacy. Here, we report a better approach to target cancer-associated endothelial cells (ECs), reverse permeability and leakiness of tumor blood vessels, and improve delivery of chemotherapeutic agents to the tumor. First, we identified deregulated microRNAs (miRs) from patient-derived cancer-associated ECs. Silencing these miRs led to decreased vascular permeability and increased maturation of blood vessels. Next, we screened a thioaptamer (TA) library to identify TAs selective for tumor-associated ECs. An annexin A2–targeted TA was identified and used for delivery of miR106b-5p and miR30c-5p inhibitors, resulting in vascular maturation and antitumor effects without inducing hypoxia. These findings could have implications for improving vascular-targeted therapy.

Authors

Lingegowda S. Mangala, Hongyu Wang, Dahai Jiang, Sherry Y. Wu, Anoma Somasunderam, David E. Volk, Ganesh L. R. Lokesh, Xin Li, Sunila Pradeep, Xianbin Yang, Monika Haemmerle, Cristian Rodriguez-Aguayo, Archana S Nagaraja, Rajesha Rupaimoole, Emine Bayraktar, Recep Bayraktar, Li Li, Takemi Tanaka, Wei Hu, Cristina Ivan, Kshipra M Gharpure, Michael H. McGuire, Varatharasa Thiviyanathan, Xinna Zhang, Sourindra N. Maiti, Nataliya Bulayeva, Hyun-Jin Choi, Piotr L. Dorniak, Laurence J.N. Cooper, Kevin P. Rosenblatt, Gabriel Lopez-Berestein, David G. Gorenstein, Anil K. Sood

The head and neck cancer immune landscape and its immunotherapeutic implications

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Abstract

Recent clinical trials have demonstrated a clear survival advantage in advanced head and neck squamous cell carcinoma (HNSCC) patients treated with immune checkpoint blockade. These emerging results reveal that HNSCC is one of the most promising frontiers for immunotherapy research. However, further progress in head and neck immuno-oncology will require a detailed understanding of the immune infiltrative landscape found in these tumors. We leveraged transcriptome data from 280 tumors profiled by The Cancer Genome Atlas (TCGA) to comprehensively characterize the immune landscape of HNSCC in order to develop a rationale for immunotherapeutic strategies in HNSCC and guide clinical investigation. We find that both HPV⁺ and HPV^– HNSCC tumors are among the most highly immune-infiltrated cancer types. Strikingly, HNSCC had the highest median Treg/CD8⁺ T cell ratio and the highest levels of CD56^dim NK cell infiltration, in our pan-cancer analysis of the most immune-infiltrated tumors. CD8⁺ T cell infiltration and CD56^dim NK cell infiltration each correlated with superior survival in HNSCC. Tumors harboring genetic smoking signatures had lower immune infiltration and were associated with poorer survival, suggesting these patients may benefit from immune agonist therapy. These findings illuminate the immune landscape of HPV⁺ and HPV^– HNSCC. Additionally, this landscape provides a potentially novel rationale for investigation of agents targeting modulators of Tregs (e.g., CTLA-4, GITR, ICOS, IDO, and VEGFA) and NK cells (e.g., KIR, TIGIT, and 4-1BB) as adjuncts to anti–PD-1 in the treatment of advanced HNSCC.

Authors

Rajarsi Mandal, Yasin Şenbabaoğlu, Alexis Desrichard, Jonathan J. Havel, Martin G. Dalin, Nadeem Riaz, Ken-Wing Lee, Ian Ganly, A. Ari Hakimi, Timothy A. Chan, Luc G.T. Morris

Subtyping sub-Saharan esophageal squamous cell carcinoma by comprehensive molecular analysis

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Abstract

Esophageal squamous cell carcinoma (ESCC) is endemic in regions of sub-Saharan Africa (SSA), where it is the third most common cancer. Here, we describe whole-exome tumor/normal sequencing and RNA transcriptomic analysis of 59 patients with ESCC in Malawi. We observed similar genetic aberrations as reported in Asian and North American cohorts, including mutations of TP53, CDKN2A, NFE2L2, CHEK2, NOTCH1, FAT1, and FBXW7. Analyses for nonhuman sequences did not reveal evidence for infection with HPV or other occult pathogens. Mutational signature analysis revealed common signatures associated with aging, cytidine deaminase activity (APOBEC), and a third signature of unknown origin, but signatures of inhaled tobacco use, aflatoxin and mismatch repair were notably absent. Based on RNA expression analysis, ESCC could be divided into 3 distinct subtypes, which were distinguished by their expression of cell cycle and neural transcripts. This study demonstrates discrete subtypes of ESCC in SSA, and suggests that the endemic nature of this disease reflects exposure to a carcinogen other than tobacco and oncogenic viruses.

Authors

Wenjin Liu, Jeff M. Snell, William R. Jeck, Katherine A. Hoadley, Matthew D. Wilkerson, Joel S. Parker, Nirali Patel, Yohannie B. Mlombe, Gift Mulima, N. George Liomba, Lindsey L. Wolf, Carol G. Shores, Satish Gopal, Norman E. Sharpless

Hepatoblastoma modeling in mice places Nrf2 within a cancer field established by mutant β-catenin

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Abstract

Aberrant wnt/β-catenin signaling and amplification/overexpression of Myc are associated with hepatoblastoma (HB), the most prevalent type of childhood liver cancer. To address their roles in the pathogenesis of HB, we generated mice in which Myc and mutant β-catenin were targeted to immature cells of the developing mouse liver. Perinatal coexpression of both genes promoted the preferential development of HBs over other tumor types in neonatal mice, all of which bore striking resemblance to their human counterparts. Integrated analysis indicated that tumors emerged as a consequence of Myc-driven alterations in hepatoblast fate in a background of pan-hepatic injury, inflammation, and nuclear factor (erythroid-derived 2)-like 2/Nrf2-dependent antioxidant signaling, which was specifically associated with expression of mutant β-catenin but not Myc. Immunoprofiling of human HBs confirmed that approximately 50% of tumors demonstrated aberrant activation of either Myc or Nfe2l2/Nrf2, while knockdown of Nrf2 in a cell line–derived from a human HB with NFE2L2 gene amplification reduced tumor cell growth and viability. Taken together, these data indicate that β-catenin creates a protumorigenic hepatic environment in part by indirectly activating Nrf2 and implicate oxidative stress as a possible driving force for a subset of β-catenin–driven liver tumors in children.

Authors

Sarah A. Comerford, Elizabeth A. Hinnant, Yidong Chen, Hima Bansal, Shawn Klapproth, Dinesh Rakheja, Milton J. Finegold, Dolores Lopez-Terrada, Kathryn A. O’Donnell, Gail E. Tomlinson, Robert E. Hammer