Rapid Development of a Registry to Accelerate COVID-19 Vaccine Clinical Trials.

Background: The unprecedented scientific response to the SARS-Cov-2 pandemic in 2020 required the rapid development and activation of extensive clinical trial networks to study vaccines and therapeutics. The COVID-19 Prevention Network (CoVPN) coordinated hundreds of sites conducting phase 2 and 3 clinical trials of vaccines and antibody therapeutics. To facilitate these clinical trials, the CoVPN Volunteer Screening Registry (VSR) was created to collect volunteer information at scale, identify volunteers at risk of COVID-19 who met enrollment criteria, distribute candidates across clinical trial sites, and enable monitoring of volunteering and enrollment progress. Methods: We developed a secure database to support three primary web-based interfaces: a national volunteer questionnaire intake form, a clinical trial site portal, and an Administrative Portal. The Site Portal supported filters based on volunteer attributes, visual analytics, enrollment status tracking, geographic search, and clinical risk prediction. The Administrative Portal supported oversight and development with pre-specified reports aggregated by geography, trial, and trial site; charts of volunteer rates over time; volunteer risk score calculation; and dynamic, user-defined reports. Findings: Over 650,000 volunteers joined the VSR, and 1094 users were trained to utilize the system. The VSR played a key role in recruitment for the Moderna, Oxford-AstraZeneca, Janssen, and Novavax vaccine clinical trials, provided support to the Pfizer and Sanofi vaccine and prophylactic antibody clinical trials, and enhanced the diversity of trial participants. Clinical trial sites selected 166,729 volunteer records for follow-up screening, and of these 47·7% represented groups prioritized for increased enrollment. Despite the unprecedented urgency of its development, the system maintained 99·99% uptime. Interpretation: The success of the VSR demonstrates that information tools can be rapidly yet safely developed through a public-private partnership and integrated into a distributed and accelerated clinical trial setting. We further summarize the requirements, design, and development of the system, and discuss lessons learned for future pandemic preparedness.

A Pilot Study of the Telomerase Inhibitor Imetelstat for Myelofibrosis.

BACKGROUND: Current drugs for myeloproliferative neoplasm-associated myelofibrosis, including Janus kinase (JAK) inhibitors, do not induce complete or partial remissions. Imetelstat is a 13-mer lipid-conjugated oligonucleotide that targets the RNA template of human telomerase reverse transcriptase. METHODS: We sought to obtain preliminary information on the therapeutic activity and safety of imetelstat in patients with high-risk or intermediate-2-risk myelofibrosis. Imetelstat was administered as a 2-hour intravenous infusion (starting dose, 9.4 mg per kilogram of body weight) every 1 to 3 weeks. The primary end point was the overall response rate, and the secondary end points were adverse events, spleen response, and independence from red-cell transfusions. RESULTS: A total of 33 patients (median age, 67 years) met the eligibility criteria; 48% had received prior JAK inhibitor therapy. A complete or partial remission occurred in 7 patients (21%), with a median duration of response of 18 months (range, 13 to 20+) for complete responses and 10 months (range, 7 to 10+) for partial responses. Bone marrow fibrosis was reversed in all 4 patients who had a complete response, and a molecular response occurred in 3 of the 4 patients. Response rates were 27% among patients with a JAK2 mutation versus 0% among those without a JAK2 mutation (P=0.30) and 32% among patients without an ASXL1 mutation versus 0% among those with an ASXL1 mutation (P=0.07). The rate of complete response was 38% among patients with a mutation in SF3B1 or U2AF1 versus 4% among patients without a mutation in these genes (P=0.04). Responses did not correlate with baseline telomere length. Treatment-related adverse events included grade 4 thrombocytopenia (in 18% of patients), grade 4 neutropenia (in 12%), grade 3 anemia (in 30%), and grade 1 or 2 elevation in levels of total bilirubin (in 12%), alkaline phosphatase (in 21%), and aspartate aminotransferase (in 27%). CONCLUSIONS: Imetelstat was found to be active in patients with myelofibrosis but also had the potential to cause clinically significant myelosuppression. (Funded by Geron; ClinicalTrials.gov number, NCT01731951.).

Intratumoral CD14+ Cells and Circulating CD14+HLA-DRlo/neg Monocytes Correlate with Decreased Survival in Patients with Clear Cell Renal Cell Carcinoma.

PURPOSE: Immunotherapeutic strategies to treat patients with renal cell carcinoma (RCC) offer new opportunities for disease management. Further improvements to immunotherapy will require additional understanding of the host response to RCC development. EXPERIMENTAL DESIGN: Using a novel approach to understanding the immune status of cancer patients, we previously showed that patients with a certain immune profile had decreased overall survival. Here, we examine in more detail the phenotypic changes in peripheral blood and the potential consequences of these changes in RCC patients. RESULTS: We found that CD14(+)HLA-DR(lo/neg) monocytes were the most predominant phenotypic change in peripheral blood of RCC patients, elevated nearly 5-fold above the average levels measured in healthy volunteers. Intratumoral and peritumoral presence of CD14 cells was an independent prognostic factor for decreased survival in a cohort of 375 RCC patients. The amount of peripheral blood CD14(+)HLA-DR(lo/neg) monocytes was found to correlate with the intensity of CD14 staining in tumors, suggesting that the measurement of these cells in blood may be a suitable surrogate for monitoring patient prognosis. The interaction of monocytes and tumor cells triggers changes in both cell types with a loss of HLA-DR expression in monocytes, increases of monocyte survival factors such as GM-CSF in tumors, and increased production of angiogenic factors, including FGF2. CONCLUSIONS: Our results suggest a model of mutually beneficial interactions between tumor cells and monocytes that adversely affect patient outcome.

Cancer Vaccines in the World of Immune Suppressive Monocytes (CD14(+)HLA-DR(lo/neg) Cells): The Gateway to Improved Responses.

Dendritic cells are an important target in cancer immunotherapy based on their critical role in antigen presentation and response to tumor development. The capacity of dendritic cells to stimulate anti-tumor immunity has led investigators to use these cells to mediate anti-tumor responses in a number of clinical trials. However, these trials have had mixed results. The typical method for generation of ex vivo dendritic cells starts with the purification of CD14(+) cells. Our studies identified a deficiency in the ability to generate mature dendritic cell using CD14(+) cells from cancer patients that corresponded with an increased population of monocytes with altered surface marker expression (CD14(+)HLA-DR(lo/neg)). Further studies identified systemic immune suppression and increased concentrations of CD14(+)HLA-DR(lo/neg) monocytes capable of inhibiting T-cell proliferation and DC maturation. Together, these findings strongly suggest that protocols aimed at immune stimulation via monocytes/dendritic cells, if optimized on normal monocytes or in systems without these suppressive monocytes, are unlikely to engender effective DC maturation in vitro or efficiently trigger DC maturation in vivo. This highlights the importance of developing optimal protocols for stimulating DCs in the context of significantly altered monocyte phenotypes often seen in cancer patients.

Transoral surgery alone for human-papillomavirus-associated oropharyngeal squamous cell carcinoma.

The aim of this retrospective study was to describe the oncologic and functional results of treating oropharyngeal squamous cell carcinoma with transoral robotic surgery and neck dissection as monotherapy. A review was performed, including all patients who underwent transoral robotic surgery and neck dissection as the only means of therapy for oropharyngeal carcinoma from March 2007 to July 2009 at a single tertiary care academic medical center. We reviewed all cases with ≥24-month follow-up. Functional outcomes included tracheostomy dependence and oral feeding ability. Oncologic outcomes were stratified by human papillomavirus (HPV) status and tobacco use and included local, regional, and distant disease control, as well as disease-specific and recurrence-free survival. Eighteen patients met study criteria. Ten patients (55.6%) were able to eat orally in the immediate postoperative period, and 8 (44.4%) required a temporary nasogastric tube for a mean duration of 13.6 days (range 3 to 24 days) before returning to an oral diet. No patient required placement of a gastrostomy tube, and all patients are tracheostomy-tube-free. Among the HPV-positive nonsmokers (12/18, 66.7%), Kaplan-Meier estimated 3-year local, regional, and distant control rates were 90.9%, 100%, and 100%, respectively. Kaplan-Meier estimated disease-specific survival and recurrence-free survival were 100% and 90.9%, respectively. No complications occurred.This study suggests that carefully selected patients with HPV-positive oropharyngeal carcinoma can be effectively treated with surgery alone with excellent functional and oncologic outcomes.

Search for chromosome rearrangements: new approaches toward discovery of novel translocations in head and neck squamous cell carcinoma.

BACKGROUND: Chromosome rearrangements that result in gene fusions have important roles in the initial steps of tumorigenesis, especially in leukemias and lymphomas, but the biological and clinical impact of gene fusions in common solid tumors are less understood. The purpose of this study was to discover novel translocations that could result in gene fusions in oropharyngeal squamous cell carcinomas (OPSCCs). METHODS: Translocations were identified using 2 different bioinformatics pipelines, SnowShoes-FTD and FusionHunter, examining data from 11 paired-end RNA sequencing (RNA-Seq) data in OPSCC. Translocations were validated by RT-PCR and Sanger sequencing analysis. RESULTS: Two novel cancer-specific translocations involving MGST3-ZMAT5 and MS4A7-C2CD3 were found in 2 of the tumor samples tested. However, these translocations were found only in the single tumor. CONCLUSIONS: We hope that this integrative methodology will elucidate key aspects of tumor biology as well as generate novel targets for cancer diagnoses and therapies.

Human papillomavirus in oropharyngeal squamous cell carcinoma: assessing virus presence in normal tissue and activity in cervical metastasis.

OBJECTIVES/HYPOTHESIS: Human papillomavirus (HPV) has been established as an etiologic and prognostic factor in oropharyngeal squamous cell carcinoma (OPSCC). HPV oncogenesis involves expression of E6/E7 oncoproteins, with downstream p53 degradation and pRb inhibition. Although much research has focused on HPV's oncogenic behavior in primary OPSCC, minimal information exists about HPV in adjacent normal and metastatic tissue. STUDY DESIGN: Retrospective cohort study METHODS: Patient-matched tumor, normal, and metastatic tissue was gathered from 42 OPSCC patients and tested with real-time quantitative polymerase chain reaction (RT-qPCR), in situ hybridization (ISH), and immunohistochemistry (IHC). RT-qPCR was performed using total RNA from fresh-frozen tissues and primers for HPV16 E6, E7, and p16 transcripts. HPV ISH was performed to detect the presence of HPV DNA and IHC to detect p16 protein. RESULTS: Primary tumor, adjacent normal tissue, and tumor metastasis from 17 OPSCC patients were analyzed. When comparing the presence of HPV16 DNA in tumor, metastatic, and normal tissue by ISH, perfect correlation is found at all subsites (P < .0001). However, active infections determined by HPV16 E6 and E7 expression using quantitative polymerase chain reaction or p16 detection by IHC, were present only in primary and metastatic tissue (P = .0012, E6; P = .02, E7). No such correlation was found in normal tissue when compared to primary or metastatic tissue. CONCLUSIONS: There is a clear pattern of active HPV expression that correlates to disease course. In HPV-positive patients, all sites including primary, metastatic, and normal tissues are DNA positive. Transcriptionally active infections were detected in primary and metastatic tissues, whereas normal tissues appear to have latent infections.

Long-term functional and oncologic results of transoral robotic surgery for oropharyngeal squamous cell carcinoma.

OBJECTIVE: To examine the long-term functional and oncologic results in patients who underwent transoral robotic surgery (TORS) as primary therapy or as part of combined therapy for oropharyngeal squamous cell carcinoma arising in the tonsil or base of tongue. PATIENTS AND METHODS: We reviewed a prospective TORS database of patients with squamous cell carcinoma arising in the tonsil or base of tongue treated between March 2007 and February 2009 to determine oncologic outcomes at 24 months or more of follow-up. The presenting tumor stage, histopathologic factors, surgical margins, and adjuvant treatment extent were evaluated. Functional outcomes included gastrostomy tube dependence and tracheostomy dependence. Oncologic outcomes included local, regional, and distant control and disease-specific and recurrence-free survival. RESULTS: A total of 66 TORS patients were followed up for a minimum of 2 years. Most (97.0%; 64 of 66) were able to eat orally within 3 weeks after surgery before starting adjuvant therapy. Long-term gastrostomy tube use was required in 3 of the 66 (4.5%) and long-term tracheotomy in 1 (1.5%). Three-year estimated local control and regional control were 97.0% and 94.0%, respectively. Two-year disease-specific survival and recurrence-free survival were 95.1% and 92.4%, respectively. CONCLUSION: With appropriate adjuvant therapy, TORS achieves excellent functional results for patients with oropharyngeal squamous cell carcinoma. Oncologic outcomes are equivalent or superior to results of other surgical and nonsurgical treatments.

Transcriptional profiling by sequencing of oropharyngeal cancer.

OBJECTIVE: To compare full transcriptome expression levels of matched tumor and normal samples from patients with oropharyngeal carcinoma stratified by known tumor etiologic factors. PATIENTS AND METHODS: Full transcriptome sequencing was analyzed for 10 matched tumor and normal tissue samples from patients with previously untreated oropharyngeal carcinoma. Transcriptomes were analyzed using massively parallel messenger RNA sequencing and validated using the NanoString nCounter system. Global gene expression levels were compared in samples grouped by smoking status and human papillomavirus status. This study was completed between June 10, 2010, and June 30, 2011. RESULTS: Global gene expression analysis indicated tumor tissue from former smokers grouped more closely to the never smokers than the current smokers. Pathway analysis revealed alterations in the expression of genes involved in the p53 DNA damage-repair pathway, including CHEK2 and ATR, which display patterns of increased expression that is associated with human papillomavirus-negative current smokers rather than former or never smokers. CONCLUSION: These findings support the application of messenger RNA sequencing technology as an important clinical tool for more accurately stratifying patients based on individual tumor biology with the goal of improving our understanding of tumor prognosis and treatment response, ultimately leading to individualized patient care strategies.

Cost considerations in the treatment of oropharyngeal squamous cell carcinoma.

OBJECTIVES: To determine the cost of treatment of oropharyngeal squamous cell carcinoma (OP SCCA) with transoral surgery with concomitant neck dissection (TOS), TOS with adjuvant radiation therapy (TOS + RT), TOS with adjuvant chemoradiation therapy (TOS + CRT), and primary chemoradiation therapy (CRT). STUDY DESIGN: Case series. SETTING: Two tertiary care teaching hospitals. SUBJECTS AND METHODS: Using the databases of 2 teaching hospitals, patients were identified who had OP SCCA treated with TOS, TOS + RT, TOS + CRT, and primary CRT in 2009 to 2010. Costs were analyzed from an institutional perspective looking at reimbursement. Patients with government payers and patients with private payers in each group were identified, and net revenue data obtained for the 3-month period from diagnosis were calculated and averaged for each group. Cost was defined as the reimbursement for all charges surrounding the 3-month episode of treatment. All revenue associated with inpatient and outpatient care, including pharmacy charges, was included. RESULTS: The mean cost of TOS (private payers/government payers) was $37,435/$15,664 (range, $22,486-$48,746/$13,325-$16,885). The mean cost of TOS + RT (private payers/government payers) was $74,484/$34,343 (range, $72,400-$84,825/$31,565-$40,810). The mean cost of TOS + CRT (private payers/government payers) was $191,780/$53,245 (range, $145,450-$217,220/$49,400-$58,325). The mean cost of CRT (private payers/government payers) was $198,285/$57,429 (range, $168,216-$298,945/$52,900-$59,545). CONCLUSION: An algorithm of transoral surgical treatment of OP SCCA with adjuvant treatment as indicated by pathology is more cost conscious than an algorithm of treatment of all OP SCCA with CRT.

Linking expression of FOXM1, CEP55 and HELLS to tumorigenesis in oropharyngeal squamous cell carcinoma.

OBJECTIVES/HYPOTHESIS: To investigate the relationship between the expression of FOXM1, CEP55, and HELLS in oropharyngeal squamous cell carcinoma to human papillomavirus (HPV), smoking, and tumor stage. STUDY DESIGN: Retrospective cohort study. METHODS: Transcriptome data were analyzed from matched tumor-normal samples taken from patients with oropharyngeal squamous cell carcinoma. Data were previously generated using deep-sequencing techniques (mRNA-Seq). Transcript levels of all three genes were validated using the NanoString nCounter system in a larger group of patients. Analyses were conducted to assess possible associations between expression levels and HPV infection status, smoking status, or tumor staging. RESULTS: FOXM1, CEP55, and HELLS were all overexpressed in oropharyngeal squamous cell carcinoma tissue when compared to normal tissue. Significant trends were found between expression levels of FOXM1, CEP55, and HELLS and tumor staging. Tumors staged 3 or greater showed significantly higher levels of expression compared with those staged 1. No significant association or trend was found between expression of any genes of interest and etiologic subgroupings (i.e., HPV, smoking). CONCLUSIONS: FOXM1, CEP55, and HELLS were all overexpressed in oropharyngeal squamous cell carcinoma. Gene expression is related to tumor stage. The significant association between the expression of these genes and advanced tumor stage suggest that they may play a role in tumorigenesis.

Tumor transcriptome sequencing reveals allelic expression imbalances associated with copy number alterations.

Due to growing throughput and shrinking cost, massively parallel sequencing is rapidly becoming an attractive alternative to microarrays for the genome-wide study of gene expression and copy number alterations in primary tumors. The sequencing of transcripts (RNA-Seq) should offer several advantages over microarray-based methods, including the ability to detect somatic mutations and accurately measure allele-specific expression. To investigate these advantages we have applied a novel, strand-specific RNA-Seq method to tumors and matched normal tissue from three patients with oral squamous cell carcinomas. Additionally, to better understand the genomic determinants of the gene expression changes observed, we have sequenced the tumor and normal genomes of one of these patients. We demonstrate here that our RNA-Seq method accurately measures allelic imbalance and that measurement on the genome-wide scale yields novel insights into cancer etiology. As expected, the set of genes differentially expressed in the tumors is enriched for cell adhesion and differentiation functions, but, unexpectedly, the set of allelically imbalanced genes is also enriched for these same cancer-related functions. By comparing the transcriptomic perturbations observed in one patient to his underlying normal and tumor genomes, we find that allelic imbalance in the tumor is associated with copy number mutations and that copy number mutations are, in turn, strongly associated with changes in transcript abundance. These results support a model in which allele-specific deletions and duplications drive allele-specific changes in gene expression in the developing tumor.

Expression profiles of viral responsive genes in oral and oropharyngeal cancers.

The association between Human Papillomavirus (HPV) DNA and oropharyngeal squamous cell carcinoma (SCC) has been supported by numerous studies strongly implicating HPV infection as a factor in the development of this cancer. In contrast, squamous cell carcinoma of the oral cavity has not been associated with HPV DNA, suggesting alternate aetiologic factors. The possibility that viral agents other than HPV could contribute to the development of oral cavity SCC should be given consideration, especially given the association of Epstein-Barr virus (EBV) with nasopharyngeal cancer. We used quantitative polymerase chain reaction (qPCR) studies to compare the expression levels of genes that may act as indicators of persistent stimulation by viral antigen in both oral cavity and oropharyngeal squamous cell cancers. Our results demonstrate that HPV-positive oropharyngeal tumours displayed gene expression patterns indicative of a viral signature and that HPV-negative oropharyngeal tumours do not display similar expression patterns. In contrast, we saw no evidence of either a viral or bacterial signature in the oral tumours examined. This would suggest that either an as of yet unidentified virus present in the oral tumour samples is not eliciting a typical immune response, or that there are no novel viral sequences or viruses present in the oral tumours examined.

Reduced hamster usage and stress in propagating Leishmania chagasi promastigotes using cryopreservation and saphenous vein inoculation.

Leishmania chagasi, a causal agent of visceral leishmaniasis, requires passage through lab animals such as hamsters to maintain its virulence. Hamster infection is typically accomplished via cardiac puncture or intraperitoneal injection, procedures accompanied by risks of increased animal stress and death. The use of the hamster model also necessitates a regular supply of infected animals, because L. chagasi parasites newly isolated from an infected hamster can be grown in culture for only several weeks before loss of function/phenotype occurs. In an effort to decrease animal usage and animal stress, experiments were performed to assess a more gentle inoculation procedure (saphenous vein inoculation) and the use of cryopreserved parasite cells for research experiments. Of 81 hamsters inoculated by the saphenous vein, 80 became infected as determined ante mortem, by display of clinical symptoms of leishmaniasis (onset of symptoms at 105 +/- 22 days post-inoculation), and postmortem by the presence of parasites within the spleen. Splenic parasite load calculated for a subset (n = 34) of infected hamsters was 124 to 26,177 Leishmania donovani infection units. Cryopreserved, and never-stored, cells were equivalent in all properties evaluated, including developmental changes in morphology during culture, culture growth rates, parasite resistance to serum-mediated lysis, and expression of developmentally regulated surface proteins major surface protease and promastigote surface antigen.

Characterization of DNA sequences that confer complement resistance in Leishmania chagasi.

Serial passage of axenically cultured Leishmania chagasi promastigotes results in a progressive diminution in resistance to complement-mediated lysis (CML), whereas high CML resistance is seen in infectious metacyclic promastigotes from the sandfly vector as well as metacyclic-like promastigotes within low-passage cultures at stationary growth phase. As we previously reported, in a screen seeking to identify novel genes involved in CML resistance: (1) a genomic cosmid library derived from DNA of CML-resistant L. chagasi promastigotes was transfected into high-passage (constitutively CML-sensitive) L. chagasi promastigotes; (2) transformants were screened for acquisition of CML-resistance; (3) multiple cosmid-transfectants exhibited partial CML resistance; and (4) the sequence for one of the cosmids (Cosmid 51) was determined. This report extends the analysis of Cosmid 51, and identifies by deletion analysis a subregion of the cosmid insert that is critical to the CML-resistance phenotype of Cosmid 51 transformants. We also report the sequence determination and initial CML-resistance activity of another cosmid that also confers partial resistance to CML.