Affordable Cancer Technologies Program Principal Investigators Meeting (Abstracts)

Jonathan Celli, PhD; Moni Kuriakose, M.D.

Motivated by the bleak situation of oral cancer in India and our UH2/UH3-supported successes with optical technologies for diagnosing and treating pre- and early cancers, a new integrated “Screen, Image and Treat Optical System” (SITOS) is proposed in the current application. This effort combines the expertise of 3 US sites in collaboration with 2 Indian clinical teams. The clinical studies in India will be led by Dr. Moni Kuriakose at Mazumdar Shaw Cancer Center, and Dr. Mohammad Akram at JN Medical College, Aligarh Muslim University. All sites were participants in the previous NCI awards as 2 individual UH2/UH3 projects focused on either detection or therapy. SITOS technology developed in this project will integrate photodynamic therapy (PDT) as a point of care treatment for oral lesions, with detection based on fluorescence and white light imaging supported by cloud-based deep-learning approach image classification. The goals will be accomplished in 3 Specific Aims. Aim 1 builds on previous successful development and clinical validation of separate low-cost devices for intraoral imaging, and intraoral PDT to produce a new, handheld, low-cost, easy-to-use, SITOS. The integrated platform enables the use of the same hardware for initial imaging, image-guided PDT and online monitoring during therapy. Incorporated is an ergonomic intraoral light delivery for PDT and preliminarily validated in optical phantoms and in vitro 3D tumor models. Aim 2A establishes conditions for optimal photosensitization of oral lesions using ex vivo and in vivo mucosa models. Based on data from 2A, Aim 2B establishes optimal PDT treatment (light delivery) parameters in a murine xenograft model. Aim 2C validates the best of these parameters in a carcinogen-induced hamster cheek pouch model which recapitulates transition from pre-malignant to malignant lesions. Aim 3 applies SITOS to identify and treat high-risk oral potentially malignant lesions (OPML), and early-stage oral cancer. Screening of patients will take place at camps and remote villages led by the clinical teams in India as in the previous UH2/UH3 effort. Patients with histologically confirmed HGD/OPML (and meeting other inclusion criteria) will be eligible. Patients will be treated using ALA photosensitization followed by image-guided PDT light delivery using SITOS. Light delivery and simultaneous image guidance will use the new intraoral probe.
The impact and relevance: The study provides, for the first time, a comprehensive low-cost approach that enables not only detection of pre-malignant/malignant oral lesions, but also an effective therapy in LMIC settings. The SITOS platform is mobile, handheld and appropriate for point of care applications. The prior experience of the team of scientists, oral cancer surgeons, oncologists and industry participation to provide the FDA photosensitizing agent (5-ALA) make the likelihood of success high.

Ayenew Ashenef, MSc

Goal: validate a new technology for detecting bad quality chemotherapy products at the point of use.

Motivation: Chemotherapy medicines form the backbone of affordable cancer treatment in low- and middle income countries (LMICs), yet LMICs often lack technical and regulatory capacity to evaluate the quality of chemotherapy products. There is currently no commercial technology to screen for bad quality chemotherapy products at the point of use in LMIC settings, and the drug regulators in Ethiopia, Malawi, Kenya, and
Cameroon do not conduct post-market surveillance testing on chemotherapy products.

Activities: The technology that will be validated, called SpotCheck, consists of an inexpensive paper test card (the chemoPAD) and a cell phone app. We will first adapt the chemoPAD to screen eight types of injectable chemotherapy drugs. The phone app’s neural network will be trained to identify products that are falsified or contain less than 65% of the stated API content. Clinical, academic, and supply chain partners in
Ethiopia, Malawi, Cameroon, and Kenya will conduct annual situation awareness and quality surveys of 320 chemotherapy products per year; the results will enable a team of researchers at U. North Carolina to model the markets for chemotherapy products and evaluate the cost-effectiveness of the SpotCheck system. After a technical performance milestone is passed, we will tailor the clinical validation of SpotCheck to suit the local
needs, clinical workflows, and regulatory capacity in each site. The validation of the SpotCheck system will proceed through a planning and ethical approval milestone (Y3 in Ethiopia and Malawi and Y4 in Kenya and Cameroon) and three clinical phases: proficiency study, clinical validation, and implementation pilot. Proficiency testing will demonstrate that oncology pharmacists and nurses can use SpotCheck with accuracy
>85% to detect SF products. Clinical validation will establish whether SpotCheck works correctly in a clinical setting on authentic products, rather than proficiency samples. The implementation pilot study will probe SpotCheck’s ability to test the drops left over in product vials after patient treatments are prepared in the hospital; this method of use would allow sustainable implementation of SpotCheck in many hospitals and clinics in low-resource settings. Technology transfer efforts will empower LMIC partners to produce the chemoPAD locally and integrate the cell phone app into regulatory reporting systems.

Impact: This project will help to fill the huge evidence gap about the quality of chemo drugs in LMICs, make it harder for manufacturers and distributors to sell bad quality products, and improve the quality of products that are used to treat patients in LMICs.

Sangeeta Desai, M.D.

Recognizing that over-diagnosis of many cancers is leading to over-treatment with adjuvant chemotherapy or with radiation therapy boost, there is a growing appreciation for the need for prognostic and predictive assays to identify those cancer patients who can benefit from therapy de-intensification. While multi-gene-expression based tests such as Oncotype DX and Decipher exist for identifying early-stage breast and prostate cancer patients who could avoid adjuvant therapies and hence mitigate side-effects and complications, the price of these tests ($3K-4K/patient) puts them beyond the reach of most patients in low- and middle-income countries (LMICs). Ironically, the need for these prognostic and predictive tests is even more acute in LMICs like India, where access to treatment resources like radiation and chemotherapy are limited and hence need to be administered judiciously to those patients who stand to receive the most benefit from them. Sophisticated digital pathomic analysis with computer vision and pattern recognition tools has been shown to “unlock” sub-visual attributes about tumor behavior and patient outcomes from hematoxylin & eosin (H&E)-stained slides alone. The Madabhushi team at Case Western Reserve University (CWRU) has extensively shown the potential for these approaches for predicting outcome and therapeutic response for breast, head and neck, lung and prostate cancer. The Madabhushi team working with collaborators Dr. Parmar and Dr. Desai at the Tata Memorial Center (TMC), the largest cancer center in India, has shown that advanced pathomic analysis is able to identify unique prognostic morphologic signatures of breast cancer that are different between South Asian (SA) and Caucasian American (CA) women 1. In addition, the CWRU group has shown that digital pathomic based image classifiers can significantly improve and even outperform the prognostic and predictive performance of expensive gene-expression assays for breast (Oncotype Dx) and prostate cancer (Decipher) 2. Building on the strong extant collaboration between CWRU and TMC 3, and a strong track record in digital image based prognostic and predictive based assays, we propose to optimize and validate an AI-enabled Digital Pathology Platform (ADAPT) for Multi-Cancer Diagnosis, Prognosis and Prediction of Therapeutic Benefit. ADAPT will involve optimizing the previously developed image assays by the CWRU group in the context of SA cancer patients. Furthermore, by integrating the AI-pathomic tools with PathPresenter, a widely used digital pathology image analysis platform, ADAPT will have a global footprint for the prognostic and predictive tools. Specifically, ADAPT will be validated for predicting outcome and benefit of adjuvant chemo- and radiation therapy in the context of estrogen receptor positive (ER+) breast cancer (BC) and triple negative breast cancer (TNBC), oral cavity squamous cell carcinoma (OC-SCC) and prostate cancer at TMC via a number of clinical trial datasets in the US (SWOG S8814, RTOG 0920, 0521) and at TMC (AREST, POP-RT). Successful project completion will establish ADAPT as an Affordable Precision Medicine (APM) solution for Indian cancer patients.

Jeanette Parkes, MBBCh

50% of cancer patients require radiotherapy during their disease course, however, only 10-40% of patients in low and middle-income countries (LMICs), have access to it. A shortfall in the specialised workforce to deliver radiotherapy has been identified as the most significant barrier to expanding radiotherapy capacity. The current radiotherapy workflow is inefficient requiring several labor intensive processes and takes weeks to months to deliver in LMICs. The growing demand for cancer treatment means that the ratio of incidence to mortality will continue to worsen without a scalable solution. Artificial intelligence (AI) based software has been developed to automate two components of the radiotherapy planning pathway 1. Delineation of anatomical areas that are at risk of tumour spread and at risk of radiation damage. 2. Definition of the position, size and shape of the radiation beams. Proposed advantages include improved treatment accuracy, as well as a reduction in the time (from weeks to less than a day) and human resources needed to deliver radiotherapy. We propose a non-randomised prospective study to evaluate the quality and economic impact of AI based automated radiotherapy treatment for cervical cancer and head and neck cancers, which are endemic in LMICs, and for which radiotherapy is the primary curative treatment modality. The sample size of 706 patients (353 for each cancer type) has been calculated based on an estimated 95% treatment plan acceptability rate. Time and cost savings will be analysed as secondary outcome measures to establish the cost and resource impact of automation using the time-driven activity-based costing model. The 48-month study will take place in six public sector cancer hospitals in India (n=2), Jordan (n=1), Malaysia (n=1), and South Africa (n=2) to ensure we include a broad range of patients and the representativeness of the findings will support implementation of the software in LMICs. If the study objectives are met, the AI based software will be offered as a not-for-profit web service to public sector state hospitals in LMICs to support expansion of high quality radiotherapy capacity, improving access, and affordability of this key modality of cancer cure and control. PUBLIC HEALTH RELEVANCE: The project is relevant to public health as it seeks to enhance the effectiveness and affordability of radiotherapy treatment in lower income settings. It is expected to improve both the health status and quality of life of men and women suffering from two major high burden cancers: cervical and head and neck cancers.

Breast cancer is an increasing challenge globally as it became the most prevalent malignancy at the end of 2020. More than 70% of all cancer mortality now occurs in low- and middle-income countries (LMICs). Histology, critical to the diagnosis and disease management for many cancers notably including breast cancer, is currently performed using techniques that are more than 100 years old. Market forces, technological advances in optics, and innovation in care strategies are opening the door for disruptive innovation that could massively reduce costs and time and improve accessibility while provide equivalent or even superior results. We propose to contribute to the field’s evolution by combining two already functioning and complementary technologies: 1) a tissue millifluidics approach (developed in PI Seibel’s lab) for hands-off core needle biopsy handling; and 2) a rapid, low-cost, direct-to-digital slide-free imaging solution (developed in PI Levenson’s lab). The goal is to implement a context-appropriate, automated instrument that can capture diagnostic-quality histopathology images from core-needle biopsies vital to high-quality breast-cancer diagnosis and staging, at time of procedure. Additional project goals include implementation of innovative rapid immunofluorescence methods for near-real-time therapy guidance; and development of AI tools for patient triage or even local diagnostic support, the latter under the direction of Dr. Mahmood (BWH), a leader in multiclass AI algorithms.
Key to success of this project is local implementation and clinical evaluation in Kumasi, Ghana, under the direction of Dr. Addai, consultant breast surgeon and CEO of Peace and Love Hospitals (established in 2002). Her group will critically assess performance, usability, and compatibility with the service environment in both a central hospital and a remote satellite clinic; clinical validation studies will eventually encompass at least several hundred patients recruited under IRB-approved protocols. Additional guidance will be provided by Dr. Dan Milner, consultant to the American Society for Clinical Pathology (ASCP), who has extensive experience in global-health-focused initiatives, as well as by our collaborating pathologists who are familiar with issues relevant to LMIC settings. Feedback will inform the design of the second-generation automated instruments to be delivered near the end of this project.

Joseph Ngonzi, M.D., Ph.D.

Goal: validate a new technology for detecting bad quality chemotherapy products at the point of use. Motivation: Chemotherapy medicines form the backbone of affordable cancer treatment in low- and middle- income countries (LMICs), yet LMICs often lack technical and regulatory capacity to evaluate the quality of chemotherapy products. There is currently no commercial technology to screen for bad quality chemotherapy products at the point of use in LMIC settings, and the drug regulators in Ethiopia, Malawi, Kenya, and Cameroon do not conduct postmarket surveillance testing on chemotherapy products. Activities: The technology that will be validated, called SpotCheck, consists of an inexpensive paper test card (the chemoPAD) and a cell phone app. We will first adapt the chemoPAD to screen eight types of injectable chemotherapy drugs. The phone app’s neural network will be trained to identify products that are falsified or contain less than 65% of the stated API content. Clinical, academic, and supply chain partners in Ethiopia, Malawi, Cameroon, and Kenya will conduct annual situation awareness and quality surveys of 320 chemotherapy products per year; the results will enable a team of researchers at U. North Carolina to model the markets for chemotherapy products and evaluate the cost-effectiveness of the SpotCheck system. After a technical performance milestone is passed, we will tailor the clinical validation of SpotCheck to suit the local needs, clinical workflows, and regulatory capacity in each site. The validation of the SpotCheck system will proceed through a planning and ethical approval milestone (Y3 in Ethiopia and Malawi and Y4 in Kenya and Cameroon) and three clinical phases: proficiency study, clinical validation, and implementation pilot. Proficiency testing will demonstrate that oncology pharmacists and nurses can use SpotCheck with accuracy >85% to detect SF products. Clinical validation will establish whether SpotCheck works correctly in a clinical setting on authentic products, rather than proficiency samples. The implementation pilot study will probe SpotCheck’s ability to test the drops left over in product vials after patient treatments are prepared in the hospital; this method of use would allow sustainable implementation of SpotCheck in many hospitals and clinics in low-resource settings. Technology transfer efforts will empower LMIC partners to produce the chemoPAD locally and integrate the cell phone app into regulatory reporting systems. Impact: This project will help to fill the huge evidence gap about the quality of chemo drugs in LMICs, make it harder for manufacturers and distributors to sell bad quality products, and improve the quality of products that are used to treat patients in LMICs.

Elizabeth A Bukusi, MPH, PhD; Megan Huchko, MPH, M.D.

Cervical cancer is the second leading cause of death for women worldwide. Alarmingly, 85% of deaths occur in low and middle-income countries (LMICs), as they lack the health care infrastructure required for cytology-based screening, referral colposcopy diagnosis, and expert physicians, which have dramatically reduced the disease burden in high income countries (HICs). Highly sensitive human papillomavirus (HPV) testing has been effective at reducing the incidence and mortality from cervical cancer when directly coupled with treatment; however, a majority of women with HPV do not have cervical precancer, making HPV testing a poor triage test as overtreatment carries risks like hemorrhage and infertility. Colposcopy followed by biopsy, the preferred triage method in HICs, is untenable in most LMIC settings due to the cost of colposcopes and pathology facilities to process and interpret biopsy results. To make matters worse, women are lost to follow up in LMIC settings when a multi-visit model for cervical cancer screening is used. Visual Inspection with Acetic Acid (VIA), the World Health Organization recommended triage test following HPV testing, has widely varied sensitivity and specificity depending on the training level of the provider. In this proposal we are proposing a single visit model for precision diagnosis and treatment in LMICs for cervical cancer prevention. Two major technological tools are needed to implement this model: a low-cost method to perform imaging of the cervix and a machine learning algorithm to automate diagnosis in the absence of a provider. We have previously developed the Pocket Colposcope, which has shown high concordance with standard colposcopy at a fraction of the cost and validated it on thousands of women across nearly every continent. We are now in the process of developing a state-of-the-art convolutional neural network (CNN), called Colposcopy Automated Risk Evaluation (CARE), trained with Pocket colposcopy images to automate the diagnostic process. Our current prototype algorithm has been highly successful at classifying cervical pre-cancers from Pocket Colposcope images retrospectively. Our goals for this proposal are fourfold: 1) improve and generalize the performance of Pocket CARE using >10,000 National Cancer Institute (NCI) standard colposcopy images; 2) generate synthetic images to address domain shifts due to environmental and personnel changes between different clinical sites; 3) embed the CARE algorithm into our existing software to enable high quality image capture with the Pocket Colposcope for automated diagnosis 4) validate the performance of Pocket CARE prospectively with a clinical study in Kisumu, Kenya, a site where Pocket CARE would ultimately be adopted. The deliverables for this proposal will be a fully validated Pocket CARE software ready for scale to different clinical scenarios based on location-specific cultural contexts and infrastructure and a comparative effectiveness of Pocket CARE to other publicly available algorithms and standard care.

Oliver Ezechi, M.D., Ph.D.

Each day, there are an estimated 28 cervical cancer deaths in Nigeria, making cervical cancer the second most common cancer among women in the entire country. Only a minority of women eligible for screening (screen-eligible) in Nigeria (30-49 years old) regularly, have received HPV screening - an essential component of comprehensive cervical cancer prevention programs recommended by the Nigerian Federal Ministry of Health and the Society of Obstetrics and Gynecology of Nigeria (SOGON). Conventional screening that relies on Pap smears and visual inspection with acetic acid (VIA) at centralized clinics, coupled with a lack of locally relevant implementation strategies have stalled progress. Innovative strategies to decentralize screening and increase women’s ownership in this process are urgently needed. In this proposal, we will adapt an existing HPV assay that combines loop-mediated isothermal amplification (LAMP) and fast one-pot lyophilization protocol within a lateral flow assay (LFA) for the Nigerian context, and then use participatory strategies (crowdsourcing open calls and learning communities) to finalize components of a single woman-centered HPV self-test kit. Crowdsourcing open calls have a group of individuals (i.e., screen-eligible women) solve all or part of a problem, then implement selected high-quality solutions. Learning communities help participants refine and optimize solutions before evaluation or use among screen-eligible women in Nigeria. Our collaborative research team has successfully used crowdsourcing open calls and learning communities to increase HIV self-testing among Nigerian youth, providing a strong foundation for the proposed research study. Our preliminary data demonstrate that our HPV self-test prototype can reliably detect HPV 16 and 18 genotypes that account for 70% of all cervical cancer cases. Once the prototype meets stringent diagnostic and trial preparedness metrics among Nigerian women, including detection of additional common HPV genotypes (31, 35, and 52), the project will move from the initial engineering phase (Specific Aim 1) to the clinical phase (Specific Aims 2 and 3). Drawing on a design and participatory action research framework, we propose the following specific aims: (1) to adapt an HPV self-test assay for point-of-care and simultaneous detection of HPV genotypes 16, 18, 31, 35, and 52 in Nigeria; (2) to use crowdsourcing open calls and participatory learning communities among screen-eligible women to finalize a single HPV self-testing implementation strategy (3) Determine whether a final revised HPV self-testing strategy increases HPV screening among 900 screen-eligible women in 18 local government areas versus usual care using a stepped-wedge, pragmatic randomized control trial. Our study will be among the first to examine how women themselves can be prime movers in optimizing, implementing and evaluating HPV self-testing implementation strategy that incorporates their unique needs to prevent cervical cancer. Our focus on open calls and tailoring HPV services for screen-eligible women resonates with NCI, NIH, and US government strategic priorities.

Robert T Ssekitoleko, MEng; Julius Mugaga, Msc; Jenna Mueller, PhD

Laparoscopic surgery is the standard of care in high-income countries for many cancer operations in the chest and abdomen. Laparoscopic surgery avoids large incisions by using a tiny camera and fine instruments manipulated through keyhole incisions, but it is generally unavailable in low- and middle-income countries (LMICs) due to high cost of installment, lack of qualified maintenance personnel, unreliable electricity and shortage of consumable items. Patients in LMICs would benefit from laparoscopic surgery, as advantages include: decreased pain, improved recovery time, fewer wound infections, and shorter hospital stays. Laparoscopic surgery would reduce recovery time, enabling patients to return to home and work more quickly, thereby mitigating impoverishing health expenditure.
KeyScope and KeyLoop (collectively called KeySuite) are laparoscopic prototypes that we have designed for the resources, needs and challenges of LMICs. KeyScope is a low-cost laparoscope that plugs into a laptop computer to display images during surgery, exists as a single unit without complicated assembly and is sterilizable by immersion in Cidex. KeyLoop is a laparoscopic retractor that lifts the abdominal wall during surgery, obviating the need for a constant power supply and medical-grade carbon dioxide. This would enable laparoscopic surgery to be performed in rural hospitals, where most patients live in LMICs, and increase access in tertiary centers where laparoscopic equipment is rare and expensive. We describe a multi-disciplinary collaboration between surgeons, engineers, oncologists, attorneys, global health experts and business executives to take this technology to the next stage.
We are performing a clinical First-in-Human study at the Uganda Cancer Institute. Ugandan surgeons will use the KeySuite devices to perform biopsies of intra-abdominal tumors. Primary outcome will be the ability to perform biopsies laparoscopically without conversion to open surgery. Secondary outcomes include: 1) device feasibility and safety data and 2) patient satisfaction.
We have demonstrated that the KeySuite devices can be constructed in Uganda, through the Duke-MUK Shipping Container Makerspace. We have created a bill of materials, manufacturing process instructions, training videos, and measures of quality control. We are currently working with a local Ugandan medical device manufacturing company and a local distribution company to establish a sustainable business model for commercialization in sub-Saharan Africa.

Colorectal cancer, “CRC”, is defined as a malignant neoplasm of the colon and abdomen. Nearly 147,950 people are diagnosed with colorectal cancer each year in the US alone. CRC has a five-year survival rate of roughly 17% for Stage IIIb and IV, referred to as “distant” cancer, and are known to have a very high rate of recurrence, even after exhibiting clear margins. Surgical resection is the gold standard of treatment in Stage IIIB and IV colorectal cancers. Despite the importance of clear margins in preventing recurrence, preoperative and intraoperative guidance technologies are severely lacking, especially given that preneoplastic lesions and isolated groups of cancer cells can be difficult to detect with the naked eye. Moreover, traditional methods of resection are primarily performed with scalpels and cauterizing tools and leave surgeons to rely on tactile information. The use of fluorescent guided surgery, FGS, has the potential to add an important layer of information by intraoperatively illuminating (fluorescing) cancerous tissue, while the use of CO2 laser technology is emerging as a superior surgical tool compared to conventional methods of in vivo ablation. To address the need for an intraoperative and interactive surgical system that enhances the identification of potentially malignant disorders and cancerous tumor margins, as well as the surgical removal of identified pathological cells rapidly and completely, we plan to develop a handheld, 3D FGS laser surgical system that integrates 1) a CO2 surgical laser, and 2) fluorophore illumination to facilitate the contemporaneous and intraoperative detection and ablation of preneoplastic and neoplastic cells and tissue to identify and remove potentially malignant disorders. The laser device and beam delivery of the 3D FGS surgical system will be adapted from our team’s existing oral surgical system, which has been cleared by the FDA for soft, osseous, and hard tissue surgery. Despite advanced technologies, countries categorized as LMICs show an increase in incidences of various cancers. The lack of preventive colonoscopies in LMICs, and the associated infrastructure, make both identification and screening of early-stage CRC difficult. The long-term objective of this work is to build a free standing CO2 laser system, for the purpose of applying this system for treating colorectal and ovarian cancers worldwide, including in LMICs. Laser ablation of tumors and photothermal coagulation of tissue speeds surgery, while offering both resection and “scanning ablation” to remove tumors and sub-mm cancerous tissue. The FGS system incorporates excitation diodes and narrow-band emission optical filters, in an interchangeable surgical handpiece, to integrate the fluorescent marker for colorectal or ovarian cancer cells. Spatial Surgical has visited and is working closely with the Massachusetts General Hospital, UMASS Medical Center, and the Philippine General Hospital. Our proposed low-cost FGS laser surgical system will be customized to various cancer types (e.g., colon and ovarian) with interchangeable handpieces, leverage existing surgical skill and techniques, and add enhanced visualization with disease-specific fluorophores to ultimately increase survival rates in patients with pre- and existing malignancies.

Albert Manasyan, M.D.

Cervical cancer is among the leading causes of cancer death in women worldwide, especially in low- and middle- income countries (LMICs). High-risk human papillomaviruses (HPV) are the main causative agents of cervical cancer and its precursor lesions; therefore, the World Health Organization (WHO) has recommended HPV DNA testing for cervical cancer screening in LMICs. Although polymerase chain reaction (PCR) methods have been widely used for HPV DNA detection, they are restricted to centralized clinical laboratories due to the need for labor-intensive procedures and expensive equipment. Here, we propose to develop a simple, rapid, highly sensitive, and specific CRISPR-on-paper diagnostic platform to simultaneously detect multiple high-risk HPV genotypes for cervical cancer screening at the point of care. This innovative diagnostic system is based on our recently developed all-in-one dual CRISPR-Cas12a (AIOD-CRISPR) assay, which combines the simplicity and high sensitivity of isothermal nucleic acid amplification with the high specificity of CRISPR detection. To develop a low-cost, multiplexed molecular detection technology, we will incorporate the AIOD-CRISPR assay into a paper-based microfluidics platform. To eliminate the need for complex electronic instruments, we will take advantage of an exothermic reaction to generate chemical heat for the CRISPR-on-paper system by using a disposable hand warmer, thus enabling instrument-free cervical cancer screening. The detection results can be read by the naked eye or reported by a programmed smartphone without the need for an expensive optical detector. We will rigorously evaluate and validate the clinical applications of our CRISPR-on-paper diagnostic system by testing clinical samples in collaboration with clinicians and healthcare workers in UConn Health and Zambia. If successful, the proposed project has an important impact on global health by providing a simple, affordable, and sensitive method for rapid screening of cervical cancer in resource-poor settings. As a platform technology, the proposed CRISPR-on-paper diagnostic system can be easily adapted to detect other emerging pathogens.

Jeff Wang, PhD; Samson Okello, M.D.

Despite substantial progress in clinical approaches to squamous cancer of the esophagus (ESCC), which causes most esophageal cancers (EC) in the world, this deadly tumor usually occurs at late disease stages, with very poor survival. Restricted availability of endoscopy (EGD), along with rarity and delays in histology, impairs detection of ESCC in LMICs, adversely impacting our ability to treat this disease effectively. Thus, in LMICs, inexpensive, safe, locally performable strategies for detecting ESCC are necessary to identify high-risk patients and refer them quickly to suitable diagnostic and therapeutic options. Therefore, a diagnostic approach featuring a retrievable swallowed sponge-on-a-string to gather esophageal specimens for molecular testing, combined with a point-of-care (POC) magnetofluidic chip for sample processing and DNA methylation detection, is proposed. The string-sponge is less expensive, more noninvasive, more convenient, and more rapid than EGD with biopsy. The magnetofluidic chip streamlines DNA purification, DNA bisulphite treatment, and PCR detection of methylation markers into a single POC apparatus. This approach does not necessitate EGD, can be performed in remote areas with portable energy supplies and does not require extensive medical training, and is thereby amenable to implementation in LMICs. Our Specific Aims are: 1: Using a sponge-capsule swallowed/tethered collection device, to construct a methylation marker-based strategy to detect ESCC. In 100 ESCC and 100 benign control patients, we propose (1) building a multivariate model containing biomarker candidates; (2) carrying out feedback-feasibility meetings with health care and endoscopy personnel at Makerere University Hospitals to fine-tune eventual POC usage; 2: In order to achieve a sample-to-answer assay, to implement DNA extraction, bisulfite treatment, and methylation-specific PCR into a magnetofluidic chip with dried reagents. We’ll use magnetofluidic techniques to streamline cell lysis, DNA extraction, bisulphite treatment, and methylation-specific PCR into a compact chip built from cheap thermoplastic materials. In addition, we’ll lyophilize reagents and use heat- deployed wax sealant plugs to permit storage at room temperature. In this fashion, we will fashion a sample-to- answer assay that is easy to use, inexpensive, and free of cold-chain steps; 3: In order to achieve fully automatic high-speed biomarker assaying, to design a small, light apparatus. We’ll engineer an instrument containing programmable magnetic actuation, temperature control, and detection of fluorescence to execute the test in a chip with very little user input. We’ll also design the apparatus to be small, light, easy to operate, portable electricity-powered, and mobile phone-controlled to ease integration with LMIC-based clinical tasking; and 4: Using our POC approach to carry out a diagnostic pilot study of ESCC in Uganda. While applying the magnetofluidic chip and apparatus used in Aim 2 and Aim 3, we’ll carry out a trial to measure specificity and sensitivity in 120 EGD-confirmed cases of ESCC and 360 benign disease control patients in Kampala, Uganda. PUBLIC HEALTH RELEVANCE: We propose an ESCC diagnostic strategy comprising a single-use swallowed retrievable sponge-capsule to collect esophageal cells for biomarker analysis, coupled with a portable microfluidic chip device for automated POC sample processing and DNA methylation detection. This strategy does not require endoscopy (EGD), can be administered in rural settings without the need for electricity or persons with medical training, and uses a portable analytic chip. The sponge is cheaper, less invasive, safer, and faster than EGD; the microchip integrates DNA extraction, bisulfite DNA conversion, and methylation analysis into a single POC device.

No woman should die from cervical cancer. We have the technical, medical and policy tools and approaches to eliminate it. Yet, one woman dies of cervical cancer every two minutes. Cervical cancer is the third leading malignancy among women in the world, after breast and colorectal cancer. Cervical cancer is also one of the tumors in which the most glaring disparities exist worldwide. The dramatic disparity in i ncidence rates between high- and low-income countries is due primarily to differential access to effective screening and pre-cancer, or preventive, treatment; similar disparities also exist within countries. Recently published hysterectomy-corrected cervical cancer mortality rates in the United States reveal a larger racial disparity in black women, that previously calculated and shows that the oldest black women have the highest cervical cancer mortality rates. Nonetheless, more than 80 percent of cases and 88 percent of deaths occur in Low and Middle Income Countries (LMICs). The World Health Organization (WHO) has developed guidelines for treatment of cervical intraepithelial neoplasia 2-3 and screen-and-treat strategies to prevent cervical cancer. In countries where multiple barriers exist for cytology based cervical cancer screening, a variety of alternative algorithms are being used, which include low-cost oncogenic HPV testing, visual inspection with acetic acid, and self-collected vaginal swabs. HPV testing is an excellent alternative to cytology for cervical cancer screening. However, HPV tests identifies women at risk for cervical cancer, but not those HPV-positive women who are most likely to have, or to develop in the near future, significant disease requiring treatment. Current practice is to triage these women by further testing with cytology and colposcopy-driven biopsies in developed countries and excision or ablation therapy in LMICs. The use of DNA biomarkers can reduce the number of women referred to colposcopy while maintaining adequate sensitivity and specificity. In this Fast Track SBIR project, we propose to demonstrate the feasibility for the commercialization of a precision methylation test, the CervicalMethDx Test, to stratify HPV+ patients for high risk of cervical cancer, as a reflextest to existing standard of care in LMICs. Our test will enable identification of HPV+ women at clinical risk for advancement from low-grade squamous intraepithelial lesions (LSIL) to Cervical Intraepithelial Neoplasia grade 3 (CIN3). In LMICs, where cervical cytology based-screening will not be implemented, optional modalities of our test will be developed in future projects to stratify HPV+ women at high risk of cervical cancer in self-collected vaginal swabs and/or urine samples. We are partnering with David Sidransky’sresearch laboratory to optimize the CervicalMethDx Test and with the ESTAMPA Study (NCT01881659) Consortium to introduce precision epigenetic services to residents of Honduras, Colombia, Argentina,Bolivia, Costa Rica, Uruguay and Paraguay. Efficient triage of HPV+ women will decrease unnecessary treatment, improve health care quality, decrease health care costs, and reduce cervical cancer mortality disparities in LMICs.

Jeffrey Martin, M.D., MPH; Aggrey Semeere, MBChB, M.Med, MAS; Ethel Cesarman, M.D., PhD; Toby Mauer, M.D.

In this project, we are developing, manufacturing, and perform a multi-site sub-Saharan African clinical validation of KS-COMPLETE — the first true point-of-care sample-to-answer diagnostic system for Kaposi’s sarcoma (KS). Our recent large-scale studies in Africa have shown that KS can be diagnosed through quantification of Kaposi’s sarcoma herpesvirus (KSHV) DNA in a skin biopsy with high sensitivity and specificity. These efforts have also resulted in the development of TINY — a robust, easy-to-use, infrastructure-free, point-of-care (PoC) technology for KSHV DNA quantification — which is being currently deployed in a multi-site evaluation. The work has also revealed that the key challenge to widespread adoption of skin biopsy-based PoC systems is the time and manual steps required to extract DNA from a skin biopsy — which can be up to 4 hours.
KS-COMPLETE will be the first “direct-to-LAMP” diagnostic system for skin punch biopsies. Similar direct-to-LAMP methods have greatly simplified PoC diagnostics for other sample matrices but the solid-phase, collagenous nature of skin has made this a challenge for biopsies. KS-COMPLETE will address this issue with our “SLICER” technology that will automatically process a punch biopsy into smaller “micro-cores” on which we can directly perform DNA quantification in TINY through our “direct-to-LAMP” approach. We hope that this approach will reduce the time to result to around 60 minutes, eliminate all the current manual and intensive sample processing steps, and is compatible with cost, robustness, infrastructure, and simplicity requirements for operation in LMICs. Clinical validation of the system will be done through our established network of KS clinical sites in Africa.
KS is one of the most common cancers in men and women in sub-Saharan Africa. KS is difficult to distinguish from other skin conditions, particularly in Africa where access to trained pathologists is limited and immunohistochemistry is practically non-existent. Early-stage and more accurate diagnosis would confer many clinical benefits. For patients who have KS, it obviates the need for the difficult to obtain, slow, and unreliable histopathology and allows for detection at earlier clinical stages resulting in better clinical outcomes. For patients with mimickers, rapid exclusion of KS allows for timely re-orienting of the diagnostic process and prevents use of potentially toxic chemotherapy. Our direct-to-LAMP diagnostic test could have significant impact beyond the diagnosis of KS as multiple other viral, mycobacterial and fungal-related skin diseases currently diagnosed through traditional pathology could be transitioned to this method and ultimately the point of care.

Cervical cancer is the fourth most common cancer in women, contributing to about 570,000 new cases and more than 300,000 deaths in 2018, most of it occurs in the low- and middle-income countries (Bray, 2018). In Indonesia, cervical cancer is the second most prevalent type of cancer among women, with 32,000 new cases and 18,000 deaths (Global Cancer Observatory, 2019). It has been shown that human papillomavirus (HPV) infection is the major causative agent of cervical cancer (Bosch, 2002). In 2017, WHO released an HPV vaccine position paper that stated HPV vaccines should be included in national immunization programs with its primary target population is 9-14 year old girls, prior to becoming sexually active (World Health Organization, 2017). Vaccination represents the most cost-effective and efficient way for the prevention of cervical cancer caused by human papillomavirus. Currently, HPV vaccine has been introduced in nearly 100 countries globally (52%), mainly in the upper-middle- and high-income countries. However, its access in the highest burden countries (low and middle income) remains lagging. Furthermore, previous data collected by global health organizations have shown continued low compliance rates for HPV vaccines, partly attributable to needle-phobia. It is predicted that this issue will persist unless a new strategy for vaccination is introduced.
In this direct to Phase II proposal, we will explore the feasibility and potential benefits of a reduced dose intradermal strategy for HPV vaccines. In this larger study, we will evaluate doses and routes of administration to determine the immunogenicity, reactogenicity, and levels of patient and caregiver acceptance for each. In settings with minimal resources, the benefits of delivery with needle-free injection are predicted to include dose savings, increased compliance, and the prevention of needle-stick injuries and disease transmission. The results from these studies will provide clinical data to answer the main problems with the current HPV vaccine schedule: 1) availability of vaccine doses; 2) cost; and 3) acceptability & compliance. This data will be a first step to facilitate future policy discussions and global health strategies regarding administration of fiHPV vaccines across Indonesia and other developing countries. The immediate next steps will include planning a larger roll-out campaign across multiple areas of Indonesia, notably in further remote areas where vaccination compliance has been historically low. Ultimately, these studies will be used as a tool to increase awareness of the PharmaJet Tropis Needle-free injection system as a method for fiHPV administration that may aid in country-wide and worldwide vaccine compliance and acceptability to schedules as recommended by the WHO and Ministries of Health.