Study Title Reducing the burden of squamous cell carcinoma in Fanconi anemia PREAMBLE Fanconi anemia (FA), named for Swiss pediatrician, Guido Fanconi, is a rare genetic disease that leads to bone marrow failure. Though considered primarily a blood disease, FA may affect all systems of the body. It is a complex and chronic disorder that is psychologically demanding. FA is also a cancer-prone disease, affecting individuals decades earlier than the general population. FA individuals have an especially high risk of developing malignant lesions, particularly solid tumors of the head and neck region and oral cavity (mouth and esophagus). The aggressive nature of these tumors combined with delayed diagnosis mandate substantial surgical intervention and radiotherapy. A non-invasive brush biopsy approach followed by conventional cytology and/or DNA cytometry and LOH assay to detect pre-cancerous lesions has proven successful in identifying early malignant lesions. Based on these results we are proposing a longitudinal screen of an international FA cohort for the early detection and monitoring of oral cancer and pre-cancer, employing soft brush biopsy combined with cytology, DNA cytometry and LOH analysis. Included in these studies will be a questionnaire to correlate presence and progression of cancer and pre-cancer with exposure to environmental risk factors such as alcohol and tobacco exposure. In cases where visible lesions are observed, we will provide the cytology and cytometry analysis results to the individual, parent of children participant and designated treating physician. The de-identified research data will be aggregated in a dataset accessible for research purposes through research databases such as the Synapse repository and analysis platform (WIRB # 20112068). The aim of the proposed study is improvement of early detection, timely diagnosis, and individualized treatment of SCC arising in the context of FA individuals, with the ultimate goal to improve treatment outcome and reduce disease burden. Secondary aims are to identify FA individuals with cancer and pre-cancer and to foster tissue sampling for the FA research community. We are seeking WIRB’s opinion regarding the feasibility of this study. Table of Content Reducing the burden of squamous cell carcinoma in Fanconi anemia .................................... 1 Chapter 1 Abbreviations: ........................................................................................................................... 3 Chapter 2 Background ................................................................................................................................ 3 Chapter 3 Study sponsors and Principal Investigators ................................................................... 4 Chapter 4 Purpose of the Study ............................................................................................................... 4 Chapter 5 Who can participate? .............................................................................................................. 5 Chapter 6 Subject recruitment/sites ..................................................................................................... 5 Chapter 7 Experimental Components and Procedure ..................................................................... 6 Chapter 8 Data Storage and Confidentiality ..................................................................................... 10 Chapter 9 Potential Risks and Discomforts...................................................................................... 10 Chapter 10 Potential Benefits ............................................................................................................... 11 Chapter 11 Conclusion ............................................................................................................................. 11 REFERENCES ............................................................................................................................................... 13 APPENDICES ............................................................................................................................................... 14 Appendix A Sponsor’s Board of Directors and Scientific Advisors .................................................... 15 Appendix B Proposed announcement Flyer............................................................................................... 17 Appendix C Information sheet for adolescents over 14 and adults ................................................... 18 Appendix D Information sheet for Participants up to age 14 .............................................................. 21 Appendix E Study Questionnaire ................................................................................................................... 24 Reducing the burden of SCC in FA - Protocol 2 Chapter 1 Abbreviations: AML: acute myeloid leukemia FA: Fanconi anemia GvHD: graft versus host disease HNSCC: head and neck squamous cell carcinoma HSCT: hematopoietic stem cell transplantation LOH: loss of heterozygosity SCC: squamous cell carcinoma WHO: World Health Organization Chapter 2 Background Fanconi anemia (FA) is an inherited disease that leads to bone marrow failure (aplastic anemia). It is primarily a recessive disorder: if both parents carry a mutation in the same FA gene their child has a 25% risk of inheriting the mutation and having the FA condition. About 5% of FA individuals do not carry a mutation on the known FA genes, suggesting that more FA genes will be discovered in the future. FA affects both males and females from all ethnic origins. The current median lifespan for a person with FA is 29 years although some individuals live into their 50s. While FA is primarily a blood disorder, it affects many other body systems, leading to a number of complications. Many individuals develop a variety of cancers such as acute myeloid leukemia (AML) or squamous cell carcinoma (SCC) at an unexpectedly early age. Individuals who have had a successful bone marrow transplant and are cured of the blood disorder still must have regular examinations to watch for signs of cancer. FA individuals have a 500-700 fold increased risk over the general population for developing SCC of the head and neck regions, most notably in the oral cavity and esophagus1,2. The causes for such elevated risk are varied and not all known. When diagnosed early these tumors can be treated successfully and relatively easily. However, when detected late, prognosis of these aggressive tumors remains poor, despite extensive surgical intervention and radiotherapy. Regular surveillance for these complications is therefore essential and FA individuals are advised to be examined regularly for early signs of abnormal lesions. Despite these guidelines for regular surveillance, not all healthcare providers are aware of the elevated risks of cancer in FA and few FA individuals seek regular medical attention for these complications. The most current method for detecting malignant lesions is through visual inspection by experienced oncologists and head/neck surgeons. One problem with this approach is the fact that FA individuals often present with visible lesions of the oral cavity from graft- versus-host disease, particularly following HSCT, and consequently the majority of visible lesions might not be pre-cancerous. Furthermore, precursor lesions are not always visible to the naked eye or they are asymptomatic and missed. Reducing the burden of SCC in FA - Protocol 3 In 2007, Dr. Brakenhoff piloted a non-invasive molecular assay to detect precursor lesions in the oral cavity. This novel method using LOH analysis has been adapted to test soft swabs of the oral mucosa. FA individuals accept and tolerate these soft-brush biopsies (including soft swabs for cytological evaluation and DNA cytometry) much better than the traditional scalpel biopsies and are more likely to comply with regular screening recommendations. Based on these observations we propose to conduct a longitudinal study to (1) raise awareness about the high cancer risk in FA, (2) collect soft-brush biopsy samples to detect, differentiate and follow-up benign vs. malignant oral lesions as determined by cytology and/or DNA Cytometry and/or LOH analysis, (3) correlate presence and progression of cancer with exposure to environmental risk factors, (4) collect material and stimulate research collaborations, and (5) provide critical educational material and data to individuals, parents and researchers to improve treatment outcome and reduce the burden of SCC in FA. Chapter 3 Study sponsors and Principal Investigators The Fanconi Anemia Research Fund, Inc. in the United States and the Deutsche Fanconi- Anaemie-Hilfe e.V. (German Parent and Patient Organization for Fanconi Anemia), are the sponsors of this project. The Board of Director and scientific advisors for FARF and for FANCONI.de are listed in Appendix A The Fanconi Anemia Research Fund is a non-profit organization created to promote discovery of effective methods to treat and someday cure problems associated with Fanconi anemia. In addition to funding relevant research projects, the foundation provides educational and support services to the FA community worldwide. The Deutsche Fanconi-Anaemie-Hilfe e.V. (FANCONI.de) is the active equivalent to FARF. Founded in 1990 by German Families with FA children FANCONI.de also supports international research projects for Fanconi anemia and provides support to family and patients worldwide. The Co-Principal Investigators for this study in the U.S. are Laura Hays, PhD., Executive Director of FARF and Amy Frohnmayer, M.A., a member of the Board of Directors of the Fanconi Anemia Research Fund. Ralf Dietrich, Executive Director of FANCONI.de and Eunike Velleuer, MD will help coordinate the study. Chapter 4 Purpose of the Study This study aims to inform FA individuals and their families of the risks of oral and oropharyngeal SCC; to collect, test and analyze samples from FA participants with follow- Reducing the burden of SCC in FA - Protocol 4 up over time; to provide materials and stimulate research collaborations and innovation and ultimately to reduce the burden of SCC in FA individuals. Chapter 5 Who can participate? This study is designed for FA individuals, irrespective of age, gender, nationality or ethnicity. We will recommend that sample collection be conducted at a dental practice or hospital for individuals with increased risk of bleeding as indicated by visible bruises and petechiae in the prior 2 weeks, or platelets lower than 20,000 if platelet count is known. Chapter 6 Subject recruitment/sites Our goal is to reach as many FA individuals as possible. To that end we have prepared an announcement flyer that we propose to distribute widely to all FA-related support groups, associations, research meetings and other events, hospitals and academic institutions (Appendix B). Specifically, we will post the announcement on the FARF website, and send it via letter mail, email, social media sites (through FARF) to FA individuals and family on the FARF mailing list or provide it to attendees at FA family meetings, FA adult meetings and/or Camp Sunshine. Camp Sunshine offers an annual meeting for individuals with FA and their families. This event takes place each summer and incorporates scientific presentations about this disease as well as support group gatherings. This meeting represents a unique opportunity to reach large numbers of people with FA in one location within a relatively short period of time. A verbal announcement will be made at the beginning of the meeting informing families about the purpose and procedures of this study and inviting people to participate. The decision to participate (or abstain) will have no effect on regular camp activity. FA individuals will be given the opportunity to participate at other times, for instance, through home visitations depending on funding availability. We will also provide information sheets, one for individuals over 14 years old (Appendix C) and one for children 7-14 years old (Appendix D)., that explain the details of the study. Interested participants will be able to review the study goals and procedure description at their leisure. Participation is voluntary and does not impact standard of care. The decision to participate or not to participate will not affect current benefit or standing. There is no penalty of loss of benefit for declining to participate. Interested participants will confirm their consent/assent to participate by signing the informed consent form. At least one designated study investigator will be present to explain the study procedures in person, respond to participant questions, and collect consent/assent. Individuals who have consented will be given a unique and random code identifier to be used instead of their names on all material collected. The sponsors and study staff will keep the identity of the individual confidential. Reducing the burden of SCC in FA - Protocol 5 The following measures will be taken to guarantee participant privacy. Nobody will be forced to do anything with which he or she feels uncomfortable. It will be made clear that participation is an invitation, not a recommendation. The intervention and interviews, including sensitive questions about substance use, will be conducted in a private setting in a separate room. Chapter 7 Experimental Components and Procedure The experimental components of this study are: A health questionnaire Non-invasive oral cavity screening: photographs and maps of oral lesions Sample collection with self-administered soft-brush biopsy procedure Cellular and molecular analysis of samples Report of cytological results of visible lesions to individuals and their treating physicians if applicable Optional additional sample collection for research and methods development Optional transfer of de-identified data onto a centralized data repository (such as Synapse) to be used in future research The procedure will take place at FA family meetings, home visits, and local meetings in academic hospitals. The sequence of events for consented participants is as follows: Step 1: Conduct interview and complete health questionnaire We will use structured interviews and a questionnaire to estimate the quality of surveillance of FA individuals by head and neck specialists according to the Fanconi Anemia Guidelines for Diagnosis and Management 3. Specifically, we will ask contact information for the participant and his/her health care provider. We will also ask demographic information and medical history as related to FA. The questionnaire is included in Appendix E Step 2: Inspect, photograph and map the lesions We will perform a careful and standardized inspection based on the WHO oral cancer diagnosis protocol and we will document any visible oral lesion in an individual “oral cavity map” (figure 1). In addition we will document the lesions using the Karl Storz Endoscopic Oral Documentation System and digital pictures 4. Note that the pictures will focus exclusively on the lesion and will not contain any facial features that can reasonably re-identify the participant. Figure 1: individual oral cavity map Step 3: Collect samples via soft-brush biopsy Reducing the burden of SCC in FA - Protocol 6 Soft brush biopsies are much more accepted than traditional surgical biopsies. For the purpose of this study, the biopsies for the LOH analysis (described below) will always be performed and the cytological brush biopsy will be used in cases where there is a visible lesion. These minimally invasive procedures can be performed by the participant him- or herself with instructions or under the guidance of trained personnel. These procedures do not require standardized conditions, making them particularly well suited for data collection in FA family meetings or homes. The diagnostic value of the brush biopsy procedure has been validated in numerous studies 5–10. Figures 2 and 3 illustrate the soft brush biopsy procedures for the LOH and cytology analyses. Figure 2: Procedure for brush sampling in the oral cavity for LOH analysis. (A) The tissue is brushed superficially five times with a sterile Rovers Orcellex Brush (Rovers Medical Devices B.V., Oss, NL). (B) The brush containing the sample is placed in a 1.5 ml reaction tube filled with a fixative (CytoLyt from Hologic, Bedford, MA, USA). Each vial is labeled with a unique individual code. (C) Generally, samples from six separate high-risk areas of the oral cavity are collected from each participant. Each location is noted on the respective vial by the letters A-F. If visible lesions at different anatomical regions are present, they will be brushed as well (labeled with the letters beginning from G). Three separate brushes are used on each area and the steps above are repeated for each brush to provide controls. Figure 3: Procedure for cytological brush biopsy. (A) If visible lesions, which have not been biopsied previously, are present, each is brushed ten times with three separate Cervi Brushes (CellPath Ltd, Newtown, UK) (B) All brushes of the same lesion are then twisted into a tube containing an alcohol-based fixing solution. Reducing the burden of SCC in FA - Protocol 7 Each collection tube is labeled with the unique individual code. Step 4: Optional saliva and tissue collection Participants will have the option to provide saliva and an additional soft brush biopsy for other research in FA. Saliva will be collected through a small absorbent saliva pad to evaluate the presence of biomarkers for oral cancer detection. Depending on how much saliva the participant has, the pad may remain in the mouth of the participant for 5-10 minutes. The collected saliva is pressed out of the pad and into a tube containing an alcohol-based fixing solution. The additional soft brush biopsy will be used to evaluate the specific bacteria (microbiome) present in the mouth. Step 5: Data analysis Coded specimen will be sent to various laboratories for analysis. The laboratories will be blinded as to the identity of the subject. Genetic changes in the oral cavity: The brush biopsies taken from six high-risk locations within the oral cavity will be sent to the laboratory of Prof. Brakenhoff, Netherlands. For participants who have not received a bone marrow transplant, an LOH analysis will be conducted. In a preliminary analysis it was shown that genetic changes detectable by loss of heterozygosity analysis (LOH) are a strong and significant predictor of cancer in the FA population. LOH analysis describes allelic loss at chromosome arms 3p, 9p, 11q and 17p. Samples from participants who have already had a bone marrow transplant will be stored until a technique has been found to distinguish between the individuals’ and the bone marrow donor’s genetic information. Cytological analysis: Brush biopsy samples from participants with visible lesions will be sent to the laboratory of Prof. Biesterfeld at the University of Dusseldorf, Germany for cytological evaluation including DNA image cytometry to determine the proportion cells with DNA content suggestive of aneuploidy if suspicious lesions are detected. This test indicates chromosomal changes, which are known to be present in pre-cancerous and cancerous tissue10. Microbiome analysis: Brush biopsy samples from participants will be sent to the laboratory of Prof. Henrich at the University of Dusseldorf, Germany (Institute for Medical Microbiology and Hospital Hygiene) for microbiological evaluation. After DNA extraction and labeling, sequencing will be performed on the HiSeq2000 Illumina platform. Species- specific differences will be analyzed using TaqMan-PCR. Biomarker of oral cancer in saliva samples: The goal of the study is to evaluate in FA a panel of salivary oral cancer genomic and proteomic biomarkers shown to correlate with oral cancer development in non-FA individuals. The saliva sample of FA participants will be assayed via. qPCR and conventional ELISA to determine the levels of 7 pre-validated salivary oral cancer transcriptomic (IL8, IL1b, DUSP1, HA3, OAZ1, S100P, SAT) and 7 proteomic (MRP14, CD59, Catalase, M2BP, Profilin, IL8 and IL1 b) biomarkers. Reducing the burden of SCC in FA - Protocol 8 Step 6: Data reporting The cytology and cytometry results will be provided to the study investigators who will use the protected database containing unique identification codes to determine the identities of participants and send them their data reports. These reports will include: a diagram of the mouth indicating location of any visible lesion(s); high resolution photos of the lesion(s); results from the cytological analysis which indicates the morphology of the cells. In case suspicious cells were identified a DNA image cytometry report will be included and participants will be strongly encouraged to visit their local physician for further investigation. Due to heightened susceptibility to cancer, all participants will be encouraged to visit their local physicians often as per recommendations outlined in the Fanconi Anemia Guidelines for Diagnosis and Management, and especially if they develop a visible lesion that does not disappear within 2-4 weeks. Figure 4: Example of a cumulative report to a treating physician from five FA individuals of cytological smears. Included are the individual mouth maps as well as the pictures of the visible lesions. Step 7: Follow up Reducing the burden of SCC in FA - Protocol 9 Follow up visits, including mouth inspection, repeated brushing and collection of an updated participant medical history, will be proposed to participants at least every second year. In addition, we will ask an update on the status of visible oral lesions twice a year. Chapter 8 Data Storage and Confidentiality We take data security, privacy and confidentiality very seriously. Each individual’s contact information will be kept secure and confidential, and separated from the research data. To protect every participant’s privacy we will not share identifiable information with researchers and laboratories performing the assays. Instead we will use a unique code identifier on the specimen and on the data. This means that no organization outside the participant, the sponsor and study staff, and regulatory agencies will be able to link the data/health information to the participant. Note that all these personnel will have completed mandatory training on protection of human subjects in research. With each participant’s permission, we will transfer electronically the de-identified, coded research data to research databases such as the Sage Bionetworks’ data repository and analysis platform, Synapse. We will make it available to selected researchers with the goal to identify biomarkers that can be used as reliable predictors for the development of oral SCC or other FA Research. The research results will not include information that can link the data to the subject. With participant’s permission, the coded specimen leftovers will be frozen and stored by the laboratories for future analysis and research or methods development. Chapter 9 Potential Risks and Discomforts The sampling of cells from the oral cavity with a small brush is generally painless but it may give a tickling sensation. Some participants may show some redness of the brushed area, which usually disappears within a few hours or 1 to 2 days. In a European study, less than 10% of participants with visible lesions reported some slight pain and bleeding of the brushed lesions for a couple of minutes. Longer lasting bleedings, swelling or infections cannot be excluded but have not been observed so far. Only individuals who have known platelet counts above 20,000 or no bruising or unusual bleeding in the preceding 2 weeks will be accepted for local brushings. FA individuals with lower platelet counts may be included in the study provided that the brushing is done in a local dental practice or hospital. Receiving results from the cytology and cytometry tests may cause emotional stress, especially if signs for pre-cancerous or already cancerous lesions are identified. The study coordinator will recommend that participants get in contact with their local physicians to exclude or confirm cancer with the help of a classical scalpel biopsy. The participant will be informed that an ultimate cancer diagnosis can only be confirmed by a specialist based on the results of the local pathologist. In some cases, the pathology will not confirm an abnormality either because the test will show changes in cells earlier than a Reducing the burden of SCC in FA - Protocol 10