Project Grant Application: Australian Head and Neck Cancer Society Research Foundation Professor Robert Ramsay, Peter MacCallum Cancer Centre, VCCC Melbourne; 5th May 2018

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1. Name of Lead Researcher: Professor Robert George RAMSAY, Differentiation and Transcription Laboratory Work Address: Peter MacCallum Cancer Centre, 305 Grattan Street, Melbourne Postal Address: Locked Bag #1, A’ Beckett Street, Melbourne 8006, AUSTRALIA. Telephone: (613) 8559 7095; Mobile: +61 414 296 352; Email: rob.ramsay@Petermac.org Roles and Positions: 2012 - Lab Head, Senior Principal Research Fellow, Peter MacCallum Cancer Centre, Member of

the Immunology Program, Cluster 4 (Renewed 2017). 2012 - Honorary Professor, Clinical Pathology Department, The University of Melbourne 2016 - Honorary Senior Research Fellow, Epworth Hospital. 2016 - Research Council, Peter MacCallum Cancer Centre 2017- Co- Head, GI Cancer Program, Peter MacCallum Cancer Centre

CV: RESEARCH Overview: I am a molecular biologist by training and specializing in the area of transcriptional regulation. I have progressively shifted his lab’s focus to the areas of tumour immunology, immune gene dysregulation and associated inflammation-mediated events that predispose to carcinogenesis. Major disease foci are colorectal (CRC), peritoneal carcinomatosis and adenoid cystic carcinoma (AdCC). The lab is tightly linked to the surgery division and participates in multidisciplinary team meetings with me being a named tram member and co-supervision of six clinical fellows. AWARDS • I received an award for outstanding achievement at the 6th World Congress on Advances in

Oncology and 4th International Symposium on Molecular Medicine, 2001, Crete, Greece. • NHMRC Senior Research Fellow Level B (2011-15), NHMRC Program Grant (2009-14). • Rothschild-Yvette Mayent-Institut Curie Award (2009) Fellowship to support Sabbatical

studies at the Curie Institute, Paris (~$40,000). • Life Membership to the Australian Society for Medical Research (2012- ). I am the tenth

person to receive this honour in the 51 year history of the Society. Other notable members are Sir Prof Gustav Nossal and Prof Peter Doherty.

• Award for Excellence in Cancer Research Education (2013) PMCC • NHMRC External Assessor ‘Outstanding Contribution’ Honour Roll 2014 • Australian Gastrointestinal Trials Group New Concepts Award 2015 ($3,000 prize: first to be

awarded to a non-clinician researcher. CURRENT COMPETITIVE RESEARCH SUPPORT 2014-2018 Project grant 4 years APP1085367 Deciphering the overlapping roles of SSB1 and

SSB2 in the regulation of haematopoiesis and intestinal homeostasis CIA: Khanna CIB: Lane CIC: Ramsay $958,728

2018-2021 Project grant 4 years APP1140147 Mechanisms of Resistance to Immunological Targeting of Primary and Metastatic Colorectal Cancers CIA: Ramsay: CIB: Desai: CIC: Heriot; CID: Michael $612,828

2016-18 TRP15008 Victorian Cancer Agency Domestic Government CIA- Ramsay $1,343,655

PUBLICATIONS REFEREED JOURNAL ARTICLES Citation analysis (according to ISI Web of Science and Google Scholar) Papers Published over Entire Career, 107 Primary, 5 Bock Chapter, 22 Invited Reviews, 2 Letters to the Editor, 1 Policy Document, 43 published conference proceedings Seven clinical trials involving my laboratory that have been conducted, initiated and/or funded since 2005; Total citations ~5,700; H-index 47; Publications since 2014 *papers relevant to project grant application.

Project Grant Application: Australian Head and Neck Cancer Society Research Foundation Professor Robert Ramsay, Peter MacCallum Cancer Centre, VCCC Melbourne; 5th May 2018

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1. Warrier, S.K., Kong, J.C., Guerra, G.R., Chittleborough, T.J., Naik, A., RAMSAY, R.G., Lynch, A.C. and Heriot, A.G. (2018) Risk Factors Associated With Circumferential Resection Margin Positivity in Rectal Cancer: A Binational Registry Study. Diseases of the Colon & Rectum 61: 433-440

2. *Kong, J.C., Guerra, G.R., Pham, T., Phillips, W., RAMSAY, R.G. and Heriot A.G. (2018) Prognostic impact of tumour infiltrating lymphocytes in primary and metastatic colorectal cancer: a systematic review and meta-analysis. Colorectal Disease, In Press

3. Guerra, G.R., Kong, J.C., Bernardi, M.P., RAMSAY, R.G., Phillips, W.A., Warrier, S.K., Lynch, A.C., Ngan, S.Y. and Heriot, A.G. (2018) Salvage Surgery for Locoregional Failure in Anal Squamous Cell Carcinoma. Dis Colon Rectum 61:179-186.

4. Carpinteri, S., Sampurno, S., Malaterre, J., Millen, M., Deane, M., Kong, J., Chittleborough, Heriot, A., Lynch, A.C. and RAMSAY, R.G. (2017) Abdominal delivery of humidified-warm carbon dioxide during open surgery ameliorates damage and hypoxia while accelerating wound healing,. British Journal of Surgery. 105:597-605

5. Hiller, J., Sampurno, S., Millen, R., Kuruvilla. N., Ho, K.W., RAMSAY, R.G., Riedel, B. (2017) Impact of celecoxib on inflammation during cancer surgery: A randomized clinical trial. Canadian Journal of Anesthesia 64:497–505

6. Dall, G., Vieusseux, J., Korach, K., Arao, Y., Hewitt, S., Hamilton, H., Boon, W.C., Simpson, E., RAMSAY, R.G. Anderson, R., Risbridger, G. and Kara Britt. (2017) Sca-1 labels a subset of estrogen responsive bipotential repopulating cells within the CD24+ CD49fhi mammary stem cell enriched compartment. Stem Cell Reports 8: 417-43

7. Dean, M., RAMSAY, R.G., Heriot A.G., Mackay J.R Hiscock, R., Lynch A.C. (2016) Warmed, Humidified CO2 Insufflation Benefits Intraoperative Core Temperature during Laparoscopic Surgery: a Meta-analysis. Asian Journal of Endoscopic Surgery 10: 128-136

8. *Millen, R., Malaterre, M., Cross, R.S., Carpinteri, S., Desai, J., Tran, B., Darcy, P., Gibbs, P., Sieber, O., Zeps, N., Waring, P., Fox, S., Pereira, L. and RAMSAY, R.G. (2016) Immunomodulation by MYB is associated with Tumor Relapse in Patients with Early Stage Colorectal Cancer. OncoImmunology 5 (7), e1149667

9. Mitra, P., Yang, R-M., Sutton, J. RAMSAY, R.G. and Gonda T.J. (2016) CDK9 Inhibitors Selectively Target Estrogen Receptor-Positive Breast Cancer Cells Through Combined Inhibition of MYB and MCL-1 Expression. OncoTarget 7: 9069-9083

10. Malaterre, M., Pereira, L., Putoczki. T., Millen, R., Paquet-Fifield, S., Germann, M., Liu, J., Cheasley, D., Sampurno, S., Stacker, S.A. Achen, M.G., Ward, R.L., Waring, P., Mantamadiotis, T., Ernst, M. and RAMSAY, R.G. (2016) Intestinal-specific Activatable-Myb initiates colon tumorigenesis in mice. Oncogene 35: 2475-2484

11. Fong, C.Y., Gilan, O.,Lam, E., Rubin, A.F., Ftouni, S., Tyler, D., Stanley, K., Sinha, D., Yeh, P., Morison, J., Giotopoulos, G., Lugo, D., Stanley, J.P., Lee, C-W., Carpenter, C., Gregory, R., RAMSAY, R.G., Lane, S.W., Abdel-Wahab, O., Kouzarides, T., Johnstone, R. Dawson, S-J., Huntly, B.J.P., Prinjha, RK., Papenfuss, A.T. and Dawson, M.A. (2015) BET inhibitor resistance emerges from leukaemia stem cells. Nature 525: 538-542

12. Cheasley, D, Pereira, L, Sampurno, S., Sieber, O., Jorissen, R., Xu, H., Germann., M., Yuqian, Y., Malaterre, J. and RAMSAY, R.G. (2015) Defective Myb Function Ablates Cyclin E1 Expression and Perturbs Intestinal Carcinogenesis. Molecular Cancer Research 13:1185-96

13. Pereira, L.A., Hugo, H.J., Malaterre, J., Xu, H, Sonza, S., Cures, A., Purcell, D.F.J., Ramsland, P.A., Gerondakis, S., Gonda, J.J. and RAMSAY, R.G. (2015) MYB elongation is regulated by the binding of NFκB p50 to an intronic RNA stem-loop structure. PlosOne 10:(4)

14. Flanagan, D.J., Phesse, T.J., Nick Barker, N., Schwab, R.H.M., Amin,N., Malaterre, J., Stange, D.E., Nowell, C.J., Currie, S.A., RAMSAY, R.G., Sansom, O, Ernst, M.R.W., Clevers, H. and Vincan, E. (2015) Frizzled7 functions as a Wnt receptor in intestinal epithelium stem cells. Stem Cell Reports 4:759-67

Project Grant Application: Australian Head and Neck Cancer Society Research Foundation Professor Robert Ramsay, Peter MacCallum Cancer Centre, VCCC Melbourne; 5th May 2018

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15. Carpinteri, S., Sampurno, S., Bernardi, M., Germann, M., Malaterre, J., Heriot, A., Chambers, B.A., Mutsaers, S.E., Lynch, A.C. and RAMSAY, R.G. (2015). Peritoneal Tumorigenesis and Inflammation are ameliorated by Humidified-Warm Carbon Dioxide Insufflation in the Mouse. Annals of Surgical Oncology Suppl 3:S1540-7

16. Sampurno, S., Cross, R.S., Pearson, H., Kaur, P., Malaterre, J. and RAMSAY, R.G. (2015) Myb via TGFβ is required for Collagen Type 1 Production and Skin Integrity. Growth Factors 33:102-12

17. *Cross, R.S., Malaterre, J., Davenport, A.J., Carpinteri, S., Anderson, R.L., Darcy, P.K. and RAMSAY, R.G. (2015) Therapeutic DNA vaccination against colorectal cancer by targeting the MYB oncoprotein. Clinical & Translational Immunology 4: e30

18. Xu, H., Yan, Y., Deb, S., Rangasamy, D., Germann, M., , Malaterre, J., Eder, N.C.,Ward, R.L., Hawkins, N.J., Tothill, R., Chen, L., Mortensen, N.J., Fox, S.P., J McKay, M.J. and RAMSAY, R.G. (2014). Cohesin Rad21 mediates LOH and is up-regulated via Wnt promoting transcriptional dysregulation in GI tumors. CELL Reports 9: 1-17

Reviews 1. Kong, J.C., Guerra, G.R., Warrier, S.K., RAMSAY, R.G. and Heriot, A.G. (2017) Outcome

and Salvage Surgery Following “Watch and Wait” for Rectal Cancer after Neoadjuvant Therapy: A Systematic Review. Diseases of the Colon & Rectum 60:335-345

2. Chittleborough, T.J., Kong, J.C., Guerra G.R., RAMSAY, R.G. and Heriot, A.G. (2017) Colonoscopic Surveillance: Quality, Guidelines and Effectiveness. ANZ Journal of Surgery 88:32-38

3. RAMSAY, R.G. and Abud, H. (2017) Exploiting induced senescence in intestinal organoids to drive enteroendocrine cell expansion. Stem Cell Investigation 4:36

4. Kong, J.C., Guerra, G.R.., Warrier, S.K., RAMSAY, R.G. and Heriot, A.G. (2017) Outcome and Salvage Surgery Following "Watch and Wait" for Rectal Cancer after Neoadjuvant Therapy: A Systematic Review Diseases of the Colon & Rectum 60:335–345

5. Ryan, J.E., Warrier, S.K., Lynch, A.C., RAMSAY, R.G., Phillips, W.A, and Heriot, A.G. (2016) Predicting pathological complete response to neoadjuvant chemoradiotherapy in locally advanced rectal cancer: a systematic review. Colorectal Disease 18:234-246

6. *Gonda, T.J. and RAMSAY, R.G. (2016) Adenoid Cystic Carcinoma can be driven by MYB or MYBL1 Rearrangements: New Insights into MYB and Tumor Biology. Cancer Discover, 6:125-127

7. *Gonda, T.J. and RAMSAY, R.G. (2015) Directly targeting transcriptional dysregulation in cancer. Nature Reviews Cancer 15:686-694

8. Bernardi, M.P., Heriot, A.G., RAMSAY, R.G. and Phillips WA (2015) Molecular Biology of Anal Squamous Cell Carcinoma: Implications for Future Research and Clinical Intervention Lancet Oncology 16:e611-e621

2. Affiliated Institutions Peter MacCallum Cancer Centre, Research Division ; Peter MacCallum Cancer Centre is a publicly funded tertiary referral hospital for Victorian patients with cancer with ~2,700 staff embedded with the Victorian Comprehensive Cancer Centre in Parkville. The Research Division is the largest organization specifically researching cancer in Australia. The Division has approximately 580 laboratory and clinical researchers of whom ~100 are graduate students. Thirty-eight group leaders form the faculty core. The closer geographical connection with the University of Melbourne (in which I have two honorary Professorial Appointments) and the VCCC continue to grow and have created better opportunities to recruit and teach students while expediting collaborations.

3. Team Members I have three post-docs associated with the lab, 6 PhD/DMSc students of whom 5 are colorectal surgeon trainees, one research assistant and a part time technical assistant. Three clinician researchers are currently writing up their theses. I have three research fellows, Drs Roth, Pereira and

Project Grant Application: Australian Head and Neck Cancer Society Research Foundation Professor Robert Ramsay, Peter MacCallum Cancer Centre, VCCC Melbourne; 5th May 2018

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Xu and an expert RA Ms Sampurno. Dr Roth has brought FACS skills, some industry training and immunological insights that underpin the MYB & RAS vaccine trials. She is the point person for the TARGOVAX trial in my lab. Along with Dr Roth I have a work-experienced PhD student (Ms Millen) who spent two years as a RA developing immunology skills in A/Prof Visvanathan’s Lab (St Vincent’s Hospital). Her project is focused on metastatic CRC and immunological modulation of the liver microenvironment. Dr Pereira is an accomplished molecular biologist who engineered the humanize MYB vaccine and he is charged with clinical compliance and its manufacture supported by the VCA and AdCC Foundation (USA). Molecular biologist Dr Xu is responsible for genomic evaluation of tumours from our patients. I support a research nurse (Ms Cain) who is responsible for the collection of clinical materials from the surgery theatre and blood samples from the wards as part of our trials activities. Funded surgery trial is supported at the Epworth Hospital (where I have an appointment) by Dr Stathopoulos. Clinician researchers with whom I closely collaborate and publish with – Dr Desai, Prof Solomon, A/Prof Michael, Prof Heriot, A/Prof Lynch, Dr Hiller complete this unique research team. Post-Doctoral Fellows; Dr Lloyd Pereira, Dr Sara Roth, Dr Huiling Xu (on secondment to Molecular Pathology, PMCC) Clinical Fellows/PhD/DMs Students; Ms. Rosemary Millen, BSc Hons, Dr Kasmira Wilson, MBBS, Dr Toan Pham, MBBS, Dr Glen Guerra, MBBS, Dr Vignesh Narasimhan, MBBS Dr Josesh Kong, MBBS (Thesis submitted), Dr Timothy Chittleborough, MBBS (Thesis submitted) Dr Meera Dean, MBBS (Thesis to be submitted, July 2018) Research Assistant; Ms Shienny Sampurno. Research Nurse; Ms Helen Cain, PMCC For this project we will be working closely with Professor Göran Stenman, Director Sahlgrenska Cancer Center, Department of Pathology and Genetics, University of Gothenburg who is an international expert in AdCC pathology and the head of the lab that discovered the role of MYB fusions in the pathogenesis of ADCC. 4. Detailed budget and timeline This project is envisaged to take two years and is broken down annually. Activity Reagents/Core costs Year One Year Two Propagation of AdCC tumouroids (aiming for 4-6 biopsy samples)

Biopsies collected from OR, processed in antibiotic-rich media and then propagated in growth factor supplemented media

$4000 $4000

Expansion of tumour infiltrating lymphocytes(TILs) and FACS analysis

Antibodies, FACS time and IL-2 mediated expansion of TILs

$2000 $2000

Measuring immune function combined with FACS analysis and checkpoint blockade assays and cytokine production

Surface marker analysis; requires a panel of 24 fluoro-tagged antibodies, and cytokine bead assay kits.

$2000 $2000

OPAL staining and analysis Multiple cell surface stains and nuclear markers CD3, CD4, CD8, FoxP3, PD-L2, PD-1.

$2000 $2000

Total $10,000 $10,000 These costs are estimates based upon the experience of processing of >80 GI cancer samples and proof-of-principle studies on an actual AdCC biopsy. These procedures are very labour intensive and usually require the efforts of several scientists at any one time.

Project Grant Application: Australian Head and Neck Cancer Society Research Foundation Professor Robert Ramsay, Peter MacCallum Cancer Centre, VCCC Melbourne; 5th May 2018

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5. Project summary

Novel Immunological Targeting of the Rare Cancer Adenoid Cystic Carcinoma Aims: (a) To establish 3D tumouroids from fresh adenoid cystic carcinoma (AdCC) biopsies or surgical resection specimens along with the expansion of tumour infiltrating lymphocytes (TILs). (b) These will be reconstituted together to evaluate the functional cytotoxicity of the TILs against the matched tumour with and without immune checkpoint blockade antibodies. (c) To define the topological landscape of immune cells within, at the AdCC tumour margin and in the stroma using multiplex immuno-fluorescence (OPAL).

Significance: AdCC is considered to be a tumour type that has a low immune infiltrate and is presumed to invoke a weak anti-tumour response. The question is whether these cells (TILs) have any function and if expanded might exhibit tumour cytotoxicity. AdCC express aberrant levels of the programmed cell death ligand, PD-L2 which is uncommon in other tumours but has the capacity to shut-down TIL activity. We have developed an assay platform and capability to directly test TIL function. In addition we are in a position to add check point blockade antibodies that are in current clinical use to modulate the interaction between PD-L1 and PD-1 receptor that resides on the cell surface of some TILs.

Expected outcomes: If this project achieves its goals we will for the first time define the abundance and function of AdCC TILs and test immunotherapy approaches to reactivate their cytotoxic function. These approached have the potential to improve current clinical trials being conducted at Peter Mac for patients with AdCC and more broadly other head and neck tumours that may have low TIL abundance that might be subject to enhancement by immunomodulation.

6. Detailed description of project Adenoid Cystic Carcinoma (AdCC) is a rare malignancy arising in the salivary gland, breast and other organs and is characterised by the display of a slow, relentlessly progressive course. Approximately 1,200 Americans and around 85 Australia are diagnosed per year (Australian Institute for Health and Welfare). Patients are often young to middle aged and their management is a long-term and expensive proposition. The mainstay treatment for AdCC is surgical resection followed by post-operative radiotherapy. This initially leads to a high rate of local control with an expected 5-year survival rate of up to 89%. However for a significant proportion of patients the slow, relentless progression of this cancer commonly leads to local relapse and the development of distant metastases. Most patients will succumb to the disease within 10-15 years. For patients with this slow recurring cancer, there are currently no treatments available and progression to fatal metastatic disease is an inevitable feature of a patient’s clinical course. Because of its rarity and distinct clinical characteristics, AdCC remains an understudied malignancy and this has resulted in few evidence-based studies aimed at developing therapies to treat AdCC.

Recently there has been an invigorated effort to understand the molecular drivers of AdCC whereby a key finding has been the discovery that the proto-oncogene MYB is the driver of this cancer 1-4. (see Figure 1). The Ramsay lab has led the field in “MYB and Cancer” 5 and has developed a strong clinical translational research program on AdCC. Thus, there is a significant clinical need to offer novel targeted treatment options for these patients and have we developed a vaccine strategy that targets MYB in patients with AdCC aimed at addressing this clinical need. This project application is to bring addition insights into the immunotherapy opportunities to treat AdCC using assays that were not developed at the time when the clinical trial was funded. For instance it was not known at the time that AdCC cell express immune modulatory PD-L2 ligand (see Figure 2) and we did not have an assay platform to integrate the functional role of PD-L2 (see Figure 3). Similarly the ability to immuno-profile multiple immune cells within the same tumour section by a new technology called OPAL was not in place (see Figure 5). But now all these elements can be explored and these form the basis of this research proposal.

Project Grant Application: Australian Head and Neck Cancer Society Research Foundation Professor Robert Ramsay, Peter MacCallum Cancer Centre, VCCC Melbourne; 5th May 2018

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Figure 1. Schematic showing the two major types of MYB and MYBL1 fusion proteins seen in AdCC, each of which are generated by translocations involving the NFIB gene. These drive very similar transcriptional programs. Gonda & Ramsay Cancer Discovery 2016;6:125-127.

New Therapeutic Opportunities for the Treatment of AdCC – a DNA Vaccine: A striking feature of the AdCC genomic landscape is its relatively “quiet” signature of structural and mutational changes, a trait that is reminiscent of paediatric haematological cancers 3. However, a sentinel molecular feature of AdCC is the high-level expression of a range of gene fusions between the oncogenic transcription factor MYB family members (MYB and A-MYB) and the developmental transcription factor NFIB (Persson et al., 2009; Mitani et al., 2011; West et al., 2011). This gene has been the focus of attention of the Ramsay lab for several decades and now finds the lab as the world leader in this field 6 (see Figure 1). We have a clinical trial starting at the VCCC that involves a vaccine that targets AdCC as well as bowel cancer (NCT03287427).

Another feature of AdCC is the high expression of the immune modulatory ligand, PD-L2 7,8. Unpublished data from our lab and that of collaborator and AdCC pathologist Prof Stenman (Sweden) indicate that PD-L2 is a target gene of MYB and A-MYB in AdCC while highly related gene PD-L1 is a MYB target gene in colorectal cancer. Both PD-L1 and PD-L2 bind to PD-1 receptor on TILs to block cytotoxic function (see Figure 4). Relatively low expression of PD-L2 in AdCC is a predictor of poor outcomes in patients 8 suggesting that immune modulation is in action in AdCC. These interactions are interrupted by anti-PD-1 antibodies such as KeytrudaTM. Our MYB vaccine is being combined with an anti-PD-1 antibody. The immune response to bowel cancer as a prototype to predict immune responses in AdCC: There is strong evidence that a poor immune response against bowel cancer, as defined by the absence of tumour infiltrating lymphocytes (TILs), particularly CD8+ T-cells, within the primary tumour is a strong predictor of patient relapse 9-11. This has led to the concept that a reawakening of the latent, immune response may lead to therapeutic opportunities to improve CRC tumour control. To this end immune checkpoint inhibitors that target the programmed death receptor, PD-1 and its ligand PD-L1, of the immune system have become exciting and effective clinical targets for the

Project Grant Application: Australian Head and Neck Cancer Society Research Foundation Professor Robert Ramsay, Peter MacCallum Cancer Centre, VCCC Melbourne; 5th May 2018

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Figure 2. (A) IHC of PD-L2 in AdCC. (B) Survival outcome of pateints with AdCC is chematic showing the two major types of MYB and MYBL1 fusion proteins seen in AdCC, each of which are generated by translocations involving the NFIB gene. These drive very similar transcriptional programs. Chang et al, OncoTargets and Therapy, 2017:10 2983–2992.

A B

PD-L2 Immunohistochemistry

treatment of some CRC. One study demonstrated the potency (~70% response rate) of immune checkpoint blockade in microsatellite unstable/high (MSI-H) CRC and other cancers using anti-PD-1 antibodies 12,13. We believe that increasing TILs in AdCC will improve tumour control. We posit that because AdCCs typically have low TIL densities that there is an opportunity to get them back or indeed induce new TILs. That is the purpose of the MYB vaccine. Novel assays that can evaluate the immune response in AdCC tumours: We have developed a novel pre-clinical platform that allows the rapid growth of primary tumour cells and the TILs that are buried within the tumour. This work has been conducted mostly with bowel cancer biopsies but

very recently we have had success in establishing the same tumouroids and TILs from AdCC biopsies. This is major breakthrough for us and based upon our international networks in the AdCC research community (AdCC Foundation USA, MYB researchers-Salivary Gland Tumors meeting in Florida, Oct 2017) this is a genuine research advance. With this platform now in place (see Figure 3).we can start to directly evaluate the immune responses to AdCC and then explore approaches to increase the response. This concept is very relevant to the MYB vaccine and other immunotherapies as we can now consider measuring the immune response at baseline and after therapies as well as test new agents in the assay before thinking about trying them in patients.

Experiments Aims (a) and (b): We will establish between four to six tumouroid lines from patients over 18 months based upon the current patient referral and treatment pattern at the VCCC. We will expand TILs from these at the same time. Interestingly although as anticipated the yield of TILs was low in a proof-of-principle sample we processed recently we can indeed expand these TILs and they exhibit cytotoxicity. We will use FACS to profile the phenotype of the TIL subsets and assess the cytokine production by these by FACS (intracellular) and by cytokine bead array (secreted). These assays are in place in the lab (Kong, et al under revision).

These assays are resource intensive but highly informative. As we have extensive experience with the techniques assessing rectal (and anal) cancer tumouroid/TIL assays we will be able to compare the AdCC profiles and activity against other cancer types. When we establish the tumouroid they become a renewable resource that can be cryopreserved and used for next generation sequencing, etc. The TILs can also be stored and intensely interrogated for immune gene expression signatures and T- cell receptor repertoire. These are important future questions that will complement the studies associated with the MYB vaccine clinical trial and will be important to the field.

Project Grant Application: Australian Head and Neck Cancer Society Research Foundation Professor Robert Ramsay, Peter MacCallum Cancer Centre, VCCC Melbourne; 5th May 2018

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Figure 3. TIL-mediated killing of patient-matched tumor cells in vitro. (A) Rectal tumor-derived tumoroids (~500 cells) embedded in Matrigel are readily generated ex vivo from surgical biopsies (i, ii) and can be combined with patient-match tumor-infiltrating lymphocytes (TILs) (iii) to allow measurement of patient-specific cytotoxicity (iv, v). (B) Morphological examination of tumoroids demonstrates their spherical structure with a smooth exterior surface on scanning electron microscopy; Three examples from different patients are presented (scale bar = 50 m). Add of TILs (rendered pink; pink arrow) to patient-matched tumoroids leads to morphological irregularities on the tumoroid cell surface and progressive structural disruption (images at 8 h). (C) Tumoroids (black arrow) at time 0 show no uptake of propidium iodide (PI) indicated by red fluorescence or activation of caspase 3/7 (apoptosis) indicated by green fluorescence in the absence of TILs. (D) By 24 h, apoptosis has initiated. (E) By 48 h, PI staining is evident consistent with tumoroid disruption and death. (F-H) Not all tumoroids (white arrow) are subject to TIL-mediated killing as demonstrated in this series of images, suggesting intrinsic resistance to immune mediated killing is a characteristic in some patients with rectal cancer. (Kong et al, under revision).

Project Grant Application: Australian Head and Neck Cancer Society Research Foundation Professor Robert Ramsay, Peter MacCallum Cancer Centre, VCCC Melbourne; 5th May 2018

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Figure 4. Model of the low immunogenicity of tumours like AdCC and the utility of immune check point blockade antibodies.

Figure 5. Multispectral IHC analysis by inform software (OPAL). This technology allows the quantitation and special evaluation of immune cell sub sets from FFPE process colorectal tumour samples. We will use this to examine AdCC biopsy sections before and after A-MYB vaccination to track changes in the immunological landscape in the context of immunotherapy.

Experiment Aim (c): Our collaborator in Sweden, Prof Goran Stenman is a world expert in AdCC pathology and together we have agreed to help us evaluate the immune landscape in tissue blocks he and we have built up. We have a very sophisticated method called “OPAL staining” that allows us to measure the abundance, activation state and location of TILs within (AdCC) tumour sections 14. We have sourced tissue blocks from Peter Mac pathology and the University of Gothenburg, Sweden. We have patient outcome data that match these FFPE blocks. OPAL allows the identification and quantitation of cell subsets that are distributed within a tissue. This has been done by us in a series of rectal cancer blocks from patients pre- and post-neoadjuvant chemoradiotherapy (not shown). An example of the images that are generated by using OPAL is shown in Figure 5. We will evaluate by OPAL the immune landscape of at least ten AdCC samples for CD, CD4, CD8, FoxP3, PD-L2 and PD-1.

Project Grant Application: Australian Head and Neck Cancer Society Research Foundation Professor Robert Ramsay, Peter MacCallum Cancer Centre, VCCC Melbourne; 5th May 2018

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Summary: The project has three aims that are directed to defining the immune landscape of AdCC by OPAL along with measuring the immune function of TIL using a bespoke assay platform developed by the Ramsay lab. These have not been done before and will provide valuable insights into the future use of immunotherapies in this very difficult to treat rare, predominately head and neck cancer. References: 1. Brayer KJ, Frerich CA, Kang H, Ness SA. Recurrent Fusions in MYB and MYBL1 Define a

Common, Transcription Factor-Driven Oncogenic Pathway in Salivary Gland Adenoid Cystic Carcinoma. Cancer Discov 2016;6:176-87.

2. Gonda TJ, Ramsay RG. Adenoid Cystic Carcinoma Can Be Driven by MYB or MYBL1 Rearrangements: New Insights into MYB and Tumor Biology. Cancer discovery 2016;6:125-7.

3. Ho AS, Kannan K, Roy DM, et al. The mutational landscape of adenoid cystic carcinoma. Nat Genet 2013;45:791-8.

4. Mitani Y, Liu B, Rao PH, et al. Novel MYBL1 Gene Rearrangements with Recurrent MYBL1-NFIB Fusions in Salivary Adenoid Cystic Carcinomas Lacking t(6;9) Translocations. Clin Cancer Res 2016;22:725-33.

5. Ramsay RG, Gonda TJ. MYB function in normal and cancer cells. Nature reviews Cancer 2008;8:523-34.

6. Gonda TJ, Ramsay RG. Directly targeting transcriptional dysregulation in cancer. Nature reviews Cancer 2015;15:686-94.

7. Sridharan V, Gjini E, Liao X, et al. Immune Profiling of Adenoid Cystic Carcinoma: PD-L2 Expression and Associations with Tumor-Infiltrating Lymphocytes. Cancer Immunol Res 2016;4:679-87.

8. Chang H, Kim JS, Choi YJ, et al. Overexpression of PD-L2 is associated with shorter relapse-free survival in patients with malignant salivary gland tumors. Onco Targets Ther 2017;10:2983-92.

9. Galon J, Costes A, Sanchez-Cabo F, et al. Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science 2006;313:1960-4.

10. Galon J, Fridman WH, Pages F. The adaptive immunologic microenvironment in colorectal cancer: a novel perspective. Cancer research 2007;67:1883-6.

11. Millen R, Malaterre M, Cross RS, et al. Immunomodulation by MYB is associated with Tumor Relapse in Patients with Early Stage Colorectal Cancer. OncoImmunology (accepted) 2016.

12. Le DT, Uram JN, Wang H, et al. PD-1 Blockade in Tumors with Mismatch-Repair Deficiency. The New England journal of medicine 2015;372:2509-20.

13. Overman MJ, McDermott R, Leach JL, et al. Nivolumab in patients with metastatic DNA mismatch repair-deficient or microsatellite instability-high colorectal cancer (CheckMate 142): an open-label, multicentre, phase 2 study. Lancet Oncol 2017;18:1182-91.

14. Dixon AR, Bathany C, Tsuei M, White J, Barald KF, Takayama S. Recent developments in multiplexing techniques for immunohistochemistry. Expert Rev Mol Diagn 2015;15:1171-86.

7. Ethics Approvals and letter of institutional support attached 8. Grant funds to Professor Robert George RAMSAY, Differentiation and Transcription

Laboratory Peter MacCallum Cancer Centre, 305 Grattan Street, Melbourne, Postal Address: Locked Bag #1, A’ Beckett Street, Melbourne 8006, AUSTRALIA. Telephone: (613) 8559 7095; Mobile: +61 414 296 352; Email: rob.ramsay@Petermac.org

Peter MacCallum Cancer Centre Locations St Andrews Place East Melbourne East Melbourne Victoria Bendigo Locked Bag 1 A’Beckett Street Box Hill Victoria 8006 Australia Moorabbin Sunshine Phone +61 3 9656 1111 Fax +61 3 9656 1400 ABN 42 100 504 883

www.petermac.org

Patron: The Honourable Linda Dessau, AM – Governor of Victoria

3rd May 2018 The Australian Research Foundation of The ANZHNCS PO Box 576 Crows Nest NSW 1585 Dear Sir/Madam, Re: Letter of Support for Prof Robert Ramsay’s application to the Australian Head and Neck Cancer Society Research Foundation Peter Mac has relocated to a new state-of-the-art facility providing world’s best cancer care and research facilities. The Cancer Research Division at Peter Mac, the largest laboratory-based cancer research effort in Australia, focuses on delivering research excellence across the range of laboratory-based and clinical disciplines. Located amongst a rich concentration of world-renowned educational, research and clinical facilities, known as the Parkville Precinct, provides an ideal collaborative environment for research. At Peter Mac alone there are more than 29 laboratories, 500 scientists, clinicians and post graduate students accessing advanced technology platforms including: Animal Care and Use Program; Bioinformatics; Cryopreservation; Experimental Animal Imaging; Flow Cytometry; Victorian Centre for Functional Genomics (VCFG); Research Laboratory Support Services; Microscopy & Histology; Molecular Genomics; Victorian Cancer Bio-Bank (Tissue Bank). Peter Mac’s Professional Services team provides administrative support for grant submissions, ethics applications, budget assessment, human resources and student coordination. In addition, we have formal mentoring procedures in place for new grant holders that involve faculty members and members of the research division executive committee. Translating scientific discoveries from the laboratory to the clinic is at the forefront of our research objectives and resources provided by our Molecular Pathology Department, Department of Biostatistics and our Clinical Trials Unit facilitate rapid research translation. Prof. Robert Ramsay has access to all of the above assistance, laboratory and support facilities including all of the above state of the art core facilities. In addition, the Ramsay lab is fully equipped to carry all the biochemical and molecular studies, and cell biology experimentations that are proposed in this project “Novel Immunological Targeting of the Rare Cancer, Adenoid Cystic Carcinoma”.

This includes incubators to growth cancer cells, biological hoods, microscopes and other relevant equipment. The equipment is available for Prof. Ramsay and his team to utilize for this project.

As Interim Associate Director Laboratory Research, I agree to host the above scheme and provide Prof. Robert Ramsay with my full support. I have approved and authorized him to submit his application. Yours sincerely

Professor Richard Pearson Interim Associate Director Laboratory Research Head, Cancer Signalling Laboratory Head, Oncogenic Signalling and Growth Control Program Sir Peter MacCallum Department of Oncology and Department of Biochemistry and Molecular Biology, University of Melbourne

Peter MacCallum Cancer Centre Locations 305 Grattan Street Melbourne Melbourne Victoria Bendigo 3000 Australia Box Hill

Moorabbin Postal Address Sunshine Locked Bag 1 A’Beckett Street Victoria 8006 Australia Phone +61 3 8559 5000 Fax +61 3 03 8559 7379 ABN 42 100 504 883

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MEMORANDUM To: Prof Rob Ramsay

From: Ethics Committee Secretariat

CC: Dr Toan Pham

Date: 26 February 2018

Re: Peter Mac Project No: 14/185 Study Title: Identification of novel prognostic markers and therapeutic targets in colorectal and adenoid cystic cancers

We are pleased to advise that the amendment submission dated 29 January 2018, has received ethical approval from the Peter MacCallum Cancer Centre Human Research Ethics Committee. The amendment also satisfies Peter Mac Research Governance requirements and may now be conducted at this site. The following documents have been reviewed and approved:

Document Version/Edition Date

Protocol 3 11 February 2018

Should you have any questions about your project please contact the Ethics Committee Secretariat on (03) 8559 7540 or ethics@petermac.org. Submission guidelines, forms and standard operating procedures are available on the ethics website: http://www1.petermac.org/Ethics/default.htm Kind Regards,

Ethics Committee Secretariat

The Peter MacCallum Cancer Centre Human Research Ethics Committee and it’s subcommittees are organised and operate in accordance with the National Health and Medical Research Council’s (NHMRC) National Statement on Ethical Conduct in Research Involving Humans (2007), and all subsequent updates, and in accordance with the Note for Guidance on Good Clinical Practice (CPMP/ICH/135/95) and the Health Privacy Principles described in the Health Records Act 2001 (Vic) and Section 95A of the Privacy Act 1988 (and subsequent Guidelines). Please be advised that the Principal Researcher and Associate Researchers named on the application did not participate in deliberative discussions or decision-making regarding the project.

Peter MacCallum Cancer Centre Locations 305 Grattan Street Melbourne Melbourne Victoria Bendigo 3000 Australia Box Hill

Moorabbin Postal Address Sunshine Locked Bag 1 A’Beckett Street Victoria 8006 Australia Phone +61 3 8559 5000 Fax +61 3 03 8559 7379 ABN 42 100 504 883

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MEMORANDUM To: Prof Sandy Heriot

From: Ethics Committee Secretariat

CC: Toan Pham & Sam McKeown

Date: 24 January 2018

Re: Peter Mac Project No: 14/61 Study Title: Rectal Tissue Bank

We are pleased to advise that the amendment submission dated 04 December 2017, has received ethical approval from the Peter MacCallum Cancer Centre Human Research Ethics Committee. The amendment also satisfies Peter Mac Research Governance requirements and may now be conducted at this site. The following documents have been reviewed and approved:

Document Version/Edition Date

Protocol 1.4 04 January 2018

Rectal Participant Information Sheet and Consent Form 2.2 04 January 2018

Salivary Gland Participant Information Sheet and Consent Form 2.3 04 January 2018

Should you have any questions about your project please contact the Ethics Committee Secretariat on (03) 8559 7540 or ethics@petermac.org. Submission guidelines, forms and standard operating procedures are available on the ethics website: http://www1.petermac.org/Ethics/default.htm Kind Regards,

Ethics Committee Secretariat

The Peter MacCallum Cancer Centre Human Research Ethics Committee and it’s subcommittees are organised and operate in accordance with the National Health and Medical Research Council’s (NHMRC) National Statement on Ethical Conduct in Research Involving Humans (2007), and all subsequent updates, and in accordance with the Note for Guidance on Good Clinical Practice (CPMP/ICH/135/95) and the Health Privacy Principles described in the Health Records Act 2001 (Vic) and Section 95A of the Privacy Act 1988 (and subsequent Guidelines). Please be advised that the Principal Researcher and Associate Researchers named on the application did not participate in deliberative discussions or decision-making regarding the project.