Royal Melbourne Hospital Biopsies of recurrent epithelial ovarian cancers for molecular profiling Can we do without them? Clare Scott MBBS PhD FRACP Medical Oncologist RMH, RWH Laboratory head, WEHI Royal Melbourne Hospital

WHY BIOPSY? Most women with high-grade epithelial ovarian cancer OC are treated with platinum/taxane chemotherapy Therapies targeting defined molecular defects are not yet approved Chemotherapy success is limited and it is imperative that we continue to drive recent molecular advances

Would you want Last Century’s medicine: surgery, carboplatin, taxol…..?

Last Century’s medicine: carboplatin, taxol… Last Century’s medicine: carboplatin, taxol….. and BTW more chemo doesn’t work (paraphrased by IMcN) ….two large randomised trials, one conducted by the Gynecologic Oncology Group (GOG) and the other by the European Organisation for Research and Treatment of Cancer (EORTC), have shown that administration of the taxane paclitaxel in combination with cisplatin significantly improves the duration of progression-free survival and overall survival in women with advanced epithelial ovarian cancer compared with cisplatin–cyclophosphamide therapy (McGuire et al, 1996; Piccart et al, 2000).

The “One size fits all approach” has failed The promise of personalized cancer care rests on our ability to truly understand and respond effectively to the biologic differences between patients How do we define these differences? Peppercorn et al J Clin Oncol 2010

Don’t treat very different subsets of epithelial ovarian cancer as if they are one disease Crum et al Curr Op Obst Gynaecol 2007 Vaughan et al, Nat Rev Ca, Oct 2011

Don’t treat one sub-type as if it is one disease Tothill et al Clinc Canc Res 2008 Helland et al PLoS One 2011 MYCN inhibitors (Norris / Haber / Chesler) ? Impact on mortality of this lethal subset of 15-20% of HG serous OC C1 C2 C3 C4 C5 C6 MYCN Serous OC Trastuzumab and Lapatinib: >50% reduction in the mortality of this lethal subset of 15-20% of Breast Cancer Perou et al Nature 2000 Breast Cancer

Design clinical trials to match treatment to molecular patterns Which means we need to design smaller trials that are tissue-driven to match a woman’s ovarian cancer to the treatment most likely to address the drivers and susceptibilities of her cancer What are the issues here?

What is Molecular Profiling What is Molecular Profiling? The study of specific patterns (“signature”) of: Protein Proteomics, IHC mRNA gene expression profiling, RNASeq, OncotypeDX, ISH for RNA DNA Sequencing, ISH for DNA amplific, WGS/NGS, incl CNV (Copy number variation) and SNPs Epigenome DNA Methylation, histone marks, ChIP-seq

Molecular Profiling The study of specific patterns, or signature of DNA, RNA and protein: ….and how this signature correlates molecular patterns with a phenotype of interest. Diverse molecular profiling platforms and strategies can be implemented during drug development to identify biomarkers useful for patient stratification.

“Detecting Gene Alterations in Cancers” Diverse molecular profiling platforms and strategies can be implemented during drug development to identify biomarkers useful for patient stratification.

Frequency of Somatic Gene Mutations in Epithelial Ovarian Cancer Approximately 10–20% of high grade ovarian cancers are associated with germline mutations in BRCA1/2 (Pal et al. 2005). Somatic alterations in BRCA1/2 and other genes associated with DNA repair are seen in approximately 50% of high grade ovarian cancers (TCGA 2011). Type I tumors have low grade serous, clear cell, endometrioid, and mucinous histological features: BRAF and KRAS somatic mutations are relatively common in these tumors, which may have important therapeutic implications. Type II tumors are high grade serous cancers of the ovary, peritoneum, and fallopian tube, high grade endometrioid and poorly differentiated ovarian cancers as well as carcinosarcomas: high levels of genomic instability with few common mutations, other than TP53, which is altered in over 90% of the cases (Kurman and Shih 2011; Landen, Birrer, and Sood 2008; TCGA 2011). PIK3CA and RAS signaling pathways are altered in 45% of the cases, but somatic mutations are rare and gene amplifications are far more common (TCGA 2011).

Frequency of Somatic Gene Mutations in Epithelial Ovarian Cancer The most common ‘actionable’ alterations with potential for small molecule targeted therapy in EOC tumors are in the PIK3CA/PTEN and KRAS/BRAF signaling pathways

Integrated genomic analyses of ovarian carcinoma The Cancer Genome Atlas Research Network high-grade serous ovarian cancer: TP53 mutations in almost all (96%); low prevalence but statistically recurrent somatic mutations in NF1, BRCA1, BRCA2, RB1, TP53, CSMD3, FAT3, GABRA6 and CDK12; 113 significant focal DNA copy number aberrations Promoter methylation events involving 168 genes 4 ovarian cancer transcriptional subtypes, 3 microRNA subtypes, 4 promoter methylation subtypes and 1 transcriptional signature associated with survival duration, BRCA1, BRCA2 and CCNE1 aberrations impact on survival Homologous recombination is defective in about half of HG-SOC NOTCH and FOXM1 signalling are involved

Design clinical trials to match treatment to molecular patterns What are the ethics to be considered by clinical resaerchers, clinicians (standard of care), patients and their supporters?

What is a biopsy? (What about ascites?) Ethics of Mandatory Research Biopsy for Correlative End Points Within Clinical Trials in Oncology What is a biopsy? (What about ascites?) Peppercorn et al J Clin Oncol 2010

Ethics of Mandatory Research Biopsy for Correlative End Points Within Clinical Trials in Oncology In the era of molecularly targeted therapies and continued poor outcomes, there is a pressing need to improve our understanding of the biology of cancer and to improve outcomes for future patients The promise of personalized cancer care rests on our ability to truly understand and respond effectively to the biologic differences between patients Peppercorn et al J Clin Oncol 2010

Ethics of Mandatory Research Biopsy for Correlative End Points Within Clinical Trials in Oncology Given the reality of constrained health care resources, there is a need to determine which patients may benefit from an intervention and which should be treated with an alternative strategy. There are moral dimensions to both our need for better treatments and better use of health care resources. Peppercorn et al

frequently under duress from their illness and Ethics of Mandatory Research Biopsy for Correlative End Points Within Clinical Trials in Oncology There is a need to acknowledge that patients with cancer seeking access to investigational therapy are frequently under duress from their illness and may be interested in trial participation primarily due to expectation of direct personal benefit. Peppercorn et al

Ethics of Mandatory Research Biopsy for Correlative End Points Within Clinical Trials in Oncology Any study using research biopsies in this population must be: well designed to address the scientific question, obtain the biopsy with minimal possible risk and ensure that research participants are fully informed of the risks, rationale, and requirements of the study, as well as of treatment alternatives. Peppercorn et al

Ethics of Mandatory Research Biopsy for Correlative End Points Within Clinical Trials in Oncology In addition, the scientific justification for a mandatory biopsy design as opposed to optional biopsy or use of clinical specimens for correlative end points must be carefully considered in trial design and review. Peppercorn et al

mandatory research biopsy for scientific end points. Ethics of Mandatory Research Biopsy for Correlative End Points Within Clinical Trials in Oncology We feel that if these guidelines are respected, an informed adult with cancer can both understand and voluntarily consent to participation in a clinical trial involving mandatory research biopsy for scientific end points. Such trials may be necessary to ultimately defeat cancer, and our patients can be valued and respected partners in this effort. Peppercorn et al

A major impediment to successful targeting of therapy in OC is poor availability to OC tissue, particularly upon OC recurrence. Biopsy of recurrent OC would massively improve our ability to attack the genetic drivers and susceptibilities of a woman’s OC and improve the possibility of “allocating” each patient to the most appropriate treatment available.

Without biopsying individual OC, improved survival may remain elusive.

When we do access HG-SOC tissue, molecular patterns of interest are obvious in vivo platinum DNA repair gene mutation No DNA repair gene mutation PDX Sensitive >/100d PARP inhibitors ? Immune therapy PDX Resistant <100d ? Model of BRCA1/2 mutation carriers resistant to PARP inhibitors ?anti-angiogenic therapy PDX Refractory PD on Platinum PARP inhibitor AND Oncogene inhibitors / BH3 mimetics Interestingly, HG-SOC #11 had an excellent response to platinum and does not harbour a DNA repair mutation, but we do know from Tothill gene expression analysis that this tumour has an immune phenotype which may account platinum sensitivity. PDX #27 is resistant to platinum, has no identified DNA repair gene defects, but may be benefiting from anti-angiogenic therapy. Monique Topp, WEHI

As we look ahead into the next century BILL GATES As we look ahead into the next century leaders will be those who empower others

by empowering future researchers and patients It is up to us to lead by empowering future researchers and patients with research repositories and processes that are relevant for this century of molecular technological advances No technology can rescue us from our cumbersome last century habits

Alternatives to solid tissue? What about at First Diagnosis? Back to the biopsy: Alternatives to solid tissue? Ascites A Blood test???? Timing? At relapse? What about at First Diagnosis?

Does everything end up in the blood? Back to the biopsy: Tumor heterogeneity Does everything end up in the blood? Just A Blood test????

Breast Cancer Circulating tumor DNA was successfully detected in 29 of the 30 women (97%) in whom somatic genomic alterations were identified; Circulating tumor DNA levels showed a greater dynamic range, and greater correlation with changes in tumor burden, than did CA 15-3 or circulating tumor cells.

Breast Cancer …when mutations could be detected in the primary tumor and subsequently in the plasma, the variation in the number of copies of circulating tumor DNA was reasonably correlated with responses to treatment. In addition, there was a significant relationship between the number of copies in blood and the ultimate prognosis of the patient.

Breast Cancer “…..could more reliably predict patients who might not need further therapy or identify those with localized breast cancer who would be adequately treated by lumpectomy alone.” “…..might be used to screen for recurrences in asymptomatic patients with previously diagnosed early-stage disease...” “….identification of new mutations in circulating tumor DNA over time might inform the clinician about tumor evolution and provide evidence to support new treatment targets not identifiable in the primary”

Pancreatic Cancer

Ovarian Cancer

Ovarian Cancer

tagged-amplicon deep sequencing (TAm-Seq) Ovarian Cancer Plasma of (OC) cancer patients contains cell-free tumor DNA that carries information on tumor mutations and tumor burden. Individual mutations have been probed using allele-specific assays, ….developed a method for tagged-amplicon deep sequencing (TAm-Seq)

a noninvasive “liquid biopsy” for personalized cancer genomics. Ovarian Cancer We identified mutations throughout the tumor suppressor gene TP53 in circulating DNA from 46 plasma samples of advanced OC patients. We demonstrated use of TAm-Seq to noninvasively identify the origin of metastatic relapse in a patient with multiple primary tumors. This low-cost, high-throughput method could facilitate analysis of circulating DNA as a noninvasive “liquid biopsy” for personalized cancer genomics.

decision-making on an individual basis. Ovarian Cancer Editor’s Summary Through several experiments, the authors were able to show that TAm-Seq is a viable method for sequencing decision-making on an individual basis. ….. once optimized, this ''liquid biopsy'' approach will be amenable to personalized genomics, where the level and type of mutations in ctDNA would inform clinical plasma of patients with less advanced cancers.

BUT it will take a while…(!!!!) to be able to glean all we need to know from the blood (a liquid biopsy) In the meantime, we need tissue biopsies to establish actionable molecular abberations (if not surgery itself…..)

ALLOCATE To move on from last century’s medicine Collecting tissue, and analyse it, to empower clinical researchers to design appropriate clinical trials for their patients to target therapy to drivers/susceptibilities of OC ALLOCATE To move on from last century’s medicine

ALLOCATE Australian Ovarian Cancer Assortment Trial Patient diagnosed with HG-EOC  Diagnostic sample collected and analysed Recurrent disease – sample collected and compared Can additional analysis inform choice of therapy? Iterative……

ALLOCATE Australian Ovarian Cancer Assortment Trial Professor Paul Waring (Principal Investigator) Professor Michael Quinn (Chair, governance committee) Dr Orla McNally (Surgical Lead) Professor David Bowtell (Genomics lead) Assoc. Prof. Linda Mileshkin (Clinical lead) Assoc. Prof. Clare Scott (Scientific lead) Professor Graham Taylor (Diagnostics lead) Dr. Jayesh Desai (CTA lead) Dr. Ben Tran (Tumour Board lead) Dr Kathryn Alsop Olga Kondrashova (PhD student)