Cancer Moonshot Connecting International Liquid Biopsy Efforts Through Academic Partnership

Abstract

The Kuhn Laboratory at the University of Southern California and the Dive Laboratory at the Cancer Research UK’s Manchester Institute are teaming up to apply new cancer cell detection technology to identify patients who will progress after initial treatment. Researchers will take a simple blood sample to identify early those patients whose cancer has returned, while analyzing circulating tumor cells (CTCs) in great detail, providing new clues on the most effective therapy for the patient’s cancer.

EARLY DIAGNOSIS OF CANCER AND CANCER RECURRENCE

Treatment of carcinomas, including those originating in the lungs, breast, prostate, colon, and ovaries, has the best chance of success if detected early. This requires both early identification of a cancer with lethal potential and monitoring of treatment response with the ability to reveal emergent treatment resistance and disease relapse. Diagnosis and monitoring require time resolution/frequency, high technical reproducibility, and validation in the context of use.

THE METASTATIC CASCADE

Cancer’s lethality derives from its ability to grow at the primary site and spread through the body. While organ-confined, local growth can be treated with curative intent, it is postulated that once distant metastases have developed the disease is more likely to be fatal. Substantial efforts in biological discovery are focused on understanding the metastatic cascade mechanistically and biologically. Mechanistically, a sequence of events occurs from intravasation via dissemination and extravasation to colonization of distant organs; biologically, disease evolution results in higher resilience to treatment approaches. The key is whether a tumor has global survival and resilience signals built in, or if these evolve over time. The primary tumor, the tumor cells within circulation, and metastatic lesions are the three tissue sites of interest to investigate. Hematogenous dissemination of tumor cells is widely assumed as a late event, although escape of cells from a primary tumor into circulation may occur early, although these “early” CTCs may not always survive and seed viable metastases,¹ raising the possibility that detection of “early” CTCs or frequent monitoring of CTCs after surgery may allow successful timely interventions.

FLUID BIOPSIES

Investigation of the fluid phase is hindered by the rarity of disease derived analytes (CTCs, circulating nucleic acids, other cellular and acellular fragments). The discovery of CTCs by Ashworth in 1869 did not gain substantial research traction until the late 1990s, when rare cell technologies began to improve. CellSearch, the technology that put CTCs “on the map,” provides robust enrichment, and enumeration of CTCs that express the epithelial surface marker EpCAM (epithelial cell adhesion molecule). In 2004, CTC enumeration had prognostic significance, with multiple CellSearch studies in various epithelial tumors confirming that increased CTC counts correlated with worse patient outcomes.² These were critical milestones, but the field remained confounded by CTC heterogeneity and EpCAM-negative CTCs consistent with epithelial to mesenchymal transition (EMT). This prompted development of marker-independent technologies with enrichment of CTCs based on physical properties, although the robustness of these approaches remains to be demonstrated. Most CTC studies have been conducted in advanced disease stages. A problem for earlier diagnosis and detection of relapse is that most CTC technologies incur cell losses during enrichment. Development of the high-definition single cell analysis (HD-SCA; Figure 1) platform removes that problem by assessing all cells in the blood sample. Coupled with the revolution in single-cell molecular analysis and bioinformatics, the information now accessible from a single CTC is staggering. The Kuhn and Dive laboratories have already established a successful collaboration exploiting the sensitivity of HD-SCA to identify and molecularly confirm rare subsets of circulating lung cancer cells undergoing vasculogenic mimicry.³

Figure 1.

The HD-SCA framework. Patient biopsies are preserved in a biorepository until immunofluorescently stained and imaged via automated scanning microscopy. Single cells of interest are further characterized by high-quality imaging, targeted proteomics, and genomic analysis.

A technically easier challenge is the detection and sequencing of circulating-tumor DNA (ctDNA). This approach has demonstrated huge potential for treatment stratification, response monitoring, and charting tumor evolution. Recently, the US Food and Drug Administration (FDA) approved the first blood-based genetic test to detect gene mutation in epidermal growth factor receptor (EGFR) of non-small cell lung cancer (NSCLC) patients. The cobas EGFR Mutation Test v2 is a companion diagnostic for the drug Tarceva (erlotinib) to determine what patients will benefit from treatment. The challenge for ctDNA-based early detection and swift reporting of disease relapse lies in increasing its sensitivity, while maintaining its specificity.

SINGLE-CELL MORPHOPROTEOGENOMICS

The use of fast and reliable automated fluorescence microscopy grants us direct measurements of multiple morphological parameters over millions of cells. Single cells are then utilized for genomic or proteomic analysis. Genetic material is use to generate copy number variation (CNV) profiles, which may provide insight into genotypic variation in tumor evolution. We are in the process of optimizing deep sequencing to identify single nucleotide variations (SNVs) focusing on the genes most relevant to a particular cancer. Specifically, for lung cancer, genomic profiling has become essential for predicting treatment response and identifying targets for new therapies.

Characterizing the CTC proteome presents the opportunity to discover novel biomarkers and facilitate our understanding of metastasis. We are integrating the technique of imaging mass cytometry (IMC), which combines mass spectroscopy and flow cytometry to detect up to 32 proteins at subcellular resolution, with the HD-SCA platform for proteomic analysis to gain a deeper level of CTC characterization.

Less than 2% of the genome is protein-coding sequences, while an abundance of nonfunctional sequences exists. Genomics represents a gene’s potential impact, while proteomics represents the functional molecules directly implicated in disease progression. The combination of genomic and proteomic analysis produces a more comprehensive molecular inventory to increase our understanding of cancer biology.

MINIMALLY RESIDUAL DISEASE

Although early stage NSCLC can be treated successfully with surgery, ~50% of cases relapse. We examined CTCs collected from the pulmonary vein draining the cancerous lung immediately before surgery⁴ and found that those patients with higher CellSearch CTC counts and/or a circulating tumor micro-embolus were more likely to relapse. CellSearch CTCs are rarely detected in peripheral blood samples in early-stage NSCLC. We will extend our pilot study by sampling peripheral blood at surgery and regular intervals thereafter using the HD-SCA platform. We will ask whether CTCs herald disease relapse, where CTC molecular analysis may inform beneficial interventions, and streamline the logistics of blood sampling in the community, avoiding the necessity for frequent hospital or general practitioner visits.

Overall survival of colorectal cancer (CRC) patients has improved with the combination of chemotherapeutic agents and targeted therapies. The challenge is selecting patients with poor prognosis and identifying the most effective treatment. Early-stage CRC patients undergo surgical resection with curative intent and are only offered chemotherapy due to risk factors such as intraoperative tumor perforation, but ~30% will relapse within 5 years. CTC detection may indicate the presence of micrometastatic lesions and have a prognostic role in early-stage patients where CellSearch CTCs identified were associated with worse survival.⁵ We have three CRC studies in which we will determine the significance of CTC number in relation to disease progression.

BLOOD PROFILING ATLAS IN CANCER (PAC)

In a partnership between government, academia, pharmaceutical, and diagnostic companies, we are participating in the generation of an open database for liquid biopsies to accelerating the development of safe and effective diagnostic tools for blood profiling to benefit patient care. The Blood PAC will aggregate datasets from CTC, ctDNA, exosomes, and other analytes from clinical studies on an open commons platform for further analysis. We have committed to sharing HD-SCA data on CTC morphology, genomics, and proteomics, as well as ctDNA, on current and future studies.

DISCUSSION

This collaboration between the Kuhn and Dive laboratories on minimal residual disease is designed to identify patients with a high likelihood of relapse to get earlier treatments, improve patient care, and deliver better outcomes in a timelier fashion. It is encouraging to begin a global collaboration within the Cancer Moonshot initiative in the ongoing effort using liquid biopsies to deliver personalized medicine for cancer patients.

Researchers will analyze blood samples from patients with NSCLC and CRC to determine those with cellular traces of cancer and who are more likely to relapse. This gives clinicians the opportunity to make better treatment decisions and quickly initiate subsequent therapy. Having the capability to monitor minimal residual disease has transformed patient care in leukemia; unfortunately, current techniques are not sensitive enough to similarly guide clinical decisions for patients with “solid” tumors. Advances in technology will empower researchers to analyze cells in greater detail and identify the most effective therapy for each patient, as well as monitor their cancer progression.

The partnership between the Dive and Kuhn laboratories will use the HD-SCA platform, conceived by Kuhn and his team, to identify rare cells more sensitively than existing techniques. Dive and her team at the Cancer Research UK Manchester Institute will be using this technology for the first time in the UK. The two teams will build and operate identical laboratories with real-time sharing of research data and experimental procedures to accelerate the development of this technology in the clinic, getting it to patients around the world as quickly as possible. The Kuhn Laboratory will focus on CRC, while the Dive Laboratory will lead the lung cancer research to refine and develop the HD-SCA technology. Our aspiration is to use the HD-SCA platform to detect early signs of cancer in otherwise healthy people via this liquid biopsy, ultimately leading to marked improvement in survival to transform cancer care.