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Oncologists’ Guide to Genomics

Stay current on the latest trends in genomics and molecular diagnostics for oncology.

Wednesday, February 20, 2019

By Catlin Nalley

Relapse is a major challenge when it comes to the treatment of patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS); oncologists are constantly searching for ways to combat this issue while improving their understanding of disease mechanisms.

"We have learned a lot about AML and MDS thanks to advances in DNA-sequencing," noted Christopher R. Cogle, MD, Professor of Medicine at the University of Florida. "And one of the things we have learned through this effort is that these diseases are much more genetically complex than we previously thought.

"With this new appreciation for the number and variety of genomic abnormalities, we are beginning to see individual AML and MDS cases as unique diseases loosely connected to one another."

Despite an increased understanding of the genomic makeup of these diseases, response rates, especially in the relapsed/refractory setting, remain poor. "In patients with relapsed or refractory AML or MDS we are seeing response rates of around 20 percent," Cogle noted. "There is an urgent need for new therapeutics or intelligently repurposed older drugs."

Recognizing the need for a deeper understanding of the complexities of these diseases, Cogle and his team pursued a new approach. "We sought to utilize computational means to interpret the high number and wide variety of abnormalities that are presenting in our MDS and AML clinic."

After finding the right software partner and validating the accuracy of the technology through retrospective analysis, the iCare1 prospective clinical study was launched. Findings from this trial were recently presented at the 2018 ASH Annual Meeting (Abstract 3086).

Study Details

Researchers utilized a genomics-informed computational biology modeling (CBM) technique to improve their understanding of the mechanisms of response or relapse after chemotherapy treatment among AML and MDS populations and to hypothesize new treatment approaches.

The investigators recruited 120 patients with AML and MDS to assess the accuracy of CBM prediction through the comparison of computer predictions of treatment response and actual clinical outcomes. Of these patients, 96 had full genomic testing profiles.

Conventional cytogenetics, whole exome sequencing, and array CGH were used to conduct genomic profiling. Disease-specific protein network maps for each patient were created by inputting somatic gene mutations into the CBM program, Cogle explained.

A digital library of FDA-approved drugs was generated for the technology by "programming each agent's mechanism of action determined from published literature," researchers outlined. "Digital drug simulations of the patient's choice of therapy were tested at varying doses and predicted efficacy of the drugs were measured as a function of a disease inhibition score, defined as the degree to which disease pathways and phenotypes (cell proliferation and viability) were mathematically returned to a mutation-free state."

Based on length of follow-up and minimum treatment exposure threshold, 50 patients were eligible for evaluation. Among these patients, 61 treatments were administered. Researchers reported that the CBM maps of relapsed samples from these patients "accurately matched the patient's nonresponse of treatment at relapse in 90 percent of patients and identified mechanisms for chemoresistance."

"By applying this computational biology method and digital drug simulation we found several things," Cogle noted. "Chief amongst them is the capability of this computational method to be employed in the clinic and provide highly accurate predictions of treatment response in our AML and MDS patients."

Implications, Next Steps

Using computers in oncology is an emerging area not only in research, but also for clinical application, noted Cogle. However, he emphasized the importance of comprehensive testing of these methods before marketing to patients with cancer.

"The iCare1 study and the work preceding it represents, firsthand, the kind of stepwise, rigorous clinical trial testing that should be done for predictive methods," Cogle elaborated. "We are not only demonstrating a feasible and accurate computational method, but we are also highlighting a path towards responsible technology development."

While the next step for the computational model tested in iCare1 is to assess it in a randomized clinical trial, Cogle and his team are also focused on educating physicians and patients about this type of technology so they are "empowered to appreciate the results that come out of computational predictions.

"Computational oncology is called a 'black box' only by those who don't know how to open the box and read what's inside," Cogle said. "My group is committed to assisting physicians and patients to better understand the methods being used so that they can determine whether or not it is an appropriate application for their case."

Looking forward, Cogle stressed the important role computational oncology will play in the field of oncology. "Interpreting one gene mutation can be challenging and time consuming, interpreting multiple gene mutations is sometimes impossible in our busy practice setting," he noted. "We have to embrace computational oncology because of limitations not only on our time, but also on our human cognitive capacity to parallel process the tangle of abnormalities that we find in cells of our cancer patients.

"Results like the ones coming out of the iCare1 study are encouraging and demonstrate that we now have access to technology that is not only feasible, but also accurately predicts treatment response."

Catlin Nalley is associate editor.

Friday, January 18, 2019

By Catlin Nalley

Recently reported data from the Angiosarcoma Project identified immune checkpoint inhibition as potentially actionable for angiosarcomas of the head, face, neck, or scalp (HFNS).

Launched by the Broad Institute in March 2017 in collaboration with Dana-Farber Cancer Institute, the Angiosarcoma Project is a patient-partnered genomics study. Patients are involved throughout the length of the project, including design, build, launch, and accrual.

Patient-reported data, medical records, archival tumor tissue, saliva, and blood samples are collected from individuals across the U.S. and Canada who have agreed to contribute to this study. This allows researchers to develop a comprehensive resource to facilitate discoveries for this rare disease.

"Angiosarcoma is an exceedingly rare cancer affecting about 300 patients a year in the U.S., has a high mortality rate, and has no standard of care; because of its rarity, there is a dearth of scientific discoveries that can lead to clinical impact," said Corrie A. Painter, PhD, Associate Director of Operations and Scientific Outreach at Broad Institute of MIT and Harvard, in a statement. "It is important to develop novel approaches for working with patients who are geographically dispersed and who may never have the opportunity to participate in a large-scale prospective research effort because they have such a rare disease."

Building the Project

The Angiosarcoma Project (ascproject.org) was developed following the success of an initial endeavor in the metastatic breast cancer space.

"The Metastatic Breast Cancer Project was launched in October 2015 and it has been very successful from a number of different metrics, including patient enthusiasm," Painter told Oncology Times. "Given the positive responses to this project, we wanted to extend the ability to work with patients affected with other cancers."

The team hypothesized that they could utilize the same platform for a rare cancer and achieve the same results they saw in a major disease. And, as a survivor of angiosarcoma, Painter knew this was a very engaged community. Posting to an online support group, Painter gauged their interest in donating left over tissue and medical records to cancer research. "Within 1 hour, 90 patients expressed their desire to participate," she recalled. "This was the rationale behind the project."

Social media remains a critical aspect of the project. "The Angiosarcoma Working Group" on Facebook is used to communicate findings and connect with patients, according to Painter. "This group represents a mixture of people—patients, researchers, doctors, and caregivers—who are highly invested in research for angiosarcoma and have come together to work on this project."

Initial Findings

Data from the initial analysis were recently presented at the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival.

Researchers performed whole exome sequencing of approximately 20,000 genes on tumor and matched germline DNA. Several genes, known to be altered in angiosarcoma, were identified in the samples, including KDR and TP53, according to study findings. Of the 321 patients registered to date, 21 percent had angiosarcomas of HFNS.

"When first reviewing the data, we noticed outliers on the tumor mutation burden (TMB) graph. We also noted that all of those cases in the first data release matched patients who had angiosarcomas of HFNS," Painter explained. "We hypothesized that the high TMB was caused by UV damage and there could potentially be a UV signature in the DNA."

From the initial cohort of 12 patients, all three with angiosarcoma of HFNS had high TMB (10 mutations per MB) and dominant UV light signature, according to the researchers.

"Our work suggests that patients with angiosarcoma of HFNS have a high mutational burden and UV light signature, and therefore may respond to checkpoint inhibition," Painter observed.

Given the potential impact of this finding, the research team sought to identify additional patients. Painter and colleagues found an additional 56 patients with angiosarcoma of HFNS who provided treatment details.

Of these patients, two had previously received immune checkpoint inhibitors. Both patients had refractory metastatic HFNS angiosarcoma and reported undergoing off-label anti-PD1 therapy, according to investigators. "Both had complete or near-complete responses following immunotherapy, and currently report stable or no evidence of disease."

Angiosarcomas of HFNS represent the largest demographic of people affected by the disease, therefore, Painter noted, these initial findings could have far-reaching implications for a significant number of patients with this rare cancer.

Looking beyond this patient population, the Angiosarcoma Project has broad implications for cancer research as a whole, especially when it comes to rare diseases. "This study serves as proof of principle that patient-partnered genomics efforts can democratize cancer research for exceedingly rare cancers," Painter noted.

Ongoing Discovery

Painter and her team plan to continue the momentum of this project. "We are still building it out in terms of samples and sequencing them," she noted. "We are getting ready to write up our findings. Additionally, we continue to look at the data and see what other insights we can gather."

And their plans go well beyond the angiosarcoma space. "Based on the success of both the Metastatic Breast Cancer and Angiosarcoma projects, we have also built and launched the Metastatic Prostate Cancer Project as well as the Gastroesophageal Cancer Project," noted Painter. "All of these have been appreciated by both the patient and scientific community. As a result we have been able to rollout an official non-profit, Count Me In (JoinCountMeIn.org).

"Our ambition is to launch projects in all major cancers and most, if not all, rare cancers as well," she continued. "Through these projects, we are going to be able to provide the biomedical community with data that will lead to discoveries across a spectrum of cancers that have had significant barriers to get patients together or the funding to actually do the type of deep analysis that we hope to make freely available.

"It is our hope that this will jumpstart a number of different labs on their way toward making discoveries that will impact the lives of patients living with cancer both today and in the future."

The Angiosarcoma Project and others like it underscore the important role patients play.

"Being a patient myself I can say, with certainty, that nobody cares more about the success of a project like this than the people who are directly impacted by it," Painter noted. "As dedicated as I was as a scientist, there was an end of the day. It may have been late, but I went to sleep and didn't have nightmares about it. When you are a patient it is 24/7 and so you will do everything in your power to drive progress.

"If scientists can find more ways to include patients, I think all of us will benefit," she concluded. "I cannot wait to see what happens when we have more established roads between scientists and patients; we are at an inflection point that is going to change everything."

Catlin Nalley is associate editor.

Thursday, December 20, 2018

While many cells in our bodies can accumulate oncogenic mutations, the majority of these events do not lead to tumor formation as these abnormal cells are eliminated by defense mechanisms. Instead, tumors arise when a mutation happens in a particular cell type that is uniquely sensitive to it. Identifying such cancer cells of origin is essential to properly target cancer.

For example, mutations in the retinoblastoma (RB) tumor suppressor gene, which normally blocks abnormal cell growth and division, gives rise to retinoblastoma. It arises in specialized retinal cells called cone cells, which collect light. Why this kind of cancer always starts in cone cells is unknown. But if scientists can get a clearer, more accurate view of the downstream effects of RB mutations in cone cells versus other cells in the retina, they may identify unique therapeutic targets that can prevent or treat retinoblastoma with laser-like precision.

But studying the effects of gene mutations in specific cell types is easier said than done. It is nearly impossible to collect pure samples made up of just one cell type. Instead, scientists often have to use a bulk sample prepared from an entire tissue.

"This only gives a sort of average picture of gene expression in individual cells since it pools thousands of cells, some of which may be unwanted no matter how much the sample is purified. The results of examining the effects of RB mutations using these kinds of samples don't accurately represent how an RB mutation affects gene expression in a particular cell type," said Maxim Frolov, Professor of Biochemistry and Molecular Genetics in the University of Illinois at Chicago (UIC) College of Medicine.

Single-Cell RNA Sequencing

Single-cell RNA sequencing lets researchers study gene expression in individual cells, eliminating the problem of contaminated cell samples and averaging. Frolov's laboratory adapted a technology called Drop-seq, which allows researchers to isolate and genetically sequence single cells. Drop-seq can sequence thousands of individual cells at the same time.

Frolov and his graduate student, Majd Ariss, assembled a Drop-seq instrument to isolate cells of the eye in developing fruit flies, which the lab uses as a model system. Then they were able to study gene expression changes caused by mutations in the RB gene in thousands of different cells in the eye compared with gene expression in cells with normal copies of the RB gene. Their results are published in Nature Communications (2018; doi:10.1038/s41467-018-07540-z).

"Since this is the first time single-cell RNA sequencing has been performed in cells of the fruit fly eye, we had to create a comprehensive map or cell atlas, accurately describing gene expression in each cell type in the normal eye. We then relied on this atlas to determine how an RB mutation affects gene expression of each cell type in the eye," Frolov explained.

Key Findings

Their analysis of eye cells with an RB mutation revealed a distinctive but small population of cells where the mutation altered gene expression and changed cell metabolism. The metabolic change sensitized the cells to apoptosis or self-induced cell death. The propensity of cells with mutations in the RB gene to undergo apoptosis is a well-known phenomenon and is eventually overcome through additional mutations during the development of cancer, which is characterized by out-of-control cell growth and division—the opposite of apoptosis.

"The metabolic changes we observed in RB mutant cells make them vulnerable in ways that might be exploited with therapeutic approaches before additional mutations hit the same cell, making them resistant to cell death," Frolov said. "Since these effects were limited to such a small group of cells, they were previously missed when whole RB mutant eye tissue was analyzed."

The Drop-seq platform took Ariss more than 3 months to build. He painstakingly followed instructions contained in a 40-page manual to generate his first single-cell RNA sequencing dataset.

"Nobody guided us on how to do single-cell sequencing as we were the first at UIC to do it," said Ariss, who is the first author on the paper.

Their Drop-seq instrument is the only one of its kind at UIC.

"It is a truly revolutionary technology that promises to shed new light on the origin of cancer and why certain cancers originate in certain cell types and not others. Only now can we begin to investigate why and how," Frolov said. "For the past year and a half, we performed over a hundred experiments and generated transcriptomes of more than a hundred thousand cells from fruit fly organs, mouse tumors, and human cell lines. We hope that more UIC researchers will use it going forward."

Tuesday, November 20, 2018

A combined evaluation of common variants with small effects and rare predisposing mutations among young female childhood cancer survivors may further stratify this high-risk population for subsequent breast cancer risk (Clin Cancer Res 2018; doi:10.1158/1078-0432.CCR-18-1775).

Female childhood cancer survivors have an increased risk of developing subsequent breast cancer compared with the general population. This increased risk has largely been attributed to cancer treatment regimens, such as chest irradiation and/or exposure to high-dose chemotherapeutic agents. Current screening of this population relies on treatments and doses used to treat childhood cancer, explained study author Zhaoming Wang, PhD, Associate Member in the Department of Epidemiology and Cancer Control at the St. Jude Children's Research Hospital in Memphis.

Wang previously found that survivors of childhood cancer have an increased risk of subsequent breast cancer if the survivors carry pathogenic or likely pathogenic (P/LP) mutations, such as mutations to the BRCA1 gene.

"Our current study attempts to investigate the contributions to the risk of subsequent breast cancer by considering the full picture of breast cancer genetic susceptibility, which includes common genetic variants with small effects (polygenic determinants) in addition to P/LP mutations (monogenic determinants)," Wang explained.

Study Specifics

Wang and colleagues utilized information from the St. Jude Lifetime Cohort Study by analyzing whole-genome sequencing data for 1,133 female cancer survivors of European ancestry. Of these survivors, 47 developed one or more subsequent breast cancers.

The researchers constructed a polygenic risk score (PRS) for individual survivors by calculating the weighted sum of 170 common breast cancer risk alleles present in each survivor's genome. Investigators also evaluated the presence of P/LP mutations in 11 breast cancer predisposition genes. Relative rates of subsequent breast cancer incidence were estimated.

Following multivariable analysis, the researchers found that survivors in the highest PRS quintile had 2.7 times the risk of subsequent breast cancer compared to survivors in the lowest quintile. Survivors treated with chest irradiation had even higher risk; those in the highest PRS quintile treated with radiation had three times the risk of subsequent cancer compared to those in lowest quintile treated with radiation.

Survivors who carried P/LP mutations had 21.8 times increased risk for subsequent breast cancer compared with those who didn't have P/LP mutations. Survivors treated with chest irradiation and who carried P/LP mutations had 10.3 times increased risk for subsequent breast cancer compared with those who didn't have P/LP mutations treated with chest irradiation.

"The PRS can identify individuals with high breast cancer risk that do not carry known pathogenic mutations," Wang noted. "Our results indicate that both polygenic determinants and large-effect rare mutations (monogenic determinants) contribute to the risk of subsequent breast cancer independently."

Notably, PRS was significantly associated with risk of subsequent breast cancer only in women less than 45 years old. "Our data supports the hypothesis that genetic risk factors play a more important role in the development of subsequent breast cancers in younger women," Wang said. "However, this observed age-specific association could be partly due to the smaller sample size of older survivors in our study."

P/LP mutations were defined as mutations to the following breast cancer predisposition genes: BRCA1, BRCA2, TP53, PTEN, CDH1, STK11, NF1, PALB2, ATM, CHEK2, and NBN.

"Our findings suggest that polygenic screening can inform personalized breast cancer surveillance in female childhood cancer survivors," said Wang. "This method can be utilized in the clinical setting to enhance the identification of high-risk survivors to enable the early detection and potential prevention of subsequent breast cancer."

In addition, "Our results indicate that personalized breast cancer surveillance strategies for survivors should incorporate prior exposure to specific anti-cancer treatments, the presence of P/LP mutations, and the cumulative presence of small-effect common variants, as represented by a polygenic risk score."

Limitations of the study include a relatively young cohort of childhood cancer survivors. Additionally, analysis was restricted to survivors of European ancestry; follow-up studies should be conducted within other non-European ethnic groups.

Monday, October 22, 2018

Despite months of aggressive treatment involving surgery and chemotherapy, about 85 percent of women with high-grade widespread ovarian cancer will have a recurrence of their disease. This leads to further treatment, but never to a cure. About 15 percent of patients, however, do not have a recurrence. Most of those women remain disease-free for years. Recently, researchers have identified an independent prognostic factor—cancer/testis antigen 45 (Cell 2018;175(1):159).

CT45 is associated with extended disease-free survival for women with advanced ovarian cancer. The team of physicians and scientists found that patients with high levels of CT45 in their tumors lived more than seven times as long as patients who lacked sufficient CT45. Data from long-term survivors averaged 2,754 days (7.5 years), compared to only 366 days for patients who had little or no CT45.

Cancer Proteomics

The study authors attribute their discovery to the emerging field of multi-level cancer proteomics. The researchers relied on minute pieces of tissue acquired from the University of Chicago ovarian cancer tissue bank, which has been following patient outcomes for 20 years.

They used pieces of these samples to isolate, identify, and characterize thousands of proteins. The most interesting of those proteins was CT45. They determined that higher levels of this biomarker were closely linked to treatment success and excellent patient outcomes.

"We believe this is the first example of mass spectrometry-based proteomics leading to the discovery of a prognostic and functionally important cancer biomarker," said co-lead author Ernst Lengyel, MD, PhD, an ovarian cancer specialist and Chairman of the Department of Obstetrics and Gynecology at the University of Chicago.

"Our goal was to find reliable biomarkers that could predict treatment response," said the study's co-lead author Matthias Mann, PhD, Chairman at the Max-Planck Institute. The team quantified more than 9,000 proteins and "identified CT45 as an independent prognostic factor for patients with high-grade serous ovarian cancer."

"Using mass spectrometry, we can identify, for the first time, almost all of the proteins in the tumor tissue of the patients," Mann said. "Our highly sensitive methods now enable us to profile thousands of proteins simultaneously, allowing us to search for the proteins that are critical to the disease by comparing the tissue samples."

It was encouraging for the research team to find the first significant biomarker in tissues from ovarian cancer patients who responded to platinum-based chemotherapy. "CT45," Lengyel said, "was completely unknown until then."

Study Details

To validate their initial findings, the researchers studied tissues collected from more than 200 patients from the University of Chicago. They found no CT45 in 82 of those patient samples, but they found high levels in 42 patients, all of whom had much longer disease-free survival.

A larger study, using sequence data from The Cancer Genome Atlas, confirmed their initial results, leading to their conclusion that "CT45 expression is a novel prognostic indicator for advanced stage high-grade serous ovarian cancer."

Since little was known about CT45's role in cellular functions, the study authors tried to understand the molecular mechanisms that improved responses to chemotherapy. They found that the standard chemotherapy for ovarian cancer, carboplatin, caused DNA damage, particularly in tumor cells expressing high levels of CT45. This lead to cell death in tissue culture and tumor reduction in treated mice.

"We suspect that CT45 plays a major role in the response of tumors to carboplatin. This gives us hope that future strategies that activate CT45 expression in the tumor could make it more sensitive to carboplatin treatment," said Marion Curtis, PhD, a postdoctoral scholar in the Lengyel laboratory.

They also found two peptides from CT45-positive ovarian cancer cells that stimulated a solid immune response against the cancer. T cells collected from a CT45-positive patient with high-grade ovarian cancer were able to kill cancer cells in vitro "in a dose-dependent manner."

"We have evidence that tumor-specific expression of CT45 stimulates the patient's immune system to fight the cancer, as would a virus or bacterial-infected cells," Lengyel added. "Our long-term goal is to find new ways to improve patient outcomes based on these exciting insights."

The clinical implications from this study "could be significant," the authors noted. Expression of CT45 improves the efficacy of platinum-based chemotherapy, and potentially immunotherapy, for patients with advanced stage ovarian cancer. "CT45 may be particularly relevant to long-term survival," they added.

"This study," researchers concluded, also "highlights the power of clinical cancer proteomics to identify targets for chemo and immunotherapy, define their mechanisms, and contribute to the development of effective cancer therapies."