Lessons From Genetic Testing in Pediatric Cancers

Alexander M. Castellino, PhD

January 28, 2016

How precision cancer medicine can be applied to pediatric cancers has been addressed in two pilot studies published online January 28 in JAMA Oncology.

Although the results led to new therapy only in a small number of patients, the findings from these studies provide room for optimism, editorialists write in an accompanying commentary.

In the future, as the cost of sequencing drops, whole-genome germline sequencing and tumor and RNA sequencing will provide insights into mutations associated with tumor heterogeneity and subclones, write the editorialists, Javed Khan, MD, and Lee J. Hellman, MD, of the National Cancer Institute, Bethesda, Maryland.

"This sequencing will usher in a new era of unprecedented comprehensive analysis of the genome for all children with high-risk, refractory, or relapsed or cancers — a necessary first step to the demonstration of the clinical utility and validity of genome-based precision therapy," the editorialists conclude.

In the first report (the BASIC3 study), researchers from Texas Children's Cancer Center at the Baylor College of Medicine categorized tumor and germline mutations in pediatric patients with solid tumors using clinical whole-exome sequencing of approximately 20,000 genes.

Tumor or germline mutations were reported in diagnostic and/or potentially actionable genes in nearly 40% of newly diagnosed patients with pediatric solid tumors, providing oncologists with clues on clinical management of the patients and providing family members with an option to follow up on cancer risk through genetic testing.

"This is the first clinical trial that made a concerted effort to analyze pediatric cancers based on exome sequencing. The results were given back to the oncologist and the family," Sharon E. Plon, MD, PhD, from the Department of Pediatrics, who is a corresponding author on the BASIC3 study, told Medscape Medical News.

"The trial represents a clinical implementation of genomics in the setting of pediatric cancers," she added.

Co-corresponding author D. William Parsons, MD, PhD, also from the Department of Pediatrics, told Medscape Medical News: "Whole-exome sequencing revealed a diversity of somatic mutations, many of which targeted cancer genes that had not been previously linked to the specific tumor type diagnosed in the patient."

In the second report (the iCat Study), advanced pediatric solid tumors were genetically profiled using gene panels to inform treatment decisions. For 31% of patients, an actionable mutation could be matched with known targeted agents. Yet, only 10% received the matched therapy.

"In children with high-risk, recurrent, or refractory extracranial solid tumors, there is the potential for tumor sequencing to have clinical implications, but there are barriers to receipt of matched therapy," the iCat researchers note.

"In this pediatric population with recurrent, refractory cancers, tumor sequencing can uncover mutations that impact how doctors diagnose patients, drug selection, or identification of appropriate clinical trials," corresponding author Katherine A. Janeway, MD, of the Dana-Farber Cancer Institute, told Medscape Medical News.

"The BASIC3 study demonstrates the breadth of data that can be generated from whole-exome sequencing, but most of the data, as yet, cannot be translated into a therapeutic regimen recommendation," Dr Khan and Dr Hellman comment in the accompanying editorial.

"The iCat study highlights the problem of implementation of genome-based precision therapy in children with cancer, given that only 3 of 31 patients actually received the recommended therapy," they add. This will be an important issue to address in future studies, they indicate.

The BASIC3 Study

The BASIC3 (Baylor College of Medicine Advancing Sequencing in Childhood Cancer Care) study reported data from 150 participants (80 boys and 70 girls; median age: 7.4 years) from Texas Children's Cancer Center. Of these, 56 children had central nervous system (CNS) tumors, and 94 had non–CNS tumors.

Researchers used peripheral blood samples for germline sequencing; frozen tumor samples were used for tumor sequencing (121/150).

Of the 121 patients who underwent tumor whole-exome sequencing, category 1 mutations (ie, mutations known to have clinical relevance for that tumor type) were identified in four (3%) patients, and category II mutations (of potential clinical utility) were noted in 29 patients (24%).

The 37 recurrent mutations in category I and II genes included CTNNB1, BRAF, KIT, KRAS, NRAS, and TSC2. Only four (11%) of these were previously detected by routine clinical molecular testing: an ALK hotspot mutation in a neuroblastoma, and BRAF V600E in three gliomas; another four are now becoming routinely tested (CTNNB1 in 3 medulloblastomas and H3F3A in a glioblastoma); 25 of the 37 mutations were oncogenic alterations or inactivating mutations in tumor suppressor genes.

Additionally, 24 of 121 patients' tumors showed mutations in category III genes — mutations in consensus genes not currently considered targetable (eg, MED12 in Wilms' tumor). The most frequent mutations were in TP53, BCOR, DDX3X, and MED12. A total of 1111 mutations in category IV genes (ie, all other somatic mutations) were also identified.

With respect to germline mutations, pathogenic or likely pathogenic mutations were identified in 15 (10%) of 150 patients. In eight patients, childhood cancer risk was associated with some of these mutations (VHL, TP53, DICERI1, MSH2, WT1, and KRAS); but in three of these, the clinical care team had not considered genetic testing, for several reasons. For example, in one case, the positive family history was not elicited — one associating Lynch syndrome with MSH2.

In five of the eight cases, germline mutations had tumor diagnoses not previously associated with the mutated gene identified in the exome sequencing (eg, BRCA1 x 2, BRCA2, SMARCA4, and CHEK2).

"This was a longitudinal study that did not try to direct treatment," Dr Plon told Medscape Medical News. "The tests were returned to the treating oncologists, and all the patients in the study are being followed to determine if the information was used to inform treatment decisions," she added.

However, Dr Plon told Medscape Medical News that families used the germline information to follow up with testing in family members. Several of the siblings who tested positive for the cancer susceptibility mutation are being followed for cancer risk through the Texas Children's Childhood Cancer Prevention and Screening Clinic, she said.

The iCat Study

The Individualized Cancer Therapy (iCat) Study was a multicenter feasibility study that determined tumor profiling from paraffin-embedded or freshly frozen tissue samples of patients aged 30 years or younger who had a recurrent, refractory, or high-risk extracranial solid tumor.

Tumor profiling was initially undertaken to detect 471 recurrent mutations in 41 cancer-related genes. This was followed by next-generation sequencing or OncoPanel sequencing that captured information on intron mutations and gene rearrangements.

Of 100 patients (60 males, 40 females; median age: 13.4 years), successful profiling was possible in 89 patients.

The expert panel rendered an iCat recommendation in 31 (31%) patients, which exceeded the 14 patients required to meet the definition of feasibility.

The most common actionable alterations leading to an iCat recommendation related to copy number alterations (eg, MYC/MYCN or cell cycle–related genes) and suspected deleterious mutations (ALK, BRAF, FGFR4, HRAS, NRAS, PIK3CA, ATM, and CTNNB1).

Only three patients received targeted therapy matched to iCat recommendations. One child with refractory, unresectable sialoblastoma with FGFR2 mutation received off-label pazopanib (Votrient, Novartis Pharmaceuticals Corporation); one child with embryonal rhabdomyosarcoma with a PIK3CA mutation received the pan-PI3K inhibitor BKM120 through a compassionate access protocol; and a patient with recurrent neuroblastoma with an ALK mutation and a MYCN high copy number gain received crizotinib (Xalkori, PF Prism CV) monotherapy.

Tumor profiling may provide a means to change a diagnosis from the one made at presentation. In the case of a stage III melanoma, the EWSR1-ATF1 mutation suggested a change in the diagnosis to cutaneous clear-cell carcinoma. The diagnostic change has important bearing for therapeutic action.

"In 43% of the tumors, researchers of the iCat study found mutations which had potential clinical implications: an actionable alteration, a clarification on the initial diagnosis, or a cancer risk syndrome," Dr Janeway told Medscape Medical News.

Lessons Gleaned

As the field of clinical care, led by oncology, is moving to precision care, the two studies suggest significant perspectives regarding its future. The editorialists indicate several important lessons learned from these studies.

First, it is important to standardize the sequencing platforms and data analysis to ensure reproducibility, they indicate. The iCat study used deep sequencing that utilized several platforms, such as OncoMap, OncoPanel, and aCGH, with profiling results read by a panel of experts.

The iCat researchers agree. "Our study did not assess which tumor profiling assays optimally balance the competing factors of minimal tissue requirement, comprehensive genomic assessment, and rapid reporting," they write in their discussion. "The rapid pace of technology development, a challenge to ongoing consistency, complicates this task," they add.

Dr Janeway explained that researchers still do not know the optimum sequencing test. Several issues remain, she indicated: the number of genes interrogated and whether one uses DNA or RNA sequencing. "We need to capture the maximum possible information without being overwhelmed with it," she said. In addition, although researchers may use similar tools to analyze their data, there is no universal agreement on how to use these tools, she explained.

The breadth and depth of sequencing are both important, according to the editorial. The BASIC3 study used whole-exome sequencing to identify mutations that were of as yet uncertain significance. The editorialists indicate that use of deep sequencing to identify subclones, as undertaken in the iCat study, is also important. The iCat study showed that with depth of sequencing, one is able to increase the number of actionable genes that can be targeted.

The editorialists also note that in both studies, tumors were sampled at a single time point. They explain that cancers resistant to therapy grow while some lesions are responding to the therapy applied. With the resistant clones having a different genetic profile, tumor profiling to compare tumors at presentation with those that recur provide information on tumor heterogeneity, clonal selection from therapy, and evolution. Because performing multiple biopsies at different time points raises an ethical concern, the use of liquid biopsies, which enable assessment of circulating tumor DNA, may provide an option, the editorialists indicate.

Dr Parsons stressed that as the spectrum of genes implicated in cancer has gotten bigger, much remains to be learned about the biology of the spectrum of variants in the gene pool. As an example, he cited the fact that BRAF V600E is a well-known BRAF mutation that has targeted therapies. However, what is known about BRAF V600E may not be applicable to other missense mutations in BRAF, he indicated. "Our ability to interpret the biological and clinical significance of many of the variant mutations detected is a significant challenge," he told Medscape Medical News.

In addition, the tiering systems, which define actionable mutations, are different, according to Dr Janeway. She explained that although iCat and BASIC3 studies yielded overlapping actionable genes, the tiering system used by each study was different. The Clinical Genomic Working Group is trying to "fix" the classification system so that it is applied universally, she indicated.

However, how does one account for the fact that in the iCat study, only 3% of patients were treated with matched therapy? Reasons advanced for not matching therapy to actionable mutations included advanced disease, the fact that the disease was not active, that the disease was well controlled on alternate therapy, and that the patient was undergoing third-line therapy. The lack of a suitable clinical trial was cited as another reason, although this was contrary to an iCat recommendation, which matched targeted therapy to an available, targeted therapy or a known, ongoing clinical trial.

The iCat researchers acknowledge that "this may have been both because we focused on profiling patients who often had advanced cancers and also because the follow-up data were collected for a relatively short period of time." This suggests that profiling tumors earlier in the disease course may increase the number of patients treated with matched targeted therapy.

"The information on the diversity of tumor mutations has significant implications and will inform the design of prospective clinical trials in precision care," Dr Parsons told Medscape Medical News.

He explained that because many of the actionable gene alterations occur with relatively low frequency, clinical trials that incorporate sequencing for precision care will need to incorporate a significant number of investigational agents that cover many molecular targets. Indeed, the planned National Cancer Institute Pediatric MATCH trial, to be run through the Children's Oncology Group, will seek to do just that.

The editorial also provides a rationale for the significance of germline sequencing as undertaken in the BASIC3 study. It assesses true tumor mutations and the likelihood that many patients may have cancer-related mutations in their germline. Indeed, the editorialists point out that in the BASIC3 study, 85% of the 10% germline mutations reported were inherited. The information provides significant implications for family members at risk for cancers that have not yet developed, they point out.

BASIC3 was an NHGRI and NCI-funded Clinical Sequencing Exploratory Research program project. The iCat study received funding from Hyundai Hope on Wheels, the Friends for Life Foundation, and the Gillmore Fund. Baylor College of Medicine and Miraca Holdings Inc have formed a joint venture with shared ownership and governance of the Baylor Miraca Genetics Laboratories, which performs exome sequencing. Several authors of the BASIC3 study have reported a governance role in this venture. The authors of the iCat Study and the editorialist have disclosed no relevant financial relationships.

JAMA Oncol. Published online January 28, 2016. Parsons et al, Abstract; Harris et al, Full text; Editorial

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