AMP case report: Detection of concurrent hematologic malignancies in solid tumor NGS testing may cause false-positive results

Discussion. NRAS and KRAS are members of the RAS family of oncogenes. Activating point mutations in these genes have been reported in a variety of tumors, including non-small cell lung cancer and hematological malignancies, mostly concentrating in codons 12, 13, and 61. RAS mutations in lung adenocarcinoma are usually mutually exclusive of other oncogenic driver aberrations including EGFR, BRAF, ERBB2, and ALK and ROS1 rearrangements, although coexistence has been rarely reported.¹ Because NRAS and KRAS are oncogenes, single activating heterozygous mutations (“one hit”) are sufficient for conferring a selective advantage, and therefore cancers with two concurrent mutations are rare. Multiple concurrent mutations in KRAS are reported only in approximately two percent of mutated colorectal carcinomas² and have also been commonly reported in pancreatic cancer.³ Double KRAS mutations in lung adenocarcinoma are exceedingly rare, although they have been reported in one case.⁴

While NRAS mutations are rare in lung adenocarcinoma and tend to be codon 61 mutations (approximately one percent),⁵ KRAS mutations are common in lung cancer (approximately 25–30 percent of cases).^5,6 KRAS variants in lung adenocarcinoma are usually in codons 12 and 13 and are less likely to be in codon 61.^5,6 NRAS mutations are more commonly identified in lung adenocarcinoma in current and former smokers.⁷ KRAS mutations also define a distinct molecular subset of lung adenocarcinoma. While KRAS mutations are found in former and current smokers and never smokers, they are rarer in never smokers and are also less common in patients of East Asian descent.^8,9 Importantly, KRAS mutations have been reported as an indicator of resistance and poor survival in patients with non-small cell lung carcinoma treated with EGFR-tyrosine kinase inhibitors.^10,11 The prognostic as well as predictive role of KRAS mutations continues to be studied in lung adenocarcinoma. Although various attempts at inhibiting KRAS have been made, there is no established therapy specific for this large subpopulation of lung cancer patients.

Genetic mutations are common in CMML and are seen in greater than 90 percent of cases.¹² RAS mutations are highly prevalent and are seen in 20–30 percent of CMML, with NRAS mutations more common than KRAS.¹³ RAS mutations in CMML have been associated with features of cell proliferation and monocytosis and with shorter survival, although some multivariate models have not substantiated that RAS mutations confer inferior outcome.^14–17 RAS variants in CMML are often associated with a myeloproliferative phenotype rather than a myelodysplastic phenotype.¹⁶

Interestingly, it is thought that the initial driver mutation in CMML is likely to be a mutation in TET or ASXL1 and then subsequent secondary mutations, including RAS mutations, may allow clonal subsets to progress.¹⁸ The patient in the case presented here had a nonsense mutation in ASXL1 identified at a notably higher VAF than the RAS variants identified in the same specimen, which probably indicates that the RAS-mutated clones were subpopulations of the larger ASXL1 CMML population. Genetic heterogeneity due to different clonal subpopulations is a well-recognized phenomenon.¹⁹ In most cases of CMML, clonal architecture is mostly linear, but split architecture with several branches arising from the same ancestor have been observed.¹⁹

Despite being found on NGS testing of the lung FNA and resection specimens, the likelihood of two KRAS variants and an NRAS variant occurring as concurrent somatic alterations in the lung adenocarcinoma is exceedingly low. Most likely, the NRAS G12D and KRAS G12D are somatic mutations in the patient’s CMML, while the KRAS G12C and TP53 variants are truly from the lung adenocarcinoma. The NRAS G12D was found at a higher VAF in the peripheral blood testing (Genoptix) than the solid tumor testing, and NRAS mutations are more common in CMML than lung adenocarcinoma. Additionally, the NRAS G12D has a higher VAF in the FNA cell block than the lobectomy resection specimen. This correlates with the observance that the FNA had significantly lower adenocarcinoma tumor cellularity (25 percent versus 70 percent), with contaminating peripheral blood (along with the patient’s CMML) constituting much of the non-adenocarcinoma cells. While the VAF of the KRAS G12D was slightly higher in the lung resection specimen than the verbally reported VAF for the same mutation in the peripheral blood, this result can be explained by proliferation of the KRAS-mutated CMML subclone between the time of the peripheral blood analysis and the lung cancer resection. The NRAS G12D and KRAS G12D therefore are thought to most likely represent false-positives in the solid tumor NGS testing. These findings could have been further confirmed with analysis of ASXL1 in the solid tumor specimens; however, this gene is not included in the 50-gene panel used in this case.

In this case, the multiple RAS mutations were a sign to further investigate the patient’s clinical history, but this finding may not always be present. In cases in which an expected variant is identified in a particular tumor type—for example, a single KRAS mutation in a lung adenocarcinoma—a false-positive result would not be as obvious. False-positives may have important clinical significance; therefore, inconsistent or unlikely results on NGS testing always warrant investigation of the patient’s history and prior molecular results. Ordering clinicians and pathologists can mitigate the possibility of false-positives owing to secondary malignancies on NGS testing by alerting molecular pathology laboratories to patients’ hematologic diagnoses and prior molecular testing, when applicable. 

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Test yourself

Here are three questions taken from the case report. Answers are online now at www.amp.org/casereports and will be published next month in CAP TODAY.

1. KRAS mutations in lung adenocarcinoma are most likely to be found in which codon(s)?

a) 12 and 13

b) 13 and 61

c) 12 and 61

d) 61

2. KRAS mutations are usually mutually exclusive of which other mutations?

a) BRAF

b) EGFR

c) ALK

d) All of the above

3. RAS mutations are found in approximately what percentage of CMML?

a) 50–60 percent

b) 20–30 percent

c) 5–10 percent

Dr. Baum is a molecular genetic pathology fellow and Dr. Marrero is a pathology resident and clinical pathology chief resident, New York Presbyterian Hospital-Weill Cornell Medicine. Dr. Borczuk is an attending pathologist and anatomic pathology vice chair, and Dr. Rennert is an attending pathologist and molecular pathology laboratory director, both at Weill Cornell Medicine, New York, NY.