RET Proto-Oncogene Testing

Number: 0319

Table Of Contents

Policy
Applicable CPT / HCPCS / ICD-10 Codes
Background
References

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Policy

Scope of Policy

This Clinical Policy Bulletin addresses RET proto-oncogene testing.

1. Medical Necessity

   Aetna considers germline RET proto-oncogene testing medically necessary when member meets any of the following criteria:

   1. Personal history:

      1. Medullary thyroid carcinoma (MTC) (familial or apparently sporadic)
      2. Pheochromocytoma (PCC) / paraganglioma (PGL) when multiple endocrine neoplasia type 2 (MEN2) is suspected
      3. Parathyroid adenoma or hyperplasia when multiple endocrine neoplasia type 2A (MEN2A) is suspected
      4. C‑cell hyperplasia on pathology
      5. Clinical features of multiple endocrine neoplasia type 2B (MEN2B) (e.g., early‑onset medullary thyroid carcinoma [MTC], mucosal neuromas, medullated corneal nerve fibers, or marfanoid habitus)
      6. Cutaneous lichen amyloidosis when MEN2A is suspected
      7. Hirschsprung disease (HD) when either of the following apply (Note: Isolated HD is not an indication for RET genetic testing):
         
         
         1. First-degree blood relative with MEN2; or
         2. Family history includes MTC, PCC/PGL, or parathyroid adenoma/hyperplasia; or
            
            

   2. Family history:
      

      1. First‑degree (i.e., parent, full-sibling, child) blood relative with MTC (Note: Sequencing of the RET gene may be considered)
      2. First-degree (i.e., parent, full-sibling, child) or second‑degree (i.e., aunt, uncle, grandparent, grandchild, niece, nephew, half-sibling) blood relative with a known germline RET pathogenic variant (Note: Targeted familial testing)
      3. Family pattern consistent with familial medullary thyroid carcinoma (FMTC) (2 or more relatives with MTC and no pheochromocytoma or parathyroid adenoma/hyperplasia).

2. Experimental, Investigational, or Unproven

   Germline RET proto-oncogene testing is considered experimental, investigational, or unproven for all other indications (e.g., non-small cell lung cancer; not an all-inclusive list) because its effectiveness for indications other than the ones listed above has not been established.

3. Related Policies

   • See CPB 0140 - Genetic Testing for policy on genetic testing for non-member relatives of Aetna members
   • See CPB 0715 - Pharmacogenetic and Pharmacodynamic Testing for RET fusion testing for members with non-small cell lung cancer (NSCLC) being considered for selpercatinib or pralsetinib, and thyroid cancer being considered for treatment with pralsetinib.

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Table:

CPT Codes / HCPCS Codes / ICD-10 Codes

Code	Code Description
CPT codes covered if selection criteria are met:
81404	Molecular pathology procedure, Level 5
81405	Molecular pathology procedure, Level 6
81406	Molecular pathology procedure, Level 7
88271	Molecular cytogenetics; DNA probe, each (e.g., FISH)
HCPCS codes covered for indications listed in the CPB:
S3840	DNA analysis for germline mutations of the RET proto-oncogene for susceptibility to multiple endocrine neoplasia type 2
ICD-10 codes covered if selection criteria are met:
C73	Malignant neoplasm of thyroid gland
C74.00 – C74.92	Malignant neoplasm of adrenal gland [when MEN2 is suspected]
C75.0 – C75.4	Malignant neoplasm of other endocrine glands and related structures
C75.5	Malignant neoplasm of aortic body and other paraganglia [when MEN2 is suspected]
C75.8	Malignant neoplasm with pluriglandular involvement, unspecified
C75.9	Malignant neoplasm of endocrine gland, unspecified
D10.30	Benign neoplasm of unspecified part of mouth
D10.39	Benign neoplasm of other parts of mouth
D35.00 – D35.02	Benign neoplasm of adrenal gland [when MEN2 is suspected]
D35.1	Benign neoplasm of parathyroid gland [when MEN2A is suspected]
D35.6	Benign neoplasm of aortic body and other paraganglia [when MEN2 is suspected]
D44.2	Neoplasm of uncertain behavior of parathyroid gland [when MEN2A is suspected]
D44.7	Neoplasm of uncertain behavior of aortic body and other paraganglia [when MEN2 is suspected]
E07.0	Hypersecretion of calcitonin
E21.0 – E21.5	Hyperparathyroidism and other disorders of parathyroid gland
E31.20	Multiple endocrine neoplasia [MEN] syndrome, unspecified
E31.21	Multiple endocrine neoplasia [MEN] type I
E31.22	Multiple endocrine neoplasia [MEN] type IIA
E31.23	Multiple endocrine neoplasia [MEN] type IIB
H18.891 – H18.899	Other specified disorders of cornea
K13.79	Other lesions of oral mucosa
L99	Other disorders of skin and subcutaneous tissue in diseases classified elsewhere [cutaneous lichen amyloidosis] [when MEN2A is suspected]
Q14.8	Other congenital malformations of posterior segment of eye
Q43.1	Hirschsprung's disease [only when first-degree blood relative with MEN2 or family history of MTC, PCC/PGL, or parathyroid adenoma/hyperplasia]
Q87.40	Marfan syndrome, unspecified
Q87.89	Other specified congenital malformation syndromes, not elsewhere classified
Z80.8	Family history of malignant neoplasm of other organs or systems [thyroid cancer]
Z83.41	Family history of multiple endocrine neoplasia [MEN] syndrome
Z83.49	Family history of other endocrine, nutritional and metabolic diseases [parathyroid adenoma/hyperplasia]
Z85.850	Personal history of malignant neoplasm of thyroid
Z85.858	Personal history of malignant neoplasm of other endocrine glands
Z86.018	Personal history of other benign neoplasm [when MEN2 is suspected]
Z86.03	Personal history of neoplasm of uncertain behavior [when MEN2 is suspected]
Z87.798	Personal history of other (corrected) congenital malformations
ICD-10 codes not covered for indications listed in the CPB:
C34.00 - C34.92	Malignant neoplasm of bronchus and lung [non-small cell lung cancer]

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Background

The RET proto‑oncogene encodes a receptor tyrosine kinase involved in cell growth and differentiation, and pathogenic variants in this gene are clinically significant in both hereditary and sporadic medullary thyroid carcinoma (MTC). Germline RET mutations are the established cause of multiple endocrine neoplasia type 2 (MEN2) syndromes and are used to determine hereditary cancer risk, guide surveillance, and inform prophylactic management strategies. In contrast, somatic RET alterations detected through tumor testing occur only within neoplastic tissue and do not indicate inherited disease but instead help characterize tumor biology and identify candidates for targeted RET‑directed therapies (Hu and Skefos, 2025). Multiple endocrine neoplasia types 2A (MEN2A) and 2B (MEN2B) are inherited in an autosomal dominant pattern with very high penetrance. The genetic defect in these disorders involves the RET proto-oncogene on chromosome 10.  Advances in the molecular genetics underlying the MEN2 syndromes have resulted in DNA testing becoming the optimal test for their detection (Ball, 2025).

Multiple endocrine neoplasia (MEN) syndromes are rare, inherited disorders in which two or more endocrine glands (e.g., pituitary, parathyroid, pancreas, or thyroid) develop noncancerous (benign) or cancerous (malignant) tumors, or grow excessively without forming tumors. There are several types of MEN syndromes: MEN type 1 (MEN1), MEN type 2A (MEN2A), MEN type 2B (MEN2B), and MEN type 4 (MEN4). Symptoms will vary depending on the affected glands.

MEN1, caused by an inactivating mutation of the MEN1 gene that encodes the nuclear protein menin, primarily features hyperplasia or, in some cases, adenomas of the parathyroid glands, leading to hyperparathyroidism, along with tumors of the pancreatic islet cells and/or the pituitary gland. MEN2A, caused by mutations in the RET proto-oncogene on chromosome 10, is characterized mainly by medullary thyroid carcinoma, pheochromocytoma, hyperplasia or occasionally adenomas of the parathyroid glands (resulting in hyperparathyroidism), and, less frequently, cutaneous lichen amyloidosis and/or Hirschsprung disease. MEN2B, which generally results from a single amino acid substitution at amino acid 918 in the RET protein leading to activation of RET proto-oncogene–mediated cellular processes, primarily presents with medullary thyroid carcinoma (MTC), pheochromocytoma, multiple mucosal and intestinal neuromas, and a marfanoid habitus. MEN4, caused by an inactivating mutation of the CDKN1B gene, is characterized mainly by adenomas and sometimes hyperplasia of the parathyroid glands (leading to hyperparathyroidism), as well as tumors of the pancreatic islet cells and/or the pituitary gland. The phenotype of MEN 4 is similar to that of MEN1 but does not include the cutaneous abnormalities associated with the latter. While these syndromes are genetically and clinically distinct, there is considerable overlap among them (Kirschner and Brock, 2025).

Genetic screening of family members of MEN2A patients is the diagnostic test of choice, and the availability of such testing has made biochemical screening for early medullary thyroid carcinoma largely obsolete. The specific RET mutation also predicts phenotypic characteristics, such as the aggressiveness of medullary thyroid carcinoma and the presence of other endocrinopathies, making it important for clinical management; however, other factors, such as older age at onset and higher tumor stage at diagnosis, may be more predictive of disease aggressiveness. Preimplantation genetic diagnosis and prenatal methods like chorionic villus sampling or amniocentesis have been used for antenatal diagnosis. In families with affected members, annual screening for hyperparathyroidism and pheochromocytoma should begin in adolescence, at 11 years of age for families with high-risk variants and at 16 years of age for those with moderate-risk variants, and continue indefinitely. Screening for hyperparathyroidism involves measuring serum calcium levels, while screening for pheochromocytoma includes inquiries about symptoms, measurement of pulse rate and blood pressure, and laboratory testing (Kirschner and Brock, 2025).

Genetic testing for RET germline mutation has shown 100% sensitivity and specificity for identifying those at risk for developing inherited medullary thyroid cancer (multiple endocrine neoplasia type 2A [MEN2A], MEN2B, or familial medullary thyroid carcinoma). Use of the genetic assay allows earlier and more definitive identification and clinical management of those with a familial risk for medullary thyroid cancer when compared to the existing standard of annual biochemical monitoring.

Medullary thyroid carcinoma (MTC) is surgically curable if detected before it has spread to regional lymph nodes. However, lymph node involvement at diagnosis may be found in up to 75% of patients for whom a thyroid nodule is the first sign of disease. Thus, there is an emphasis on early detection and intervention in families that are affected by the familial cancer syndromes of MEN types 2A and 2B and FMTC, which account for 25% of medullary thyroid cancer. After genetic counseling, most family members who test positive undergo surgery to remove the thyroid gland. First-degree relatives of those with MTC that appears to be sporadic in origin also undergo the biochemical test to verify that the patient's tumor is not caused by an inheritable form of this disease.

RET screening for the detection of genetic mutations is essential for effectively managing MTC. All individuals diagnosed with MTC, regardless of whether they have a family history of multiple endocrine neoplasia type 2 (MEN2), should undergo genetic analysis of the RET gene to confirm or exclude the inheritance of the disease. Additionally, RET screening is essential for assessing first-degree relatives of the proband to determine their potential carrier status, as well as to understand the relationship between the type of mutation and the aggressiveness of the disease, which informs the appropriate timing for prophylactic thyroidectomy. In cases of sporadic MTC, identifying somatic RET mutations also holds prognostic value, allowing for the selection of individuals who require more vigilant follow-up. The presence of somatic mutations in specific codons of the RET gene is associated with greater aggressiveness and a worse prognosis (Taccaliti et al., 2011).

Clinical variants of MEN2A may include cutaneous lichen amyloidosis (CLA) and, less commonly, Hirschsprung disease (HD). HD is a congenital disorder characterized by defective migration and colonization of enteric neural crest cells (ENCCs), which results in aganglionosis of the distal intestine and leads to partial or complete functional obstruction. Affected infants generally present with obstipation and abdominal distention. The genetic basis of HD is complex, with more than 40 genes implicated in its pathogenesis (Belkind‑Gerson and Pekarsky, 2025). RET mutations account for approximately 50% of familial HD cases (Tomuschat and Puri, 2015). HD may also occur as part of several monogenic syndromes, such as Bardet–Biedl, cartilage‑hair hypoplasia, Mowat–Wilson, Smith‑Lemli‑Opitz, and Waardenburg syndromes. In addition, HD may occur as part of specific RET‑associated cancer predisposition syndromes, most notably MEN2A. Evaluation for RET mutations is recommended in infants with HD who also have a family history of MEN2 (Lopez, 2025). Although RET coding variants are observed in roughly 50% of familial and 20% of sporadic HD cases, and RET regulatory variants contribute to risk in nearly 48% of all patients, these variants typically exhibit low penetrance in isolated HD cases. Therefore, effective risk prediction and genetic counseling depend on family history, sex, aganglionic segment length, and systematic assessment for syndromic features—particularly when a syndromic form of HD is suspected—targeted testing of RET, EDNRB, and additional syndrome‑associated genes may be informative (Tilghman et al., 2019).

Fialkowski et al. (2008) stated that MEN2A is a genetic syndrome manifesting as MTC, hyperparathyroidism, and pheochromocytoma (PCC). MEN 2A results from mutations in the RET proto-oncogene. Hirschsprung disease (HD) is a congenital condition characterized by a blockage of the large intestine due to poor muscle movement in the bowel. HD is a rare manifestation of MEN 2A and has been described in known MEN 2A families. These investigators described two MEN 2A families that were only identified after the diagnosis of HD. Kindred 1: A boy presented in infancy with HD. Genetic screening revealed a C609Y mutation, which is consistent with MEN 2A. Evaluation of his sister, father, and grandmother revealed the same mutation. All three had thyroidectomies demonstrating C-cell hyperplasia. The grandmother had a microscopic focus of MTC. Kindred 2: An infant boy and his sister were diagnosed with HD as neonates. Genetic testing demonstrated a C620R gene mutation consistent with MEN 2A. Total thyroidectomies revealed metastatic MTC in the father and C-cell hyperplasia in both children. The authors concluded that HD can be the initial presentation of MEN 2A. They strongly recommend that genetic screening be performed in patients presenting with HD, looking for the known RET mutations associated with MEN 2A. If a mutation consistent with MEN 2A is detected, genetic screening of all first-degree relatives in the kindred is recommended.

In a case report, Pandey et al. (2011) emphasized that all patients with a history of HD should consider screening for RET mutations (it should be noted that RET mutations are the predominant but only one of a number of possible causes of HD), as there is a well-established association between HD and MEN 2A. If present, this could facilitate early diagnosis of MEN 2A with resultant thyroidectomy prior to the onset of MTC or at least prior to the development of metastatic disease.

Vaclavikova et al. (2012) noted that inactivating germline mutations in the RET proto-oncogene are the major genetic cause of HD. In some cases, HD can be associated with MTC that is commonly caused by activating RET mutations. These investigators performed retrospective and prospective genetic analyses of 157 patients with HD operated on between December 1979 and June 2011; DNA was isolated from peripheral leukocytes. Patients with HD, as well as family members, were tested for RET mutations by direct sequencing and single-strand conformation polymorphism methods. RET mutations were detected in 16 patients (10%). Association with MTC was found in two families; the other eight families had a mutation with potentially high risk of MTC development, and four novel mutations were detected. Total colonic aganglionosis was noted to have a high mutation detection rate (40%). Three patients underwent total thyroidectomy (two had clinical manifestations of MTC, and one had C-cell hyperplasia). The authors concluded that these findings showed the benefit of systematic RET mutation screening in HD patients in order to identify the risk of MTC in the preclinical stage of the disease. All patients should be tested for RET mutations at least in exon 10, and now additionally in exons 11 and 13 as well.

Pheochromocytomas (PCC) and paragangliomas (PGL) are rare neuroendocrine tumors with up to 32% of patients having a germline mutation in one of the known common susceptibility genes, including RET. RET mutation carriers usually have other clinical characteristics of MEN2A or MEN2B (Fishbein et al., 2013). Although most PCCs are sporadic, genetics in the development of these tumors is becoming more and more essential these days. RET proto-oncogene has shown to play an important role in the pathogenesis of PCCs. In MEN2 patients, the PCCs are usually of adrenal localization, benign and bilateral in more than 50% of patients. Karasek et al. (2010) reported a higher malignancy risk in children with MEN2B‑associated PCCs compared with MEN2A or sporadic disease. MEN2-related tumors overexpress phenylethanolamine N-methyltransferase, thus the biochemical phenotype is consistent with hypersecretion of epinephrine in large amounts, resulting in an early clinical phenotype characterized by attacks of palpitations, nervousness, anxiety, and headaches rather than more common patterns of hypertension to be seen in other hereditary tumor. The increased plasma and urinary levels of catecholamine O-methylated metabolite of epinephrine–metanephrine in MEN2 patients distinguish them from those with VHL and SDHx mutations (Karasek et al., 2010).

Multiple endocrine neoplasia type 2 (MEN2) encompasses three phenotypes—MEN2A, familial medullary thyroid carcinoma (FMTC), and MEN2B—and is clinically diagnosed when characteristic endocrine tumors are present. MEN2A is clinically diagnosed by the presence of two or more characteristic endocrine manifestations in an individual or their close relatives (medullary thyroid carcinoma [MTC], pheochromocytoma [PCC], or parathyroid adenoma/hyperplasia). FMTC is clinically diagnosed when two or more family members have MTC without clinical or biochemical evidence of PCC or parathyroid disease. MEN2B is clinically diagnosed by early-onset MTC accompanied by mucosal neuromas of the lips and tongue, medullated corneal nerve fibers, distinctive facies with enlarged lips, and a marfanoid habitus. Molecular confirmation is established through identification of a heterozygous germline gain‑of‑function RET pathogenic variant, and RET genetic testing is recommended for all individuals who meet clinical diagnostic criteria because genotype‑specific RET variants determine surveillance intervals, prophylactic thyroidectomy timing, and family-based risk assessment. Diagnostic evaluation includes biochemical testing for elevated serum calcitonin (for MTC), plasma free metanephrines or 24‑hour urine metanephrines (for PCC), and serum calcium/PTH (for hyperparathyroidism). Molecular testing options include RET single‑gene sequencing, select‑exon sequencing for MEN2A/FMTC hotspots (exons 10–11, 13–16), targeted variant testing for MEN2B (e.g., p.Met918Thr), or full‑gene sequencing/multigene hereditary tumor panels when initial testing is nondiagnostic (Eng and Plitt, 2023).

According to the National Comprehensive Cancer Network (NCCN) guidelines on "Neuroendocrine and Adrenal Tumors" (Version 3.2025), multiple endocrine neoplasia type 2 (MEN2) is an autosomal dominant hereditary cancer syndrome caused by a germline activating (gain‑of‑function) pathogenic variant in the RET proto‑oncogene located on chromosome 10q11.2, resulting in constitutive activation of the RET tyrosine kinase receptor. MEN2 encompasses the clinical phenotypes MEN2A, familial medullary thyroid carcinoma (FMTC), and MEN2B. MEN2A is clinically diagnosed by the presence of two or more MEN2A‑associated manifestations—medullary thyroid carcinoma (MTC), pheochromocytoma (PCC), or parathyroid adenoma/hyperplasia—in a single individual or among close relatives, and may also be associated with cutaneous lichen amyloidosis or Hirschsprung disease. FMTC represents a variant within the MEN2 spectrum characterized by familial MTC in the absence of PCC or parathyroid disease. A clinical diagnosis of MEN2B includes MTC, PCC, mucosal neuromas of the lips and tongue, medullated corneal nerve fibers, distinctive facies with enlarged lips, marfanoid habitus, and may include alacrima (inability to cry tears). The NCCN Guidelines recommend genetic counseling and germline RET testing for individuals with a clinical diagnosis or suspicion of MEN2, as well as evaluation of at‑risk relatives. NCCN further emphasizes that all patients with MEN2 should be evaluated for a coexisting pheochromocytoma prior to anesthesia or any invasive procedure because of the risk of catecholamine‑mediated complications.

Familial medullary thyroid carcinoma (FMTC) is defined specifically by the inheritance of MTC without associated endocrinopathies such as pheochromocytoma or hyperparathyroidism. While FMTC is a variant of the MEN2 family (caused by RET gene mutations), it is distinguished from MEN2A and MEN2B, which do include these other tumors (Moo-Young et al, 2016). FMTC should be suspected in families with more than one individual diagnosed with MTC in the absence of pheochromocytoma or parathyroid adenoma/hyperplasia (Eng and Plitt, 2023).

The NCCN recommends that genetic counseling and testing be offered to individuals with a clinical diagnosis of MEN2, including familial medullary thyroid cancer (MTC). Familial MTC, now considered a variant of MEN2A. is typically multifocal and preceded by C-cell hyperplasia. Familial MTC that arises in the absence of other endocrine malignancies or disorders is generally the least aggressive form.

In summary, RET proto-oncogene tests can identify familial disease-causing RET point mutations in members of families known to be affected by inherited MTC. For individuals in families with defined RET point mutations, the results of these tests can guide decisions regarding prophylactic thyroidectomy or the need for continued monitoring. Additionally, RET proto-oncogene tests can help differentiate sporadic tumors from familial cancers in patients with MTC who do not have a prior family history of the disease, as well as in their first-degree relatives if a germline RET mutation is identified. Furthermore, RET proto-oncogene tests hold clinical value for individuals with Hirschsprung disease.

Non-Small Cell Lung Cancer

Yoshida et al. (2013) noted that the recent discovery of ROS1 gene fusion in a subset of lung cancers has raised clinical interest because ROS1 fusion-positive cancers are reportedly sensitive to kinase inhibitors. To better understand these tumors, these researchers examined 799 surgically resected non-small cell lung cancers (NSCLCs) by reverse transcriptase polymerase chain reaction (PCR) and identified 15 tumors harboring ROS1 fusion transcripts (2.5% of adenocarcinomas). The most frequent fusion partner was CD74, followed by EZR. The affected patients were often younger, non-smoking female individuals, and they had overall survival (OS) rates similar to those of the ROS1 fusion-negative cancer patients. All the ROS1 fusion-positive tumors were adenocarcinomas except for one, which was an adenosquamous carcinoma. Histologic examination identified at least a focal presence of either solid growth with signet-ring cells or cribriform architecture with abundant extracellular mucus in 53% of the cases. These two patterns were reportedly also characteristic of anaplastic lymphoma kinase (ALK)-rearranged lung cancers, and these data suggested a phenotypic resemblance between the ROS1-rearranged and ALK-rearranged tumors. All tumors except for one were immune-reactive to thyroid transcription factor-1. Fluorescence in situ hybridization (FISH) using ROS1 break-apart probes revealed positive rearrangement signals in 23% to 93% of the tumor cells in ROS1 fusion-positive cancers, which were readily distinguished using a 15% cutoff value from 50 ROS1 fusion-negative tumors tested, which showed 0% to 6% rearrangement signals. However, this perfect test performance was achieved only when isolated 3' signals were included along with classic split signals in the definition of rearrangement positivity. Fluorescence in situ hybridization signal patterns were unrelated to 5' fusion partner genes. All ROS1 fusion-positive tumors lacked alteration of epidermal growth factor receptor (EGFR), KRAS, HER2, ALK, and RET genes.

Lira et al. (2014) stated that approximately 7% of NSCLCs harbor oncogenic fusions involving ALK, ROS1, and RET. Although tumors harboring ALK fusions are highly sensitive to crizotinib, emerging preclinical and clinical data demonstrated that patients with ROS1 or RET fusions may also benefit from inhibitors targeting these kinases. Using a transcript-based method, these investigators designed a combination of 3' overexpression and fusion-specific detection strategies to detect ALK, ROS1, and RET fusion transcripts in NSCLC tumors. They validated the assay in 295 NSCLC specimens and showed that the assay is highly sensitive and specific. ALK results were 100% concordant with FISH (n = 52) and 97.8% concordant with IHC (n = 179) [sensitivity, 96.8% (95% confidence interval [CI]: 91.0% to 98.9%); specificity, 98.8% (95% CI: 93.6% to 99.8%)]. For ROS1 and RET, these researchers also observed 100% concordance with FISH (n = 46 and n = 15, respectively). They identified 7 ROS1 and 14 RET fusion-positive tumors and confirmed the fusion status by RT-PCR and FISH. One RET fusion involved a novel partner, cutlike homeobox 1 gene (CUX1), yielding an in-frame CUX1-RET fusion. ROS1 and RET fusions were significantly enriched in tumors without KRAS/EGFR/ALK alterations. ALK/ROS1/RET/EGFR/KRAS alterations were mutually exclusive. The authors concluded that as a single-tube assay, this test showed promise as a more practical and cost-effective screening modality for detecting rare but targetable fusions in NSCLC.

Wijesinghe et al. (2015) noted that ROS1 and RET gene fusions were recently discovered in NSCLC as potential therapeutic targets with small-molecule kinase inhibitors. The conventional screening methods for these fusions are time-consuming and require samples of high quality and quantity. These researchers described a novel and efficient method by coupling the power of multiplexing PCR and the sensitivity of mass spectrometry. The multiplex mass spectrometry platform simultaneously tests samples for the expression of 9 ROS1 and 6 RET fusion genes. The assay incorporated detection of wild-type exon junctions immediately upstream and downstream of the fusion junction to exclude false-negative results. To flag false positives, the system also comprised two independent assays for each fusion gene junction. The characteristic mass spectrometric peaks of the gene fusions were obtained using engineered plasmid constructs. Specific assays targeting the wild-type gene exon junctions were validated using complementary DNA from lung tissue of healthy individuals. The system was further validated using complementary DNA derived from NSCLC cell lines that express endogenous fusion genes. The expressed ROS1-SLC34A2 and CCDC6-RET gene fusions from the NSCLC cell lines HCC78 and LC-2/ad, respectively, were accurately detected by the novel assay. The assay is extremely sensitive, capable of detecting an event in test specimens containing 0.5% positive tumors. The authors concluded that the novel multiplexed assay is robustly capable of detecting 15 different clinically relevant RET and ROS1 fusion variants.

Rossi et al. (2017) stated that immunohistochemistry (IHC) is a widely tested, low-cost, and rapid ancillary technique available in all laboratories of pathology. This method is generally used for diagnostic purposes, but several studies have investigated the sensitivity and specificity of different immunohistochemical antibodies as a surrogate test in the determination of predictive biomarkers in NSCLC, particularly for epidermal growth factor receptor (EGFR) gene mutations, ALK gene, and ROS1 rearrangements. In this review, a critical examination of the works comparing the consistency of IHC expression and conventional molecular techniques to identify genetic alterations with predictive value in NSCLC was discussed. Summarizing, data on sensitivity and specificity of antibodies against ALK and ROS1 are very consistent, and the time has come to trust in IHC at least as a cost-effective screening tool to identify patients with rearranged tumors in clinical practice. On the other hand, mutant-specific antibodies against EGFR demonstrated good specificity but low-to-fair sensitivity, raising some caution on their employment as robust predictive biomarkers. A brief comment on preliminary experiences with antibodies against BRAF, RET, HER2, and c-MET was also included.

Hess and colleagues (2021) noted that contradictory and limited data are available regarding the presentation and outcomes of patients with RET-fusion positive metastatic NSCLC as compared to patients without RET fusions. In an observational study, these researchers employed a linked electronic health records (EHR) database to genomics testing results and compared characteristics, tumor response, progression-free survival (PFS), and overall survival (OS) outcomes by RET fusion status among patients with metastatic NSCLC treated with standard therapies. Adult patients with metastatic NSCLC with linked EHR and genomics data who received systemic anti-cancer therapy on or after January 1, 2011, were eligible. Adjusted, using all available baseline covariates, and unadjusted analyses were performed to compare tumor response, PFS, and OS between patients with RET-fusion positive and RET-fusion negative disease as detected by next-generation sequencing (NGS). Tumor response outcomes were analyzed using Fisher's exact test, and time-to-event analyses were conducted using the Cox proportional hazards model. There were 5,807 eligible patients identified (RET+ cohort, n = 46; RET- cohort, n = 5,761). Patients with RET fusions were younger, more likely to have non-squamous disease, and be non-smokers, and they had better performance status (all p < 0.01). In unadjusted analyses, there were no significant differences in tumor response (p = 0.17) or PFS (p = 0.06), but OS was significantly different by RET status (hazard ratio [HR], HR = 1.91, 95% CI: 1.22 to 3.0, p = 0.005). There were no statistically significant differences by RET fusion status in adjusted analyses of either PFS or OS (PFS HR = 1.24, 95% CI: 0.86 to 1.78, p = 0.25; OS HR = 1.52, 95% CI: 0.95 to 2.43, p = 0.08). The authors concluded that patients with RET fusions had different baseline characteristics that contributed to favorable OS in unadjusted analysis. However, after adjusting for baseline covariates, there were no significant differences in either OS or PFS by RET status among patients treated with standard therapy prior to the availability of selective RET inhibitors.

The National Comprehensive Cancer Network guidelines on "Lung cancer screening" (Version 1.2026) does not provide a recommendation for RET proto-oncogene testing.

Pheochromocytoma / Paraganglioma

Pheochromocytoma is a rare, usually noncancerous (benign) tumor that develops in cells in the center of an adrenal gland, leading to attacks of raised blood pressure, palpitations and headache. Paraganglioma is a tumor of the tissue composing the paraganglion, a small round body containing chromaffin cells, found near the aorta and in the kidney, liver heart and gonads.

Brito and associates (2015) noted that the presence of germline mutations in sporadic pheochromocytomas and paragangliomas (SPPs) may change the clinical management of both index patients and their family members. However, the frequency of germline mutations in SPPs is unknown. In a systematic review, these researchers described the frequency of germline mutations in SPPs and determined the value of testing index patients and their family members for these mutations. They searched databases through June 2012 for observational studies of patients with SPPs who underwent germline genetic testing. The criteria used to define sporadic tumors were

1. the absence of a family history of PCC/PG
2. the absence of syndromic features
3. the absence of bilateral disease, and
4. the absence of metastatic disease.

These investigators included 31 studies including 5,031 patients (mean age of 44 years). These patients received tests for any of these 10 mutations: SDHAF2, RET, SDHD, SDHB, SDHC, VHL, TMEM127, MAX, isocitrate dehydrogenase (IDH) mutation and NF1. The overall frequency of germline mutation in SPP was 551 of 5,031 (11%); when studies with patients fulfilling 4 criteria for sporadic tumors were used, the frequency was 171 of 1,332 (13%). The most common germline mutation was SDHB 167 of 3,611 (4.6%). Little outcome data were available to assess the benefits of genetic testing in index cases and family members. The authors concluded that the frequency of germline mutations in SPPs is approximately 11 to 13% and the most common mutations affect less than 1 in 20 patients (5%). They stated that the value of testing for germline mutations in patients with SPPs and their family members is unknown, as the balance of potential benefits and harms remains unclear.

The NCCN Panel on "Neuroendocrine and adrenal tumors" (Version 3.2025) state MEN2, caused by germline activating mutations in the RET proto‑oncogene, is associated with the development of pheochromocytoma, which—when present—is commonly bilateral.

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References