Algorithm of genetic diagnosis for patients with head and neck paraganglioma—update

Paragangliomas are rare tumors originating from the paraventricular bodies of the autonomic nervous system located in the adrenal glands, chest, abdomen, pelvis and head and neck. Tumors of this type account for 0.5% of head and neck cancers, 0.03% of all cancers and their incidence is estimated at 1–30/100,000 per year. Head and Neck Paragangliomas (HNPGL) are localized in carotid body, tympanic cavity or jugular foramen. It is established that HNPGL may be associated with mutations of the SDH complex, with SDHD being the most prevalent. However, SDHB, SDHC and SDHAF are also potential causes. The aforementioned mutations are influenced by various risk factors, including young age, a positive family history of paraganglioma, the presence of metastases and gender The purpose of this study is to summarize the results of genetic testing performed on patients with head and neck paraganglioma and to create an up-to-date genetic diagnosis algorithm for patients with HNPGL based on previous studies published in the literature that can be used in daily practice. Several papers observed that among SDHD mutation carriers, most or all of those studied had HNPGL, and SDHB mutations were more frequently found in the presence of metastasis. Based on the results, it was concluded that there is no basis for genetic testing for VHL in patients without a positive family history. In each algorithm proposed by different authors, proposals for rational genetic diagnosis were analyzed based on the studies cited by the author and the analyses included in our paper. For the analysis of the treatment algorithms, the following were included: Martin, Mannelli, Neumann, Gupta. Subsequently, publications related to the genetic diagnosis of HNPGL were analyzed to verify the proposed algorithms in light of the latest genetic studies and to establish an updated diagnostic management scheme.


Introduction
Paragangliomas are rare tumors originating from the paraventricular bodies of the autonomic nervous system (1) located in the adrenal glands, chest, abdomen, pelvis and head and neck.Tumors of this type account for 0.5% of head and neck cancers, 0.03% of all cancers (2), and their incidence is estimated at 1-30/100,000 per year (3).Paragangliomas can originate  Identification of studies via databases and registers.PCC, Pheochromocytoma.
Interestingly, this study also found higher levels of methoxytyramine, the O-methylated metabolite of dopamine in benign HNPGLs, which contrasts the prior suggestion of an increase in methoxytyramine and dopamine in metastatic PGLs (10).The tumors are most often benign, but 10-15% may metastasize to bone, liver, lung or lymph nodes (1,(11)(12)(13).The current World Health Organization classification divides these tumors into non-metastatic and metastatic (13)(14)(15) and recommends using this term instead of "malignant tumors." They can occur as sporadic, multiple or as part of genetically determined syndromes (3,7,(16)(17)(18).Genetic testing in patients with paragangliomas is complementary to diagnosis and allows management to be targeted to detect potential multiple tumors or metastases.In addition, tests performed in family members can lead to the diagnosis of paraganglioma at an early stage, while they are still asymptomatic, and implement appropriate management.It is established that HNPGL may be associated with mutations of the SDH complex, with SDHD being the most prevalent.However, SDHB, SDHC and SDHAF are also potential causes.The aforementioned mutations are influenced by various risk factors, including young age, a positive family history of paraganglioma, the presence of metastases and gender (1,3,5,12).
To date, no such work has been conducted for the Polish population, and in determining the indications for diagnosis it is necessary to rely on the results of studies for groups of patients from other countries.Nevertheless, at present, the extension of diagnostics in patients with HNPGL by genetic testing should be an essential part of the treatment process.To this end, bearing in mind also the potential costs of testing, it is worth using a diagnostic algorithm.

Aim of the study
The purpose of this study is to summarize the results of genetic testing performed on patients with head and neck paraganglioma and to create an up-to-date genetic diagnosis algorithm for patients with HNPGL based on previous studies published in the literature that can be used in daily practice.

Methodics
Databases from Medline, Embase, Scopus, Google Scholar from 2000 to X 2023 were searched.Inclusion criteria included papers Data were downloaded on genetic test results and their correlation with clinical data.The genetic diagnostic algorithms proposed so far were analyzed.Algorithms related to diagnostic imaging or proposed treatment were not included in the study.
The selection of publications for the study is shown in Figure 1.

Results
The results of genetic testing for HNPGL are shown in Table 1, and papers with repeated patient groups are omitted.The most common mutations found were in the SDH complex (2,22,25,27,28), with mutations within SDHD most common for HNPGL, and SDHB, SDHC and SDHAF2 mutations less common.The papers showed that the main risk factors for mutations were young age, a positive family history of PCC/PGL, multifocality (12,20,24), metastatic tumors (lymph nodes, bone, liver, lung) and gender (22).However, there remained a group of patients without risk factors who also had genetic mutations (17).
Several papers observed that among SDHD mutation carriers, most or all of those studied had HNPGL (8,12,18,19,22,29,30), and SDHB mutations were more frequently found in the presence of metastasis (12,18,19,22).Based on the results, it was concluded that there is no basis for genetic testing for VHL in patients without a positive family history (12,31).
In each algorithm proposed by different authors, proposals for rational genetic diagnosis were analyzed based on the studies cited by the author and the analyses included in our paper.For the analysis of the treatment algorithms, the following were included: Martin (32), Mannelli (19), Neumann (20), and Gupta (31).Subsequently, publications related to the genetic diagnosis of HNPGL were analyzed to verify the proposed algorithms in light of the latest genetic studies and to establish an updated diagnostic management scheme.
The first of these algorithms, proposed by Martin et al. (32), deals with the diagnosis of paragangliomas in general, without dividing them into HNPGL or other tumors, and only proposes genetic diagnosis with SDHD, SDHB and SDHC testing in one aspect, without considering possible risk factors.With the development of genetic testing, new and different schemes have emerged, making diagnosis more targeted, depending on the primary location and the presence of additional factors (19) (Figure 2).
Frontiers in Neurology 05 frontiersin.org In the above scheme, a positive family history and multifocal tumor were taken as risk factors for mutations, but the age of the patients was not taken into account.In the description included in the paper, it is suggested in the aspect of age, to take 50 years as a cut-off point and above this age to abandon genetic testing, since only 5% of patients in this group showed mutations.Other authors (20,31) suggest 40 years or even 35 years as the cutoff age (12, 20) (Figure 3).
In the scheme proposed by Neumann et al. (20), genetic testing was to be performed only in patients with risk factors such as positive family history, previous pheochromocytoma, multiple tumors, malignant tumors, age under 40 years or gender.As shown in later studies, also patients without the aforementioned risk factors may carry genetic mutations, and genetic diagnosis should be performed for this group as well (31, 33).The publication of subsequent studies resulted in the algorithm described by Gupta et al. (31), shown in Figure 4.
The algorithm proposed by Gupta applies only to HNPGL, dates back to 2019, and proposes two stages of testing to reduce diagnostic costs.In the first stage, the authors recommend genetic testing according to the risk factors found and an algorithm for patients without such factors, with the firąst stage additionally distinguishing age groups, unlike previous authors who proposed genetic testing for the entire SDHx group (19,20,32).This approach seems reasonable because, as shown in earlier work, 2 mutations are not found in the same patient (20).However, in light of the results of other studies, it seems reasonable to supplement the above algorithm with SDHAF2 testing, in patients younger than 40 years and in multifocal tumors (4, 21) (Figure 5).

Discussion
Over the past few years, one can observe changes related to the approach to genetic diagnosis in paragangliomas.Undoubtedly, this is related to the rapid expansion of knowledge about different types of mutations and their impact on tumor biology.Not insignificant is the fact of developing diagnostic methods and improving accessibility to such tests.Based on the data collected from the literature, it can be noted that initially genetic testing was recommended for patients with HNPGL only in groups with risk factors, i.e., patients with young age, positive family history of pheochromocytoma, in multiple, metastatic tumors (formerly called malignant).As research has evolved, some recommendations have evolved or new ones have emerged.
Young age for different authors did not always mean the same metric age.Depending on the author, it ranged from 35 to 50 years of age (12,19,20,22,34).Burnichon   syndromes associated with paraganglioma formation (1,7,13,33,35,36).In contrast to multiple tumors, mutations in SDHB have been found in this group of patients when the ability to give metastasis to other organs such as lymph nodes, bone, liver (1,11,12).Due to the more malignant clinical course in such cases, Zhu et al. (34) authors suggested including family members in genetic and clinical care in situations where SDHB mutations were found.
In the genetic diagnostic algorithm, Gupta (31) proposes separating the group of patients with multiple tumors into those with and without metastasis, and if metastatic features are found, perform first-line testing for SDHB.In light of the presented study results, it seems reasonable to include such a procedure in the genetic  Updated algorithm based on Gupta's proposal. 10.3389/fneur.2024.1437027 Frontiers in Neurology 08 frontiersin.orgdiagnostic algorithm.When planning diagnosis of family members can furthermore take into account the gender of the patients, which is important in determining the mutation carrier.Transmission of the mutation from the father increases the risk of behavior, while in the case of carrier in the mother, there is no need for screening in children (4,16,23).Some controversy surrounds genetic testing in patients with isolated eardrum paraganglioma (Fisch A type).In a 2009 Burnichon study, 330 patients were studied with tumors in the tympanic cavity and none of them had mutations typical of HNPGL (12).However, the paper did not specify the number of patients with this type of paraganglioma.Based on the results, it was concluded that patients with isolated eardrum HNPGL did not require genetic testing.In subsequent studies, this group of patients was excluded from genetic diagnosis (12).In 2012, Piccini et al. (17) included these patients in their study and obtained positive mutation results in 6/16 patients, of which 3 patients with type A paraganglioma were accompanied by multiple lesions and 3 had isolated tumors of the tympanic cavity.Also, isolated eardrum HNPGL were diagnosed in a study by Heesterman (23), where patients with SDHD mutations were observed.Therefore, it seems reasonable to include patients with tympanic PGLs in future genetic studies, not only in situations where they are part of multifocal tumors.
In light of the findings presented regarding the necessity of genetic testing in HNPGL in patients with risk factors.There remains, however, a group of patients with isolated tumors not burdened by such factors.Previous publications have shown mutations in this group of patients as well, such as in the work of Mannelli (19) where 14.3% of patients with a negative family history and isolated tumors were found to have mutations.This study was extended several years later by Piccini (17) where mutations were found in 18.8% of patients without risk factors.Also among the patients studied by Chen et al. (22) were patients with SDHD mutations without risk factors.Therefore, a modern diagnostic algorithm should include all patients with HNPGL.
In the analyses presented so far, studies for the Polish population have been included in a multicenter study by Neumann et al. (20), who included 23 patients from Poland.However, the results were analyzed collectively for the entire group of 598 patients with HNPGL, so it is difficult to draw conclusions for our country's population on this basis.In 2008, a description of 2 cases of familial HNPGL in Polish patients was presented (37).In 2015, a paper describing 14 patients with multiple head and neck chaperones was also published based on the Polish group (38).In none of these papers, however, genetic testing was performed, so it is reasonable to initiate genetic testing also in the Polish population in order for the management algorithm to be based not only on data from the literature but also on the results of the study.

Conclusion
A genetic diagnosis of paraganglioma should be an integral part of the diagnostic process.The use of this diagnostic tool enables the early detection of pathological lesions and the initiation of treatment in family members of patients, thereby reducing the risk of complications.Furthermore, the rationalization of genetic diagnostics has an impact on the cost and efficiency of the process, which may lead to the applicability of diagnostic algorithms in clinical practice.

FIGURE 2 Flow
FIGURE 2Flow chart suggested for genetic analysis in patients affected by Pheos or PGLs.The genes reported in the boxes are those more likely to be found mutated according to clinical picture.MTC, Medullary thyroid carcinoma; HMGB, hemangioblastomas; CC, cancer/cysts.

TABLE 1
Genetic testing in patients with HNPGL in order of publication years.SDHB missense, SDHB truncating.SDHB less frequent in HNPGL, more frequent in PPGL.SDHB and SDHD in slightly younger PPGL patients.In this study, the PPGL and HNPGL groups were considered together.risk of PPGL and metastasis SDHB truncating increases the risk of PPGL and malignancy by 20% describing genetic findings in patients with head and neck paragangliomas.Original papers, review papers, and case reports and family studies describing a genetic background or family history of HNPGL were included in the review.Search words used-head and neck paraganglioma (HNPGL) genetic spectrum, HNPGL genetic classification, hereditary HNPGL, SDH mutations in HNPGL, genetic overview of HNPGL, genetic testing in HNPGL.We searched for all results related to the genetic background of HNPGL as well as included review papers that proposed algorithms for genetic diagnosis.Papers examining paragangliomas outside the head and neck region were excluded from the analysis.Also, analyses investigating the molecular mechanisms of mutation effects on PGL development, without clinical analysis, were not included in this paper.Due to the rarity of HNPGL, case reports and family line studies were included in the paper.
Single tumors, negative family history of HNPGL 14.3% with mutations, 82% of germline mutations detected in patients under 50 years of age Neumann 2009 (20) 598 patients from different countries, the largest study involving only HNPGL, SDHD, SDHC, SDHB 183 (30.6%) patients-germline mutation in SDHx gene, frequency (decreasing): SDHD, SDHB, SDHC Six factors were found to suggest the presence of a germline mutation: age (<40 years), gender, number of tumors (single or multiple), tumor biology (malignant or benign HNP), previous chaperone tumors and family history of HNPGL or pheochromocytoma Bayley 2010 (21) 443 patients with seemingly sporadic PCC/PGL Tested SDHAF2 SDHAF2 mutation testing is warranted for young patients with HNPGL if no mutations in SDHD, SDHB, and SDHC are found SDHAF2-early onset of disease and high risk of multifocal, some patients were asymptomatic, tumors were detected through family coverage, early onset-diagnosis at an average age of 33.Patients inheriting mutation from mother, do not have PGL disease ("cancer free carriers") Piccini 2012 (17) 79 patients with HNPGL 114 tumors (33 patients previously described in Mannelli 2009) SDHA, SDHB, SDHC, SDHD, SDHAF2, VHL, MAX, and TMEM127 Division into 3 groups:Positive family history-all-had SDHD mutations.Multiple HNPGL, family history negative-all-were carriers of SDHD Without risk factors (SDHD, VHL, SDHB, SDHAF)-18.8%carried germline mutations 16 cases of eardrum tumors, 5 showed mutations of which 3 were isolated tumors and 3 had other HNPGL.-Theonly study to show mutations in et al. determined a young age for patients <35 years old, with SDHD, SDHB and SDHC mutations (12).Mannelli et al. did not recommend genetic testing in patients >50 years