Abstract
Introduction.
Ameloblastoma is an aggressively growing, highly recurrent odontogenic jaw tumor. Its association with BRAFV600E mutation is an indication for BRAFV00E-inhibitor therapy The study objective was to identify a sensitive low-cost test for BRAFV600E-positive ameloblastoma. We hypothesized that immunohistochemical staining (IHC) of formalin-fixed paraffin-embedded (FFPE) tissues for BRAFV600E mutation is a low-cost surrogate for BRAFV600E gene sequencing when laboratory resources are inadequate for molecular testing.
Methods.
Tissues from 40 ameloblastoma samples were retrieved from either FFPE blocks, RNAlater™ stabilization solution (RSS) or samples inadvertently pre-fixed in formalin before transfer to RNAlater™ (FFRSS). BRAFV600E mutation was assessed by Direct Sanger Sequencing (DSS), Amplification Refractory Mutation System (ARMS)-PCR and IHC.
Results.
BRAFV600E mutation was detected by IHC, ARMS-PCR and DSS in 93.33%, 52.5% and 30% of samples respectively. Considering DSS as standard BRAFV600E detection method, there was significant difference between the three detection methods (χ2(2)=31.34, p< 0.0001). Sensitivity and specificity of IHC were 0.8 (95% CI: 0.64–0.90) and 0.9 (95% CI: 0.75–0.99) respectively, while positive predictive value (PPV) and negative predictive value (NPV) were 0.9 and 0.8 (Fischer’s test, p<0.0001) respectively. Sensitivity and specificity of ARMS-PCR detection method were 0.7 (95% CI: 0.53–0.80) and 0.9 (95% CI=0.67–0.98) respectively, while PPV and NPV were 0.9 and 0.6 respectively (Fischer’s test, p<0.0001).
Conclusion.
Low-cost and less vulnerability of IHC to tissue quality make it a viable surrogate test for BRAFV600E detection in ameloblastoma. Sequential dual IHC and molecular testing for BRAFV600E will reduce equivocal results that could exclude some patients from BRAFV600E-inhibitor therapies.
Keywords: ameloblastoma, recurrence, diagnostics, BRAF, targeted therapy
INTRODUCTION
Ameloblastoma is the most common odontogenic tumor of epithelial origin1 and the most prevalent odontogenic tumor in developing countries2. Ameloblastoma has a locally-invasive and aggressive growth pattern, about 70% recur depending on mode of treatment and 2% metastasize to other sites3,4. Up to 80% of ameloblastomas display genetic mutation of BRAF, a serine/threonine protein kinase activating the MAPK/ERK-signaling pathway3. This results in constitutive activation of BRAF protein downstream of MEK/ERK with resultant neoplastic transformation. There is a subset of ameloblastomas with mutations in non-MAPK signaling genes like smoothened (SMO), a G protein-coupled receptor and signaling effector component of sonic hedgehog (SHH) signaling pathway3,5.
Over 90% of the BRAF mutations have a substitution of valine for glutamate at codon 600 (V600E)6,7. The high BRAFV600E mutation in ameloblastomas causes dysregulation of MAPK/ERK-mediated cell proliferation and cell survival similarly observed in other BRAF-mutated tumors like melanoma, colorectal and thyroid cancers where these functions are constitutively activated8,9. Additionally, BRAFV600E mutation blocks apoptosis and regulate proliferation and aggressiveness of thyroid cancers as well as growth of aggressive colorectal cancer phenotypes. A systematic review with meta-analysis clearly presented several studies that claimed that BRAFV600-mutated ameloblastomas occur more in younger individuals, develop more in the mandible relative to the maxilla and display longer delayed recurrence than BRAF-wild type tumors10. All these point to BRAFV600E as a potential diagnostic and prognostic marker as well as therapeutic target.
Direct Sanger sequencing (DSS) is a well-accepted method for diagnosis of BRAFV600E mutation in patients with melanoma and thyroid cancer because several FDA approved BRAF inhibitor therapies reduce disease progression11. However, sensitivity of DSS to detect BRAFV600E mutated alleles from wild-type alleles is variable because tumor cell proportion in the sample material affects DSS sensitivity. Genetic methods like high resolution melting (HRM) analysis, pyrosequencing, amplification refractory mutation system (ARMS) or allele-specific PCR, and next generation sequencing (NGS) are other testing methods12. Interestingly, immunohistochemistry (IHC), a widely available low-cost technique is a highly sensitive and specific detection method for BRAFV600E in melanoma and thyroid cancer13,14 which makes IHC a very useful adjunctive test for BRAFV600E in ameloblastoma.
Globally it is yet to be conclusively determined whether BRAFV600E mutation is a determinant of ameloblastoma’s aggressive growth, response to targeted therapies and post-surgical recurrence because genetic testing of ameloblastomas for diagnostic and prognostic purposes is not standard practice worldwide especially in sub-Saharan Africa. Considering its prevalence and high recurrence in developing countries like those in sub-Saharan Africa2, it is imperative to assess BRAFV600E mutational status of ameloblastoma for therapeutic purposes and application of a low-cost technique for ameloblastoma mutational analysis15. The objectives of this study were to assess whether IHC is a sensitive low-cost surrogate test for BRAFV600E genetic testing of ameloblastoma and whether tissue handling and/or inadvertent tissue mishandling affect the test results. We hypothesized that immunohistochemical (IHC) staining of formalin fixed paraffin embedded (FFPE) tissues for BRAFV600E mutation can be a surrogate for gene sequencing when tissue quality is compromised, and laboratory resources are inadequate for molecular testing.
MATERIALS AND METHODS
Ameloblastoma tissue samples
A total of n=40 combined fresh surgical and archival ameloblastoma tissue samples were collected and tested for BRAFV600E mutation. Our protocols were approved by the Office of Regulatory Affairs Institutional Review Board, University of Pennsylvania, Philadelphia PA, and the Ethical Committees of Lagos University Teaching Hospital (LUTH), Lagos and University College Hospital (UCH), Ibadan, Nigeria. Patient demographic information and final histopathological diagnosis of ameloblastomas were recorded. Samples included fresh surgical tissues stored in RNA stabilizing solution [RNALater™, (RSS, n=15)], another set of RSS samples inadvertently fixed in formalin before transfer to RNALater™ (FFRSS, n=14) and archival formalin-fixed paraffin-embedded tissues (FFPE, n=11) (Figure 1). Five 10μm sections were stripped from FFPE blocks and tumor sections were isolated for DNA/RNA extraction using established protocols.
Figure 1. Ameloblastoma tissue processing and BRAFV600E detection.
Distribution of ameloblastomas based on tissue processing and BRAFV600E detection methods. Tissues inadvertently transiently pre-exposed to formalin before transfer to RNA stabilizing solution were coded as formalin-fixed tissues stored in RNA stabilizing solution (FFRSS). [N/A =not available tissue blocks to process IHC detection]
BRAFV600E mutational analysis
BRAFV600E mutation was assessed by Direct Sanger Sequencing (DSS), Amplification Refractory Mutation System (ARMS-PCR)/allele-specific PCR and immunohistochemistry (IHC). FFRSS were included in the analysis to evaluate the effects of formalin crosslinking of biomolecules on BRAFV600E detection. Ten RSS samples were not tested by IHC because the corresponding FFPE blocks were not available.
Genomic DNA extraction
Genomic DNA was extracted from all three tissue types (FFPE, FFRSS and RSS) using QIAamp DNA FFPE Tissue Kit (Qiagen, Maryland, USA). Five 10μm FFPE sections were deparaffinized with xylene and DNA was extracted following manufacturer’s instructions. For tissues stored in RNAlater™ (FFRSS and RSS), 25mg of tissue was minced and washed twice with PBS and the same protocol as for FFPE samples was followed without deparaffinization and heating at 90°C steps. Samples incompletely lysed after 4 hours of incubation were digested overnight with 20μl excess proteinase K and 50μl of ATL lysis buffer. For samples still incompletely lysed after overnight incubation, the lysate was centrifuged at 14000rpm for 3 minutes and the supernatant was used in subsequent steps. DNA was eluted in 75μl elution buffer for all samples and quantified using Nanodrop spectrophotometer (Fisher Scientific, Waltham, MA. USA).
ARMS-PCR and Sanger sequencing
The following primers were used for ARMS-PCR and Sanger sequencing: forward primer (FP) 5′-CTCTTCATAATGCTTGCTCTGATAG-3′, reverse primer (RP) 5′-GCCTCAATTCTTACCATCCAC-3′, forward wild type primer (FoWT) 5′-GTGATTTTGGTCTAGCTACAGT-3′ and reverse mutant primer (RMu) 5′-CCCACTCCATCGAGATTTCT-3 as previously described16 ARMS-PCR was performed in 25 μl final volume containing 1X PCR master mix (DreamTaq Green PCR Master Mix (2X), K1081, Thermo Scientific, Carlsbad, CA. USA), 400 nM FP, 200 nM RP, 800 nM FoWT and 200 nM RMu. 50ng of genomic DNA for tissue stored in RNAlater™ and 100–250ng of genomic DNA from FFPE specimens were used in the PCR reaction. 1U of Taq polymerase was added in excess per reaction for the FFPE DNA samples with low purity (less than 1.5 for absorbance ratio of 260/230 and 260/280) to overcome PCR inhibition by impurities. PCR conditions were as follows: initial denaturation for 5 minutes at 95°C, 40 cycles of denaturation for 20 seconds at 95°C, annealing at 60°C for 20 seconds and extension for 20 seconds at 72°C with a final extension for 10 minutes at 72°C. PCR products were finally evaluated in 2% agarose gel.
For DSS, 25μl PCR reaction was performed with forward and reverse primer, following the same conditions as outlined for ARMS-PCR procedure. PCR products were analyzed in 2% agarose gel for the presence of target amplicon. Purification of PCR products was done using ExoSap kit (ExoSAP-IT™ 78201.1.ML, Applied Biosystems, Waltham, MA. USA) as per the manufacturer’s instructions. Bidirectional sanger sequencing was performed with the purified PCR products and the sequence was analyzed by FinchTV (Version 1.4.0, Geospiza Inc, Denver CO. USA).
Immunohistochemical analysis of BRAFV600E mutation
FFRSS and RSS tissues were paraffin-embedded before being tested by IHC. For all three tissue processing conditions (FFPE, FFRSS and RSS), 5 μm tissue sections were deparaffinized in 100% xylene and rehydrated in graded ethanol, followed by antigen retrieval using the Antigen Unmasking Solution, Citric Acid Based (H-3300, Vector Laboratories, Newark, CA. USA) at 95°C for 20 minutes. Sections were incubated in BLOXALL® Endogenous Blocking Solution (SP-6000, Vector Laboratories, Newark, CA. USA) for 10 minutes at room temperature to inactivate endogenous peroxidase. Then, tissue sections were incubated with normal blocking solution (VECTASTAIN® Elite ABC-HRP Kit, PK-6200, Vector Laboratories, Newark, CA. USA) at room temperature for 1 hour, followed by overnight incubation at 4°C with B-Raf (V600E) primary antibody (MA5–24661, Invitrogen, Carlsbad, CA. USA) at 1:100 dilution. Subsequently sections were incubated at room temperature for 30 minutes with VECTASTAIN® biotinylated universal secondary antibody and ABC reagents (Vector Laboratories, Newark, CA. USA) respectively. Sections were stained with DAB Substrate Kit (SK-4100, Vector Laboratories, Newark, CA. USA) and counterstained with hematoxylin (26030–20, Electron Microscopy Sciences, Hatfield, PA. USA). Incubation with primary antibody was omitted in negative control tissues. Cytoplasmic staining based on BRAFV600E immunoreactivity was considered positive regardless of intensity of staining as previously described17
Statistical analysis
Categorical variables were presented as numerical data with percentages and results were summarized and compared using contingency tables. Effect of each detection methods on ameloblastoma BRAFV600E results was analyzed using Chi-square and Fisher’s exact tests; and sensitivity, specificity with confidence intervals were computed assuming DSS a priori reference test. All statistical analysis were performed with Prism10 version 10.0.2 (GraphPad Software, Boston MA. USA). All p values were two-sided, and statistical significance was set at p<0.05.
RESULTS
Patient and tissue sample characteristics
Demographically, 95% of ameloblastoma patients were of the Black race or Afro-descendants, male to female ratio was 0.6:0.4 and 60% were between ages of 21–50 years. Primary ameloblastoma constituted 87.5% of the ameloblastomas and 92.5% were mandibular tumors. Histologically, 67.5% were conventional ameloblastomas, 27.5% unicystic and 5% ameloblastic carcinomas (Table 1)
Table 1.
Demographic and clinico-histopathological profile of ameloblastoma patients
| Demographic and Clinico-histopathological Profile of Ameloblastoma Patients | ||||
|---|---|---|---|---|
| Ameloblastoma types | Conventional N (%) | Unicystic N (%) | Ameloblastic carcinoma N (%) | Total N (%) |
| Gender | ||||
| Male | 16 (69.5) | 5 (21.8) | 2 (8.7) | 23 (57.5) |
| Female | 11 (64.8) | 6 (35.2) | 0 | 17 (42.5) |
| Age groups | ||||
| <21 years | 3 (30) | 7 (70) | 0 | 10 (25) |
| 21–50 years | 19 (79.2) | 3 (12.5) | 2 (8.3) | 24 (60) |
| >50 years | 5 (83.3) | 1 (16.7) | 0 | 6 (15) |
| Mean age ±SD = 37.2±17.8 | ||||
| Racial profile | ||||
| Blacks | 25 (65.8) | 11 (29) | 2 (5.2) | 38 (95) |
| White | 1 (100) | 0 | 0 | 1 (2.5) |
| Asian | 1 (100) | 0 | 0 | 1 (2.5) |
| Tumor type | ||||
| Primary | 24 (68.6) | 10 (28.6) | 1 (2.8) | 35 (87.5) |
| Recurrent | 3 (60) | 1 (20) | 1 (20) | 5 (12.5) |
| Tumor location | ||||
| Maxilla | 0 | 3 (100) | 0 | 3 (7.5) |
| Mandible | 27 (73) | 8 (21.6) | 2 (5.4) | 37 (92.5) |
| Total N (%) | 27 (67.5) | 11 (27.5) | 2 (5) | 40 (100) |
Results of BRAFV600E detection by immunohistochemistry, direct sanger sequencing and amplification refractory mutation system
BRAFV600E mutation was detected by DSS (30%, 12 of 40 samples), ARMS-PCR (52.5%, 21 of 40 samples) and IHC (93.3% based on 28 of 30 samples with available paraffin-fixed tissues) (Figure 1). Representative nucleotide sequences of four ameloblastoma samples with BRAFV600E mutation compared with BRAF-wild-type in Figure 2 display the mutated nucleotide sequences at c.1799T>A. Also, shown in Figure 2 are representative agarose gel electrophoresis of ARMS-PCR amplicons for four ameloblastoma samples compared with control samples. Clearly shown in the gel are the 144bp fragments indicative of BRAFV600E mutation. Comparative presentation of the demographic profile of patient samples, clinico-pathological status and BRAFV600E detection results are listed in Table 2. Both DSS and ARMS-PCR did not detect BRAFV600E mutation in FFPE tissues (Table 2). Considering that tissue blocks were not available for 10 patient samples, IHC detected BRAFV600E mutation in 18 of 19 (94/7%) ameloblastoma samples irrespective of whether the tissues were fixed in formalin pre- or post-exposure to RNA stabilizing solution (Figure 1). A representative composite histomicrograph of immunoreactive ameloblastoma samples compared with controls is presented in Figure 3. Only 30 of 40 samples were comparatively tested for BRAFV600E mutation by all three detection methods because FFPE tissues were not available for 10 samples (Table 2). Reliability of the three testing methods for detection of BRAFV600E in ameloblastomas is summarized in Table 3. BRAFV600E mutation was detected 93.33% by IHC, 43.33% by ARMS-PCR and 23.33% by DSS. There was significant difference between the three BRAFV600E detection methods [χ2(2)=31.34, p<0.0001]. Direct comparison of IHC with either ARMS-PCR (p<0.0001) or DSS (p<0.0001) was highly significant (Table 3). DSS is the gold standard molecular method used for detecting BRAFV600E in cancers because of its high specificity. To determine sensitivity and specificity of the diagnostic tests, we considered DSS a priori gold standard detection method to reference IHC and ARMS-PCR BRARV600E results. Sensitivity and specificity of IHC detection method in reference to DSS were 0.8 (95% CI: 0.64–0.90) and 0.9 (95% CI: 0.75–0.99) respectively, positive predictive value (PPV) and negative predictive value (NPV) were 0.9 and 0.8 (Two-sided Fischer’s exact test, p < 0.0001). Sensitivity and specificity of ARMS-PCR detection method were 0.7 (95% CI: 0.53–0.80) and 0.9 (95% CI=0.67–0.98) respectively, PPV and NPV were 0.9 and 0.6 respectively (Two-sided Fischer’s exact test, p < 0.0001). Direct comparison of ARMS-PCR results in reference to DSS was not statistically significant (Table 3).
Figure 2. Representative BRAFV600E detection by direct sanger sequencing and ARMS-PCR.
Representative direct sanger nucleotide sequences (left panel) of four ameloblastoma samples (#12, 35, 39 and 40) with BRAFV600E mutation compared with BRAF-wild-type (#19) display the mutated nucleotide sequences at c.1799T>A. Right panel shows representative agarose gel electrophoresis of ARMS-PCR amplicons for four ameloblastoma samples(#27, 29, 39 and 40) compared with negative and positive control tissues. The 144bp fragments are indicative of BRAFV600E mutation
Table 2. Comparison of BRAFV600E detection methods.
The table summarizes reliability of the three BRAFV600E testing methods. Differences between the three BRAFV600E detection methods was statistically significant [χ2(2) = 31.34, p < 0.0001]. Direct comparison of IHC with ARMS-PCR (p < 0.0001) or IHC with DSS (p < 0.0001) was statistically significant.
| Mutational Analysis of Ameloblastomas | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Sample ID # | Gender | Age (years) | Race | Histologic type | Primary/Recurrent | Tumor site | Tissue type | Direct Sanger Sequencing (DSS) | ARMS-PCR | IHC |
| 1 | Male | 46 | Black | Ameloblastic carcinoma | Recurrent | Mandible | FFRSS | Positive | Positive | Positive |
| 10 | Female | 26 | Black | Unicystic | Primary | Mandible | FFRSS | Positive | Positive | Positive |
| 11 | Female | 71 | Black | Conventional | Primary | Mandible | FFRSS | Positive | Positive | Positive |
| 12 | Male | 46 | Black | Conventional | Primary | Mandible | FFRSS | Positive | Positive | Positive |
| 37 | Female | 35 | Black | Conventional | Primary | Mandible | RSS | Positive | Positive | Positive |
| 39 | Female | 77 | Asian | Conventional | Primary | Mandible | RSS | Positive | Positive | Positive |
| 40 | Female | 74 | White | Conventional | Primary | Mandible | RSS | Positive | Positive | Positive |
| 2 | Female | 42 | Black | Conventional | Primary | Mandible | FFRSS | Negative | Positive | Positive |
| 3 | Male | 24 | Black | Conventional | Primary | Mandible | FFRSS | Negative | Positive | Positive |
| 4 | Female | 19 | Black | Unicystic | Primary | Mandible | FFRSS | Negative | Positive | Positive |
| 7 | Male | 18 | Black | Conventional | Primary | Mandible | FFRSS | Negative | Positive | Positive |
| 13 | Male | 45 | Black | Conventional | Primary | Mandible | FFRSS | Negative | Positive | Positive |
| 38 | Female | 35 | Black | Conventional | Primary | Mandible | RSS | Negative | Positive | Positive |
| 5 | Male | 45 | Black | Conventional | Primary | Mandible | FFRSS | Negative | Negative | Positive |
| 6 | Male | 37 | Black | Conventional | Primary | Mandible | FFRSS | Negative | Negative | Positive |
| 8 | Male | 29 | Black | Conventional | Primary | Mandible | FFRSS | Negative | Negative | Positive |
| 9 | Male | 19 | Black | Unicystic | Primary | Mandible | FFRSS | Negative | Negative | Positive |
| 14 | Male | 20 | Black | Conventional | Primary | Mandible | FFRSS | Negative | Negative | Positive |
| 15 | Male | 32 | Black | Ameloblastic carcinoma | Primary | Mandible | FFPE | Negative | Negative | Positive |
| 16 | Female | 12 | Black | Unicystic | Primary | Maxilla | FFPE | Negative | Negative | Positive |
| 17 | Male | 67 | Black | Conventional | Primary | Mandible | FFPE | Negative | Negative | Positive |
| 18 | Female | 45 | Black | Conventional | Primary | Mandible | FFPE | Negative | Negative | Positive |
| 20 | Female | 14 | Black | Unicystic | Primary | Mandible | FFPE | Negative | Negative | Positive |
| 21 | Male | 37 | Black | Conventional | Recurrent | Mandible | FFPE | Negative | Negative | Positive |
| 22 | Male | 38 | Black | Unicystic | Primary | Mandible | FFPE | Negative | Negative | Positive |
| 23 | Female | 71 | Black | Unicystic | Primary | Mandible | FFPE | Negative | Negative | Positive |
| 24 | Male | 16 | Black | Unicystic | Primary | Maxilla | FFPE | Negative | Negative | Positive |
| 25 | Male | 18 | Black | Unicystic | Primary | Maxilla | FFPE | Negative | Negative | Positive |
| 19 | Male | 35 | Black | Conventional | Primary | Mandible | FFPE | Negative | Negative | Negative |
| 36 | Female | 27 | Black | Conventional | Primary | Mandible | RSS | Negative | Negative | Negative |
| 31 | Male | 44 | Black | Conventional | Primary | Mandible | RSS | Negative | Positive | N/A |
| 34 | Male | 42 | Black | Conventional | Primary | Mandible | RSS | Negative | Positive | N/A |
| 27 | Male | 49 | Black | Conventional | Primary | Mandible | RSS | Positive | Positive | N/A |
| 30 | Male | 46 | Black | Conventional | Recurrent | Mandible | RSS | Positive | Positive | N/A |
| 32 | Female | 13 | Black | Unicystic | Primary | Mandible | RSS | Positive | Positive | N/A |
| 33 | Female | 24 | Black | Conventional | Primary | Mandible | RSS | Positive | Positive | N/A |
| 35 | Male | 11 | Black | Conventional | Primary | Mandible | RSS | Positive | Positive | N/A |
| 26 | Female | 37 | Black | Conventional | Recurrent | Mandible | RSS | Negative | Positive | N/A |
| 28 | Female | 62 | Black | Conventional | Primary | Mandible | RSS | Negative | Negative | N/A |
| 29 | Male | 40 | Black | Unicystic | Recurrent | Mandible | RSS | Negative | Negative | N/A |
FFRSS= Formalin fixed ameloblastoma tissue stored in RNAlater™
RSS = Fresh ameloblastoma tissue stored in RNAlater™
FFPE = Formalin fixed plastic embedded ameloblastoma tissue block
ARMS = Amplification-refractory mutation system
IHC = Immunohistochemistry
N/A = FFPE not available
Figure 3. Expression of BRAFV600E in ameloblastomas.
Composite histomicrograph shows FFPE ameloblastoma tissue (left panel) and RSS-exposed ameloblastoma tissue (right panel). Representative BRAFV600E immunoreactive stromal tissues (brown staining, top panel), accompanying negative controls (middle panel) and hematoxylin/eosin stained (lower panel) sections indicate that tissue samples stored in RNA stabilizing solution can still be fixed and tested for BRAFV600E by IHC.
Table 3. Comparison of BRAFV600E detection methods.
The table summarizes reliability of the three BRAFV600E testing methods. Differences between the three BRAFV600E detection methods was statistically significant [χ2(2) = 31.34, p < 0.0001]. Direct comparison of IHC with ARMS-PCR (p < 0.0001) or IHC with DSS (p < 0.0001) was statistically significant.
| Comparison of BRAFV600E Detection Methods (n = 30 samples) | |||
|---|---|---|---|
| BRAFV600−ve | BRAFV600E+ve | % BRAFV600E+ve **** | |
| Immunohistochemistry (IHC) | 2 | 28 | 93.33 |
| ARMS-PCR | 17 | 13 | 43.33 |
| Direct Sanger sequencing | 23 | 7 | 23.33 |
| **** χ2 test (2, n=90 BRAFV600E analyses) = 31.34, p < 0.0001 | |||
| IHC **** (Reference: Direct Sanger sequencing) | IHC**** (Reference: ARMS-PCR) | ARMS-PCR (Reference: Direct Sanger sequencing) | |
| Sensitivity (95% CI) | 0.8 (0.64 – 0.90) | 0.7 (0.53 – 0.80) | 0.7 (0.43 – 0.82) |
| Specificity (95% CI) | 0.9 (0.75 – 0.99) | 0.9 (0.67 – 0.98) | 0.6 (0.42 – 0.72) |
| Positive Predictive Value (95% CI) | 0.9 (0.79 – 0.99) | 0.9 (0.79 – 0.99) | 0.4 (0.27 – 0.61) |
| Negative Predictive Value (95% CI) | 0.8 (0.59 – 0.88) | 0.6 (0.39 – 0.73) | 0.8 (0.59 – 0.88) |
| **** Fisher’s exact test (two-sided) | p <0.0001 | p <0.0001 | ns |
DISCUSSION
Genetic mutations are frequently identified in many types of odontogenic tumors and BRAFV600E mutation is commonly found in ameloblastoma18. Although many molecular testing methods are available to detect BRAFV600E mutation, they are not cost effective. We used three different techniques (IHC, ARMS-PCR and DSS) to identify a sensitive, specific, and low-cost method to detect BRAFV600E mutation in ameloblastoma samples. Tested were tissues from FFPE blocks, tissues inadvertently fixed in formalin before transfer to RNA stabilization solution-RNAlater™ (FFRSS), and those immediately preserved in RNAlater™ (RSS). Albeit FFRSS samples were included to account for inadvertent errors that often occur with tissue handling and processing making vital surgical tissue samples unusable for molecular biology techniques. The age and gender distribution of our study patients align with established demographic profile of ameloblastoma patients19,20. However, majority of the patients were overwhelmingly Afro-descendants (95%). Ameloblastoma is highly prevalent in Africa15 but there is paucity of information on genetic profile of ameloblastomas from Afro-centric patients. This could be due to limited laboratory infrastructure and resources for genetic analysis as well as a significantly high cost. Among 30 of 40 samples that could be practically tested with all three detection methods, BRAFV600E was significantly positive by IHC (93.3%) compared to DSS (23.3%) and ARMS-PCR (43.33%) (Table 3). This is encouraging considering that IHC is an easier, low-cost laboratory procedure compared to molecular biology techniques and the B-Raf (V600E) monoclonal antibody used in this study reacts to BRAF V600E mutant without any cross reactivity with wild type BRAF.
DSS is the gold standard molecular technique for detecting BRAFV600E in cancers because of its high specificity. It is noteworthy that direct comparison of IHC either with DSS or ARMS-PCR produced similar specificity and PPV values which suggests that IHC is a reliable BRAFV600E detection method. The discordance and superiority of IHC over the other two molecular methods could be due to their requirement for high quality starting DNA material and a need for precise tissue handling and processing. While FFRSS-processed samples were amenable to IHC technique, they were not suitable for DSS and ARMS-PCR due to DNA degradation caused by the inadvertent though transient (less than 1 hour) formalin fixation. DNA from FFPE samples is used in routine diagnostic tests for the mutation profiling in tumor patients21. However, formalin fixation causes DNA degradation due to fragmentation and DNA crosslinking which can potentially affect downstream PCR amplification process22 making the ARMS-PCR and DSS comparatively less sensitive than IHC. The sensitivity of ARMS-PCR and DSS increases when tissues are stored in RSS, but the tumor cell fraction also has an impact on the sensitivity of DNA based BRAFV600E testing methods23. It is noteworthy that formalin crosslinking of biomolecules did not impact the detection of BRAFV600E protein by IHC technique which makes it more sensitive than DNA-based BRAFV600E mutation testing methods for ameloblastoma24
Our data showed that IHC successfully detected BRAFV600E mutation in 94.7% of ameloblastoma samples that were formalin-fixed either pre- or post-storage in RNA stabilizing solution (Figure 1) which suggests that tissue samples stored in RNA stabilizing solution can still be fixed and tested for BRAFV600E by IHC (Figure 3). The ease of formalin fixation procedure combined with a less delicate immunostaining protocol makes IHC a strong option for BRAFV600E testing of ameloblastomas especially in geographical regions with inadequate genetic laboratory infrastructure and supplies.
Therapeutic application of BRAFV600E inhibitors such as vemurafenib, dabrafenib and trametinib for BRAF mutated tumors like melanoma and thyroid cancers as well as ameloblastomas suggest that genetic testing of these tumors is a vital part of treatment planning7,25. The standard of care for ameloblastoma is surgical resection3, but most patients in developing countries show up with large sized ameloblastomas1,26 which present a surgical challenge. There are indications that BRAFV600E inhibitors can be a neo-adjuvant therapy to shrink large ameloblastomas before resection so that morbidity and mortality can be greatly reduced3. This makes pre-surgical testing for BRAFV600E mutation highly essential, however molecular diagnostic testing especially on a large scale are costly and require expensive laboratory equipment. While DSS is highly specific, it has low sensitivity because BRAFV600E detection from starting DNA sample requires that a higher percentage (20 – 60%) of tumor cells must be present within the diagnostic samples tested14. But IHC has the advantage of fewer critical procedural steps and does not require extensive laboratory infrastructure. A study that compared diagnostic accuracy of IHC with molecular tests for BRAFV600E detection reported that sensitivity of IHC compared to molecular tests ranged from 0.71 to 1.0027. Other studies have also reported high sensitivity, specificity, PPV and NPV of IHC tests for BRAFV600E in ameloblastoma28,29. Our data of 0.8 sensitivity of IHC is in line with these studies28 27,29. Our data also showed concordance between IHC and ARMS-PCR and no significant differences between DSS and ARMS-PCR. Therefore, our study supports the proposition30 that sequential dual testing of ameloblastomas starting with the relatively low-cost IHC test will allow more samples to be readily tested for BRAFV600E mutation. Furthermore, samples that result in negative or equivocal IHC results should be verified with a molecular test at the disposal of the diagnostic facility, either DSS or real-time PCR.
Our study has some limitations. First, the sample size of ameloblastoma tissues is small. Secondly, 25% (10 of 40) samples were not assessed by IHC, a factor that could have yielded additional IHC-negative samples and different statistical outputs. Third, majority of the samples were collected from Afro-descendants, so the racial implications of our results could not be assessed. However, this study still provides valuable information about BRAFV600E mutation in ameloblastomas of patients from a predominantly Black community17. In conclusion, the high sensitivity of IHC technique and cost effectiveness suggest that IHC is a viable surrogate testing method for detection of BRAFV600E mutation in ameloblastoma where the resources and infrastructure are inadequate for molecular genetic approaches.
Since commercially available antibodies with strong reactivity to BRAFV600E mutant without cross reactivity with wild type BRAF have been used diagnostically with high sensitivity in ameloblastoma and other BRAFV600E mutant tumors9,14,27–29, we propose sequential dual testing of ameloblastoma for BRAFV600E mutation starting with the low-cost IHC test followed by a molecular test to verify negative or equivocal IHC results. The ability to ‘rule-in’ or ‘rule-out’ BRAFV600E mutant tumors should help identify candidate ameloblastoma for BRAFV600E inhibitor therapy and neo-adjuvant therapy of large sized ameloblastoma. To avoid excluding some patients from BRAF inhibitor therapy, the use of a highly sensitive and specific BRAFV600E detection method is vital.
Acknowledgement
This work was supported by grant R01CA259307 (awarded to S.O.A.) by the United States Department of Health and Human Services/National Institutes of Health, Bethesda, MD. We thank Prasath Jeyaraman for technical support and Penn Vet Comparative Pathology Core which is part of the Abramson Cancer Center Support Grant (P30 CA016520).
Footnotes
Ethics Approval Statement.
Study protocols were approved by the Office of Regulatory Affairs Institutional Review Board, University of Pennsylvania, Philadelphia PA, and the Ethical Committees of Lagos University Teaching Hospital (LUTH), Lagos and University College Hospital (UCH), Ibadan, Nigeria.
Contributor Information
Arularasan Anbinselvam, Department of Oral Medicine, School of Dental Medicine, University of Pennsylvania, Philadelphia PA.
Abdul-Warith O. Akinshipo, Department of Oral and Maxillofacial Pathology/Biology, Faculty of Dental Sciences, University of Lagos, Lagos, Nigeria.
Akinyele O. Adisa, University of Ibadan and University College Hospital Ibadan, Ibadan, Nigeria.
Olajumoke A. Effiom, Department of Oral and Maxillofacial Pathology/Biology, Faculty of Dental Sciences, University of Lagos, Lagos, Nigeria.
Xinhe Zhu, Department of Oral Medicine, School of Dental Medicine, University of Pennsylvania, Philadelphia PA.
Kehinde E. Adebiyi, Department of Oral Pathology & Oral Medicine, Faculty of Dentistry. Lagos State University College of Medicine Lagos.
Godwin T. Arotiba, Department of Oral and Maxillofacial Surgery, Faculty of Dental Sciences, University of Lagos, Lagos, Nigeria
Sunday O. Akintoye, Department of Oral Medicine, School of Dental Medicine, University of Pennsylvania, Philadelphia PA.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author, [SOA], upon reasonable request.
References
- 1.Bassey GO, Osunde OD, Anyanechi CE. Maxillofacial tumors and tumor-like lesions in a Nigerian teaching hospital: an eleven year retrospective analysis. Afr Health Sci. 2014;14(1):56–63. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Oginni FO, Stoelinga PJ, Ajike SA, et al. A prospective epidemiological study on odontogenic tumours in a black African population, with emphasis on the relative frequency of ameloblastoma. Int J Oral Maxillofac Surg. 2015;44(9):1099–1105. [DOI] [PubMed] [Google Scholar]
- 3.Effiom OA, Ogundana OM, Akinshipo AO, Akintoye SO. Ameloblastoma: current etiopathological concepts and management. Oral Diseases. 2018;24(3):307–316. [DOI] [PubMed] [Google Scholar]
- 4.DeVilliers P, Suggs C, Simmons D, Murrah V, Wright JT. Microgenomics of ameloblastoma. J Dent Res. 2011;90(4):463–469. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Mishra P, Panda A, Bandyopadhyay A, Kumar H, Mohiddin G. Sonic Hedgehog Signalling Pathway and Ameloblastoma - A Review. J Clin Diagn Res. 2015;9(11):ZE10–13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Brown NA, Betz BL, Weigelin HC, Elenitoba-Johnson KS, Lim MS, Bailey NG. Evaluation of allele-specific PCR and immunohistochemistry for the detection of BRAF V600E mutations in hairy cell leukemia. Am J Clin Pathol. 2015;143(1):89–99. [DOI] [PubMed] [Google Scholar]
- 7.Kurppa KJ, Caton J, Morgan PR, et al. High frequency of BRAF V600E mutations in ameloblastoma. J Pathol. 2014;232(5):492–498. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Holderfield M, Deuker MM, McCormick F, McMahon M. Targeting RAF kinases for cancer therapy: BRAF-mutated melanoma and beyond. Nat Rev Cancer. 2014;14(7):455–467. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Zhao J, Liu P, Yu Y, et al. Comparison of diagnostic methods for the detection of a BRAF mutation in papillary thyroid cancer. Oncol Lett. 2019;17(5):4661–4666. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Mamat Yusof MN, Ch’ng ES, Radhiah Abdul Rahman N. BRAF V600E Mutation in Ameloblastoma: A Systematic Review and Meta-Analysis. Cancers (Basel). 2022;14(22). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Subbiah V, Kreitman RJ, Wainberg ZA, et al. Dabrafenib plus trametinib in BRAFV600E-mutated rare cancers: the phase 2 ROAR trial. Nat Med. 2023;29(5):1103–1112. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Ihle MA, Fassunke J, Konig K, et al. Comparison of high resolution melting analysis, pyrosequencing, next generation sequencing and immunohistochemistry to conventional Sanger sequencing for the detection of p.V600E and non-p.V600E BRAF mutations. BMC Cancer. 2014;14:13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Rashid FA, Tabassum S, Khan MS, et al. VE1 immunohistochemistry is an adjunct tool for detection of BRAF(V600E) mutation: Validation in thyroid cancer patients. J Clin Lab Anal. 2021;35(2):e23628. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Colomba E, Helias-Rodzewicz Z, Von Deimling A, et al. Detection of BRAF p.V600E mutations in melanomas: comparison of four methods argues for sequential use of immunohistochemistry and pyrosequencing. J Mol Diagn. 2013;15(1):94–100. [DOI] [PubMed] [Google Scholar]
- 15.Patel P, Effiom OA, Akinshipo A-WO, Akintoye SO. Differential Profile of Primary and Recurrent Ameloblastomas Among Afro-descendants and Non-Afro-descendants—a Systematic Review. J Racial and Ethnic Health Disparities. 2023. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Huang T, Zhuge J, Zhang WW. Sensitive detection of BRAF V600E mutation by Amplification Refractory Mutation System (ARMS)-PCR. Biomarker Research. 2013;1(1):3. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Owosho AA, Ladeji AM, Adebiyi KE, et al. BRAF V600E mutation-specific immunohistochemical analysis in ameloblastomas: a 44-patient cohort study from a single institution. Eur Arch Otorhinolaryngol. 2021;278(8):3065–3071. [DOI] [PubMed] [Google Scholar]
- 18.Togni L, Zizzi A, Mazzucchelli R, Santarelli A, Rubini C, Mascitti M. Identification of BRAF V600E mutation in odontogenic tumors by high-performance MALDI-TOF analysis. Int J Oral Sci. 2022;14(1):22. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Hresko A, Palyvoda R, Burtyn O, et al. Recurrent Ameloblastoma: Clinical Manifestation and Disease-Free Survival Rate. Journal of Oncology. 2022;2022:e2148086. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Goh YC, Siriwardena BSMS, Tilakaratne WM. Association of clinicopathological factors and treatment modalities in the recurrence of ameloblastoma: Analysis of 624 cases. Journal of Oral Pathology & Medicine: Official Publication of the International Association of Oral Pathologists and the American Academy of Oral Pathology. 2021;50(9):927–936. [DOI] [PubMed] [Google Scholar]
- 21.Javey M, Reinsch C, Feldkamp M, et al. Innovative Tumor Tissue Dissection Tool for Molecular Oncology Diagnostics. J Mol Diagn. 2021;23(4):399–406. [DOI] [PubMed] [Google Scholar]
- 22.Gilbert MT, Haselkorn T, Bunce M, et al. The isolation of nucleic acids from fixed, paraffin-embedded tissues-which methods are useful when? PloS one. 2007;2(6):e537. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Lade-Keller J, Romer KM, Guldberg P, et al. Evaluation of BRAF mutation testing methodologies in formalin-fixed, paraffin-embedded cutaneous melanomas. J Mol Diagn. 2013;15(1):70–80. [DOI] [PubMed] [Google Scholar]
- 24.da Silva Marcelino BMR, Parise GK, do Canto AM, et al. Comparison of Immunohistochemistry and DNA Sequencing for BRAF V600E Mutation Detection in Mandibular Ameloblastomas. Appl Immunohistochem Mol Morphol. 2021;29(5):390–393. [DOI] [PubMed] [Google Scholar]
- 25.Heikinheimo K, Kurppa KJ, Elenius K. Novel targets for the treatment of ameloblastoma. J Dent Res. 2015;94(2):237–240. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Anyanechi CE, Saheeb BD. A review of 156 odontogenic tumours in Calabar, Nigeria. Ghana Med J. 2014;48(3):163–167. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Martins-de-Barros AV, Anjos RSD, Silva CCG, Silva E, Araujo F, Carvalho MV. Diagnostic accuracy of immunohistochemistry compared with molecular tests for detection of BRAF V600E mutation in ameloblastomas: Systematic review and meta-analysis. J Oral Pathol Med. 2022;51(3):223–230. [DOI] [PubMed] [Google Scholar]
- 28.Chang JYF, Lu PH, Tseng CH, Wang YP, Lee JJ, Chiang CP. Factors affecting the accuracy of anti-BRAF V600E immunohistochemistry results in ameloblastomas. J Oral Pathol Med. 2023;52(4):342–350. [DOI] [PubMed] [Google Scholar]
- 29.Mendez LD, Wolsefer NS, Asa SL, Wasman J, Yoest JM, Stojanov IJ. The diagnostic utility of BRAF VE1 mutation-specific immunohistochemistry in ameloblastoma. Mod Pathol. 2022;35(11):1570–1577. [DOI] [PubMed] [Google Scholar]
- 30.Martinuzzi C, Pastorino L, Andreotti V, et al. A combination of immunohistochemistry and molecular approaches improves highly sensitive detection of BRAF mutations in papillary thyroid cancer. Endocrine. 2016;53(3):672–680. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author, [SOA], upon reasonable request.