Nasopharyngeal Angiofibroma: Karyotyping Profile and Florescent In-Situ Hybridization Analysis with C-Myelocytomatosis, Tumor Suppressor p53 and CEP-X/Y Probes

Abstract

The chromosomal characterization of nasopharyngeal angiofibroma (NPF) of Indian origin seems important as it is consistently absent in literature. Routine karyotyping (G-banding) and fluorescence in situ hybridization (FISH) analysis was undertaken using CMYC, TP53 CEPX/Y probes in 9 cases of NPF wherein chromosomal characteristics were correlated with clinical parameters. Karyotype profile of venous sample from every case was normal. Following FISH analysis, 5 (55%) cases showed deleted CMYC allele while 4 (44%) showed deleted TP53. In addition, loss of Y centromere was evident in 1 case. Despite definite trends, no significant correlation with clinical parameters and FISH expression/ G-banding karyotyping could be appreciated. The peripheral blood karyotype cannot predict any significant parallel picture in solid tumour. Solid tumour karyotyping and FISH have a definite potential to screen the genetic abnormalities. While high cost of FISH probes and the required expertise may limit its routine clinical use, this technique can be combined with other molecular tools for better results.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12070-024-05171-z.

Nasopharyngeal angiofibroma (NPF) is the most common benign tumour of nasopharynx in young males. This has evolved in last few decades and has shown wide variation in its clinical [1] and molecular epidemiology [2] across the globe. NPF of south-east Asia origin is probably different from the west [3]. The debatable etiology of this heterogeneous entity is well accepted and its well established androgen predominance has shown inconsistencies in past decades. However, its exclusive presence in males suggests a definite role of Y chromosome. The chromosomal literature on NPF is almost exclusively from the west. The chromosomal aberrations published from the west [4] have revealed abnormalities in 18 chromosomes as per comparative genomic hybridization (CGH) with gains in chromosomes 4q, 6q, 8q and complete loss of Y chromosome in more than 50%. Neither any chromosomal study on NPF has been published from Indian subcontinent, nor has the Y-chromosome been characterized so far. Hence chromosomal characterization of NPF of Indian origin seems important. Accordingly, this paper intends to investigate routine karyotype along with fluorescence in situ hybridization (FISH) analysis using C-Myc (C-myelomatosis), TP53 expression and sex-chromosome markers (CEPX/Y). An attempt is made to further analyze chromosomal characteristics with clinical parameters. These specific markers were selected with simple rationale that one is a marker of proliferation, another of tumour suppression and the third as centromere marker of sex chromosome. C-Myc protein is a transcription factor and key regulator of gene expression that modulates cell growth, proliferation, apoptosis, and differentiation. The TP53 is a tumour suppressor gene also referred as “guardian of genome”. It modulates cellular responses to stress, cell cycle arrest, DNA repair, and apoptosis. On the other hand, CEPX and CEPY probes have been used in FISH to detect specific regions on the X and Y chromosomes, respectively. The aim of these centromere probes is to study sex chromosome abnormalities/ constitution, and diagnose disorders related to sex chromosome aneuploidy. For this exclusive adolescent male disease, sex chromosome characterization seemed important.

Materials and Methods

A total of 9 prospective cases of NF were recruited in this chromosomal study from the Department of Otorhinolaryngology and Head-Neck Surgery of a tertiary care university hospital of North India. Following approval by the ethics committee of IRB, a written informed consent was obtained from every patient. Apart from routine anamnesis the clinical parameters were recorded. These included symptomatology with duration, detailed otorhinolaryngological examination, specifics of facial profile (frog face, broadened nose, telecanthus, malar fullness, cheek swelling), palatal bulge, proptosis, preoperative haematological investigations, radiological assessment (staging as per Fisch [5], Mishra [6], Radkowski [7] criteria), weight of tumour (through electronic balance), volume of tumour (through water displacement method), and intraoperative haemorrhage following transpalatal resection. Apart from routine histology, venous sample was obtained for Karyotyping from every case and tumour tissue subjected to Fluorescence in-situ hybridization (FISH) analysis at Department of Hematology laboratory of the collaborating institute.

Karyotyping was carried out through established laboratory protocols. In summary the venous blood in heparin vacutainer was obtained and PHA (Phyto-haemagglutinin) stimulated culture was set up. Chromosomes were captured in metaphase by using colchicine and further treated with trypsin (to digest envelope of histones- proteins) before staining with Giemsa stain. Giemsa banding or G-banding is a technique that produces a visible picture of all chromosomes (by staining condensed chromosomes) referred to as karyotype. Heterochromatic (more condensed) regions are rich in adenine and thymine but are relatively gene-poor. These stain more darkly in G-banding, while less condensed (Euchromatin) that is rich in guanine and cytosine and also more transcriptionally active appear as light bands. The pattern of bands is numbered on each arm of the chromosome from the centromere to the telomere. This numbering system allows any band on the chromosome to be identified and described precisely. Microscopic photographing was performed after chromosome preparation and staining according to MIC (Medical Information Centre) cooperative group. Cytogenetic analysis was undertaken in 20 such cells in metaphase. The analysis primarily detects structural and numeric aberrations of chromosomes. The FISH test was done on the formalin-fixed paraffin-embedded (FFPE) tumour specimens. The ‘TP53 gene deletion probe’ was used for identifying deletion of TP53 gene, ‘CMYC gene rearrangement probe’ for rearrangement of c MYC gene, and Centromeric probes of chromosomes X/Y were used for enumeration of X/Y chromosomes. In summary the standard laboratory protocol included paraffin pre-treatment (Slide aging, Wax removal), tissue rehydration, heat & enzyme pre-treatment, tissue dehydration, addition of probes, co-denaturation, overnight hybridization, post wash and counter staining before visualisation.

The data was tabulated using Microsoft excel software and its analysis undertaken through SPSS software (version 26). A correlational analysis was undertaken between the various recorded clinical parameters and FISH results so obtained for CMYC, TP53 and CEP X/Y. Pearson’s and Spearman correlation tests were used to define the respective correlation coefficients and their p values. A p value < 0.05 was considered as significant.

Observations

The clinical and molecular summary of the patient series is depicted in Table 1. All subjects were males with average age of presentation being 15y (SD 3.12). The duration of nasal blockage varied from 1 to 48 months (average 9.66, SD 14.71) and that of epistaxis also ranged from 1 to 48 months (average 10.83, SD 15.60). The palatal bulge was seen in 4 cases while facial profile was altered in only 2 cases. Average weight of resected tumour was 13.92 g (SD 18.18) whereas mean volume 35 ml (SD 18.76). It is important to note that 5 of 9 cases (55%) showed deleted CMYC allele while rest 4 showed normal CMYC-FISH appearance. Similarly, 4 of 9 cases (44%) showed deleted TP53 allele while rest 5 showed its normal FISH appearance. In this exclusive male population only a single case revealed loss of Y centromere on FISH analysis, rest being normal.

Table 1.

Clinical and molecular summary

	Case 1	Case 2	Case 3	Case 4	Case 5	Case 6	Case 7	Case 8	Case 9
Age (y)	22	13	14	12	12	16	15	14	17
Nasal blockage: duration (months)	48	4	2	6	4	12	1	4	6
Epistaxis: duration (months)	48	2.5	5	24	3	2	1	4	8
Epistaxis severity	2	1	1	1	2	1	2	2	1
Facial profile	N	Altered	N	Altered	N	N	N	N	N
Palatal bulge	-	+	+	+	+	-	-	-	-
Mishra stage	IIIC	IIC	IIA	IIC	IIC	IIB	IIC	IIA	IIB
Radkowsky stage	IIIB	IIA	IA	IIB	IIB	IIB	IIB	IA	IIA
Fisch stage	IVB	II	I	II	II	II	IIIA	I	II
Haemorrhage (ml)	487.83	1102.92	364.96	661.21	675.25	278.25	1011.81	383.33	366.48
Tumour weight(gm)	14.68	42.95	36.49	57.64	42.52	16.21	51.54	18.11	7.22
Tumour volume (ml)	19	43	32	64	45	24	58	22	8
Haemoglobin (gm%)	14.8	9.4	12.7	14.7	9.7	13.98	11	10.8	12.5
Platelet count	3.13	3.5	2.54	4.8	4.28	1.5	2.8	2.41	1.47
Prothrombin time	14.9	15.5	3.5	2.54	4.8	4.28	1.5	2.8	2.41
Thrombin time (sec)	14	13	13	24	24.6	14	17	14	13
INR	1.11	1.15	1.13	0.96	1.87	1.37	1.33	1.11	1.2
APTT	26	27	27	26	28	26	32	23	30.5
CMYC profile	Del	Del	Del	N	N	Del	N	N	Del
TP53 profile	Del	Del	N	N	N	Del	Del	N	N
CEPX/Y profile	N	ND	N	N	N	‘Y’ loss	ND	N	N

Del: Deletion, N: normal, ND not done

The karyotype images were normal in all the cases. It is important to note that this karyotype analysis was obtained from venous blood sample and not from the tumour tissue as such since the facility for solid tumour karyotyping did not exist in our collaborating laboratory. Figure 1 depicts the FISH profile of CMYC as assessed during interphase. FISH analysis using c-MYC break-apart probe depicts presence of nuclei with single yellow/fusion signal, rather than with two yellow/fusion signals. This indicates a loss of one of the copy of CMYC gene in the tumour nuclei.

Fig. 1.

FISH profile of CMYC Note the presence of nuclei with single yellow/fusion signal, indicating loss of copy of c-MYC gene

Figure 2 depicts FISH profile of TP53 as assessed during interphase. The FISH analysis using TP53 deletion probe normally shows presence of nuclei with two green and 2 red signals. However, the presence of nuclei with one red and one green signal indicates loss of one of the copy of TP53 gene, possibly resulting from monosomy of chromosome 17.

Fig. 2.

FISH profile of TP53 Note that the presence of nuclei with one red and one green signal indicates loss of one copy

Figure 3 depicts FISH profile of CEP XY as assessed during interphase. Following use of CEP XY probe, normal findings include presence of nuclei with one green and one red signal. The absence of Y-chromosome is suggested by presence of only single signal.

Fig. 3.

FISH profile of CEP XY Note that absence of Y-chromosome is suggested by presence of only single signal. Normal findings include presence of both one green and red signals

The correlation analysis (Table 2) revealed relationship between many variables but importantly the FISH findings did not correlate with any of the clinical parameters except for TP53 with duration of nasal blockage (or duration of disease). A palatal bulge per se likely signifies a large size of tumour in terms of both weight (Pearson’s coefficient 0.921, p0.003; Spearman’s coefficient 0.808, p < 0.001) and volume (Pearson’s coefficient 0.824, p0.02; Spearman’s coefficient 0.588, p < 0.001), while altered facial configuration correlated with volume only (Pearson’s coefficient 0.790, p 0.03; Spearman’s coefficient 0.612, p < 0.001). Of all the staging systems the FISCH staging system correlated with age of patient (Pearson’s coefficient 0.762, p 0.04; Spearman’s coefficient 0.426, p < 0.001), duration of nasal blockage (Pearson’s coefficient 0.920, p 0.003; Spearman’s coefficient 0.852, p < 0.001), and duration of epistaxis (Pearson’s coefficient 0.853, p 0.01; Spearman’s coefficient 0.418, p < 0.001), while the MISHRA staging correlated with duration of epistaxis (Pearson’s coefficient 0.767, p 0.04; Spearman’s coefficient 0.093, p < 0.001) only. Importantly CEP XY expression did not correlate with any parameters possibly due to a single (small) sample. Further correlations between demographic factors and inter-relationship between coagulation profiles are depicted in Table 2.

Table 2.

Correlation summary

	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
1		+							+
2	+		+						+											+
3		+					+		+
4
5												+			+
6												+
7			+					+	+
8							+		+
9	+	+	+				+	+
10											+		+		+
11						+						+		+		+
12					+	+				+	+			+		+
13
14										+	+	+
15					+
16										+	+	+		+					+
17
18
19																+
20		+
21

1 Age, 2 Duration of nasal blockage, 3 Duration of epistaxis, 4 Epistaxis severity, 5 Facial profile, 6 Palatal bulge, 7 Mishra stage, 8 Radkowsky stage, 9 Fisch stage, 10 Haemorrhage, 11 Weight, 12 Volume, 13 Haemoglobin%, 14 Platelet count, 15 Prothrombin time, 16 Thrombin time, 17 INR, 18 APTT, 19 CMYC, 20 TP53, 21 CEPXY. ‘+’ indicates significant (p < 0.05) correlation whereas blank spaces did not show any correlation. ‘±’ indicates borderline correlation (p = 0.05)

Discussion

The only gross whole-chromosomal aberration encountered in this study was absence of Y-chromosome (revealed through CEPY FISH probe) in a single case, but such absence was not reflected in the respective karyotype obtained from the venous blood sample. Since the karyotyping was not undertaken from solid tumour tissue, a normal karyotype from blood sample is not unexpected. Hence the karyotype with G-banding from blood sample is unlikely to be a surrogate marker for similar such karyotypic change in tumour tissue. Most of the genetic aberrations in NPF are seen at gene level rather than at chromosome level. Karyotype in general reflects the chromosomal status per se and identifying a gene in whole chromosome picture is like finding a ‘needle in a hay stack’. Still considering multiple genes in multiple combinations involved in tumorigenesis, the karyotype G-banding from tumour tissue may still be considered a cytogenetic screening tool for gross aberration.

C MYC expression is induced by extracellular signals (EGF, PDGF, Wnt signalling etc.) that activates intracellular signalling pathways (Ras/Raf/MEK/ERK, PI3K/Akt) leading to activation of transcription factors that increase transcription of CMYC by binding to the promoter region of gene. Moreover, MYC gene is also transcriptionally regulated by Wnt/β-catenin or Notch signalling etc. The translated CMYC protein dimerizes with Max and such functional transcription factor complex CMYC/Max regulates (1) genes of cell cycle progression (Cyclin D1) and cyclin-dependent kinases (CDKs), (2) expression of ribosomal RNA (rRNA) genes, ribosomal proteins, and translation initiation factors, further enhancing the protein synthesing capacity, (3) expression of genes involved in glycolysis, and mitochondrial biogenesis, (4) pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) genes. However negative regulation of CMYC is through Mad/Max Complex or Ubiquitin-Proteasome Pathway.

The CMYC expression has already been demonstrated in our geographical population [8] but this paper represents somewhat qualitative effect rather than quantitative graded influence as documented earlier [8]. A loss of one CMYC possibly denotes altered proliferation in haploid state wherein the presence of other allele might have been normal rather than oncogenic. No correlation was evident with staging or duration of the disease and hence possibly the FISH expression of just the presence vs. absence of CMYC is not robust enough to categorize the disease severity. Although no significant correlation could be established with FISH expression of CMYC with other clinical parameters but an influencing trend in age of presentation and duration of disease (nasal blockage) could be appreciated. Those cases with deleted CMYC showed late age of presentation (mean 16.4y, SD 3.50 vs. mean 13.25y, SD 1.5 for normal CMYC) along with prolonged duration of nasal blockage (mean 14.4 m, SD 19.15 vs. mean 3.75 m, SD 2.06 for normal CMYC). The comparison was however not significant with small sample size in both groups.

TP53 gene referred as “guardian of the genome,” modulates responses to stress, (DNA damage, oncogene activation, hypoxia) and facilitates cell cycle arrest, DNA repair, senescence, or apoptosis. With DNA damage, phosphorylation of p53 prevents interaction with MDM2, that usually degrades p53. Abnormal activation of oncogenes (like Ras, Myc) triggers the ARF (alternative reading frame) protein, that binds to MDM2, thereby enhancing p53 levels. Hypoxic conditions also activate p53 through AMP-activated protein kinase (AMPK) and HIFs (hypoxia-inducible factors). The p53 during cell cycle arrest results in transcriptional activation of p21. This inhibits CDK2 and CDK4/6, leading to cell cycle arrest at G1/S and G2/M checkpoints, providing sufficient time to repair DNA damage. Further, p53 induces DNA repair genes viz. GADD45 (growth arrest and DNA-damage-inducible 45) and XPC (xeroderma pigmentosum). Moreover, with irreparable DNA damage, p53 induces apoptosis by upregulating BAX and other pro-apoptotic genes. Not the least p53 can also facilitate release of cytochrome-C from the mitochondria (by entering the later and interacting with BAX/ Bcl-2) to subsequently activate intrinsic apoptotic pathway. Finally, p53 can promote cellular senescence by upregulating p21 and PML (promyelocytic leukemia protein). The control of p53 activity is through negative regulation by MDM2 or through post-translational modifications such as phosphorylation (by ATM, ATR, Chk1/2 in response to stress signals, that stabilizes and enhance its transcriptional activity of p53), acetylation (by histone acetyltransferases to enhance its ability to bind DNA and activate transcription) or methylation and ubiquitination (instrumental in fine-tuning p53 stability and activity). TP53 mutation in tumorigenesis that often result in loss of its tumor-suppressive functions and many mutations, result in non-functional or dominant-negative p53 protein that poorly modulates cell cycle arrest or apoptosis.

Although no significant correlation could be established with FISH expression of TP53 with clinical parameters but a similar influencing trend was appreciated as in CMYC (age of presentation, duration of nasal blockage) along with dissimilarity in intraoperative haemorrhage. The cases with deleted TP53 presented relatively late (mean16.5y, SD 3.87 vs. mean 13.8y, SD 2.04 for normal TP53) with prolonged duration of nasal blockage (mean 16.25 m, SD 21.66 vs. mean 4.4 m, SD 1.67 for normal TP53) and enhanced intraoperative haemorrhage (mean 720.20 ml, SD 400.34 vs. mean 490.24 ml, SD 162.71 for normal TP53). Again the comparison was not significant with small sample size in both groups.

The CEPX and CEPY FISH probes target centromeric regions of X and Y chromosomes respectively and particularly diagnose sex chromosomal aneuploidy. Females reveal two signals for CEPX probe (indicating two X chromosomes), while male presents with one such signal. On the other hand, only a single signal for the CEPY probe is seen in male, and no such signal in female. The usual application of both these probes is established in sex determination, detection of aneuploidy, chimerism/ mosaicism studies and cancer diagnostics. Hence a normal female karyotype (46,XX) shows 2 green signals (CEPX) with no red signal (CEPY), while a normal male (46,XY) reveals 1 green and 1 red signal. The current study revealed an absent Y chromosome in FISH analysis. The overall genes on Y chromosome are primarily involved in male-specific functions and characteristics. The loss of Y chromosome in the tumour tissue may indicate a dominant influence of X chromosome genes per se. Although archival NPF has been known for its androgenic predominance since decades but the modern molecular profile does not reveal the same high expression of androgenic receptor (AR) in NPF [8] universally. A higher predominance of oestrogen receptors (than AR) has been documented [9] and many studies [10, 11] have stressed on careful such selection of hormone therapy in NPF. Accordingly, the loss of Y chromosome as encountered in our series may possibly suggest a predominant ER effect in tumour tissue in that particular case. This however could not be verified with immunohistochemistry. However, the non-visualization of Y-chromosome in FISH analysis cannot be taken as its complete loss since the FISH probe CEP Y signifies the centromere region only. Hence at the most, genes adjoining the centromeric region may be implicated in such cases rather than assuming the loss of all the genes of Y-chromosome. In accordance it can be argued if such aberrations can be hereditary in male off-springs but still needs to be established.

Although centromere itself is gene-poor, but its nearby vicinity contain key genes related to male fertility and spermatogenesis viz. TSPY (Testis-Specific Protein, Y-Linked), DAZ (Deleted in Azoospermia), RBMY (RNA Binding Motif Protein, Y-linked) and USP9Y (Ubiquitin Specific Peptidase 9, Y-Linked). Some other important genes with varied functions include EIF1AY (Eukaryotic Translation Initiation Factor 1 A, Y-linked), PRKY (Protein Kinase Y-linked), AMELY (Amelogenin, Y-linked), ZFY (Zinc Finger Protein, Y-linked), RPS4Y1 (Ribosomal Protein S4, Y-linked 1), RPS4Y2 (Ribosomal Protein S4, Y-linked 2), TXLNGY (Taxilin Gamma Pseudogene, Y-linked), CDY1/CDY2 (Chromodomain Protein, Y-linked), BPY2 (Basic Protein Y 2), HIST1H2BBY (Histone Cluster 1 H2BB, Y-linked), TBL1Y (Transducin Beta Like 1, Y-linked), VAMP7Y (Vesicle-Associated Membrane Protein 7, Y-linked), TMSB4Y (Thymosin Beta 4, Y-linked), KDM5D (Lysine Demethylase 5D), UTY (Ubiquitously Transcribed Tetratricopeptide Repeat Containing, Y-linked), NLGN4Y (Neuroligin 4, Y-linked), AZFa, AZFb, AZFc Regions (Azoospermia Factor Regions) and SRY-Box 21. Considering fertility-related genetic profile of Y chromosome, the probable genes in context to NPF are those related to growth and immune function such as SHOX (Short Stature Homeobox), IL3RA (Interleukin-3 Receptor Alpha) and CSF2RA (Colony Stimulating Factor 2 Receptor Alpha). Much research of Y-chromosome needs to be done as this tumour is exclusively encountered in males.

Karyotyping/ G-banding in cytogenetics has now been replaced by more sophisticated comparative genomic hybridization while facilities for solid tumour cytogenetics is largely unavailable in our country. The later seems important since G-banding karyotype from peripheral blood does not correlate karyotype in solid tumour. The high cost of FISH probes and the required expertise may limit its routine clinical use; however, this technique can augment other molecular tools for genetic research.

Electronic Supplementary Material

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Author Contributions

This is a Master’s thesis project of VP under chief guidance of AM. MKS was co-guide whose laboratory was used to analyse tissue samples and generate cytogenetic images. MKS was instrumental in providing the images of FISH and karyotyping. VP helped in molecular data generation. All the clinical data was collected at one institute while cytogenetic workup was undertaken at another institute. The complete data was analysed jointly and manuscript prepared thereafter primarily by AM.

Declarations

Competing Interests

None.