Baseline characteristics of the study participants
Mean (±SD) age of the studied patient cohort was 59.24 (±10.16) years for BC and 60.29 (±11.45) for CRC, 48.9 (±13.91) for healthy female controls and 49.01 (±17.14) for healthy male controls. The baseline characteristics of the BC and CRC patients are summarized in Table 1 and 2 respectively. The majority of both BC and CRC patient population represents the Sinhalese ethnicity and were over 40 years in age.
Analysis of TP53 sequence variants
A total of 16 sequence variants were found in 30 BC patients and 15 sequence variants were found in 21 CRC patients. In healthy male and female controls 6 and 8 variants were found respectively. Table 3 illustrates the characteristics of each variant and detailed in-silico and functional analysis are given in supplementary table 2.
Two novel frameshift variants were found in exon 8. The first of these, c.848_849delGC (Fig. 1), was observed in a 55 year old female BC patient with triple negative, poorly differentiated invasive ductal carcinoma, which results in an Arginine to Histidine substitution at the site of the deletion followed by other downstream amino acid changes and truncation of the protein at codon 303. The second frameshift variant, c.851_855delCAGAG (Fig. 1), was found in a 62 year old female BC patient with human epidermal growth factor receptor-2 (HER2) negative invasive ductal carcinoma with Ki67 index 32% and it result in a coding change from Threonine to Arginine at the site of deletion and other downstream amino acid encoding changes, plus a premature stop codon at position 302. Both the predicted truncated proteins are similar in length, with tetramerization and negative regulatory domains lost in both proteins.
A novel 3-base pair in-frame deletion was identified in exon 5. c.431_433delAGC (Fig. 2), resulting in the loss of the Glutamine amino acid residue at position 144 present in the β strand of the DNA binding domain and produces a 392 amino acid, shorter by one amino acid compared with the 393 amino acid full length protein. It was detected in a 66-year-old female CRC patient with moderately differentiated adenocarcinoma who had a previous history of cervix cancer. This patient also had a pathogenic KRAS codon 12 variant sequence (c.35G>A; rs121913529).
A reported nonsense variant, c.637C>T in exon 6 was found in both BC and CRC patients, resulting in a change from Arginine to a stop codon at 213. This truncated protein lacks part of the DNA binding domain, the tetramerization domain and negative regulatory domain, which would make the p53 protein non-functional. The BC patient was 47 years old with estrogen receptor (ER)/ progesterone receptor (PR) positive, HER2 equivocal invasive ductal carcinoma. The CRC patient was a 58 year old female with moderately differentiated adenocarcinoma.
There were four reported pathogenic missense variants, c.400T>G, c.730G>T, c.743G>A, c.840A>T in exons 5, 7 and 8 observed only in BC patients. Variants c.400T>G, c.730G>T and c.840A>T were observed in one patient each, while c.743G>A present in a CpG site was observed in 3 patients and 2 of them had triple negative invasive ductal carcinoma.
There were four reported pathogenic missense variants, c.524G>A, c.581T>G, c.733G>A, c.844C>T in exon 5, 6, 7 and 8 respectively observed only in patients with CRC. A c.524G>A variant was observed in a 67 year old male patient with moderately differentiated adenocarcinoma. He also had the pathogenic KRAS codon 61 variant (c.183A>T; rs17851045). A c.581T>G substitution was found in a 65 year old female with well differentiated adenocarcinoma. The missense variant c.733G>A was found in a 62 year old male patient with moderately differentiated adenocarcinoma and in a 71 year old female with tubular adenocarcinoma. A c.844C>T substitution was reported in a 66 year old female with moderately differentiated adenocarcinoma, who had a previous history of ovarian cancer. She also carried the pathogenic KRAS codon 12 variant (c.34G>C; rs121913530).
A likely pathogenic variant c.626G>A in exon 6 was observed in a 48 year old BC patient with ductal carcinoma. There were also 2 silent variants with uncertain significance, of which, c.63C>T appeared in 2 BC patients and in 1 female healthy control and c.459C>T appeared in 1 CRC patient and in a healthy control. Another silent variant c.903A>G observed in exon 8 in 1 BC patient is categorized as likely benign.
The codon 72 variant (p.R72P) in exon 4 is a well known TP53 polymorphism. In the present study, R/R, R/P and P/P genotype distribution was 12 (40%), 11 (36.67%), 7 (23.33%) respectively in BC patients and 12 (40%), 13 (43.33%) and 5 (16.66%) respectively in healthy controls. No significant difference was observed (p = 0.78) in the prevalence of different genotypes in either the BC patients or the healthy controls. The genotypic distribution of R/R, R/P and P/P was 6 (28.57%), 9 (42.86%), 6 (28.57%) respectively in CRC patients and 6 (28.57%), 10 (47.62%) and 5 (23.81%) respectively in healthy controls. Similarly, there was also no significant difference (p value = 0.93) observed in the prevalence of different genotypes between the CRC patients and healthy controls
Immunohistochemistry
The results obtained from immunohistochemistry analysis of 13 BC and 14 CRC tissue samples for IHC, were categorized into three; widespread IHC positive tumour nuclear staining involving either the entire or a segment of a tissue section (Pattern-A), rare/ scattered positive cells (Pattern-B) and complete absence of IHC positive signal (Pattern-C) (Fig. 3).
Status of TP53 gene and the p53 protein expression
For BC patients, positive IHC staining was detected in 7/13 (53.85%) cases. Three of these tumour sections showed pattern A, while 4 showed Pattern-B. All 3 BC cases that showed Pattern-A had TP53 missense variants while the 4 cases that showed Pattern-B had no detectable pathogenic variants of TP53. Among the 6 cases with immuno-negativity (Pattern-C), one had a silent variant and the remaining 5 patients had wild-type TP53.
For CRC patients, positive IHC staining was observed in 11/14 (78.57%) cases. Four of these tumours showed pattern A, while 7 showed pattern-B. Among the 4 CRC samples that showed pattern A, 3 had a TP53 missense variant each, while the remaining case had no detectable pathogenic variants. All 7 cases that showed pattern-B had no detectable pathogenic TP53 variants. Among the 3 cases with pattern-C, one had a non-sense variant, another one had a silent variant and the remaining patient showing immuno-negativity had wild-type TP53.
Comparison of p53 protein expression with the expression p21 and MDM2
Among the 3 BC samples showing p53 IHC staining with pattern A; 1 showed pattern B staining and the remaining 2 showed pattern C for p21. From the 4 samples with pattern B of p53 expression, 3 showed pattern B, while 1 showed pattern C for p21. All 6 samples with pattern C IHC for p53 also showed pattern C for p21. However, expression of MDM2 for all samples showed pattern C IHC staining, regardless of the expression pattern of p53 or p21 protein (Table 4-1)
Among 4 CRC samples with pattern A p53 IHC staining, 2 showed pattern B and the remaining 2 showed pattern C staining for p21. All 7 samples with pattern B p53 IHC staining showed pattern B staining for p21. Among 3 with pattern C p53 IHC staining, 2 showed pattern C staining for p21 and the remaining one showed pattern A for p21 IHC staining. In the case of MDM2, among the 4 samples with p53 IHC staining with pattern A, 1 showed pattern B and the remaining 3 showed pattern C. From the 7 samples with pattern B of p53 expression, 4 showed pattern B, while 3 showed pattern C for MDM2. All 3 samples with pattern C IHC for p53 also showed pattern C for MDM2 (Table 4-2).