Changes in p53 Expression By Immunohistochemistry during Azacitidine Therapy in Patients with MDS Can Predict Overall Prognosis

Introduction

The TP53 gene encodes the tumour suppressor and cell cycle regulatory protein and is found to be mutated in a variety of carcinomas. Mutation in TP53 gene is associated with resistance to conventional therapy, disease progression and overall poor prognosis in solid tumours and haematological malignancies including Myelodysplastic Syndromes (MDS). TP53 mutated sub-clones in MDS have been demonstrated by deep sequencing technology in prior studies. Strong nuclear staining of p53 protein by immunohistochemistry has been used as a surrogate marker for TP53gene mutation in haematological and other malignancies.

Methods

We analysed sequential marrow samples for p53 expression on 35 patients with MDS from a single institution pre and post Azacitidine therapy. Formalin fixed, paraffin embedded marrow biopsies were stained with DO-7 mouse p53 monoclonal antibody. 1000 haematopoietic cells were examined under the high power and p53 expression was determined as per Modified Quick Score.

Results

Median age of the patients was 70 and WHO subgroups were identified as follows: 7 RCMD, 1  5q- syndrome, 1 MDS/MPN, 8 CMML, 6 RAEB-1, 6 RAEB-2 and 6 t-MDS. Cytogenetic risk as per IPSS-R/CPSS showed 17 (50%) lower risk, 4(12%) intermediate risk and 13(38%) higher risk groups. Patients received a median 13 cycles of Azacitidine. Marrows were assessed prior to treatment and after 3-6 cycles. Median overall survival of the study group was 20 months and transformation to AML occurred in 13 patients (37%). At diagnosis, 27 patients (77%) were p53 negative and 8 patients (23%) were p53 positive. At reassessment, 24 patients (69%) remained p53 negative while 6 patients (17%) remained p53 positive. Two patients (6%) became p53 negative and 3 (8%) became p53 positive following Azacitidine treatment. Median overall survival of patients who remained p53 negative during Azacitidine treatment was 28 months compared to 11 months in patients who remained p53 positive, p=0.005. Similarly, median overall survival of patients who remained or became p53 negative was 28 months compared to 18 months for those who remained or became p53 positive during Azacitidine therapy, p=0.012. p53 expression at diagnosis or changes in p53 expression during Azacitidine treatment did not correlate with transformation to Acute Myeloid Leukaemia (AML) or time to progression to AML. Among the p53 positive group, patients who had more than 10% p53 expression had lower overall survival compared to those who were <10% positive (14 vs. 27 months) In addition, changes in p53 positivity pre and post treatment did not seem to influence overall survival or transformation to AML in this small cohort.

Conclusions

In conclusion, our study showed that p53 expression changes in some patients during treatment with Azacitidine. Persistently positive p53 expression correlates with poor survival but does not correlate with transformation to AML. Analysis of p53 expression by immunohistochemistry is a clinically useful tool without employing expensive gene sequencing techniques and is readily available for routine practice. This method may be particularly useful in predicting outcomes for a subgroup of MDS patients on Azacitidine.

Table 1: Comparison of Median Overall Survival between p53 Positive and Negative groups during Azacitidine Therapy

Group	N, %	Median OS
P53 Negative at Diagnosis P53 Positive at Diagnosis	27, 77% 8, 23%	23 months 14 months	p= 0.049 (95% CI)
Positive p53 >10% at Diagnosis Positive p53 <10% at Diagnosis	10, 29% 23, 66%	14 months 27 months	p=0.039 (95% CI)
Remained p53 Neg Remained p53 Pos	24, 69% 6, 17%	28 months 11 months	p= 0.005 (95% CI)
Remained or became p53 Neg Remained or became p53 Pos	26, 74% 9, 26%	28 months 18 months	p= 0.012 (95% CI)
Decrease in p53 post-Azacitine(≤1%) Increase in p53 post-Azacitidine(≥1%)	20, 57% 13, 37%	19 months 20 months	p=0.294 (95% CI)

Figure 1: Comparison of Overall Survival between Patients who remained p53 positive and p53 negative after Azacitidine