|Year : 2013 | Volume
| Issue : 3 | Page : 195-199
P-glycoprotein expression as a predictor of response to neoadjuvant chemotherapy in breast cancer
S Vishnukumar1, G Umamaheswaran2, D Anichavezhi2, S Indumathy2, C Adithan2, K Srinivasan1, D Kadambari1
1 Department of Surgery, Jawaharlal Institute of Postgraduate Medical Education & Research, Pondicherry, India
2 ICMR Centre for Advanced Research in Pharmacogenomics, Department of Pharmacology, Jawaharlal Institute of Postgraduate Medical Education & Research, Pondicherry, India
|Date of Web Publication||23-Sep-2013|
Department of Surgery, Jawaharlal Institute of Postgraduate Medical Education & Research, Pondicherry
Source of Support: None, Conflict of Interest: None
Background: Chemoresistance is an important factor determining the response of tumor to neoadjuvant chemotherapy (NACT). P-glycoprotein (P-gp) expression-mediated drug efflux is one of the mechanisms responsible for multi-drug resistance. Our study was aimed to determine the role of P-gp expression as a predictor of response to NACT in locally advanced breast cancer (LABC) patients. Materials and Methods: P-gp expression was performed by real-time quantitative polymerase chain reaction [qRT-PCR] in 76 patients with LABC. Response to adriamycin-based regimen was assessed both clinically and with contrast enhanced computed tomography (CECT) scan before and after NACT. The significance of correlation between tumor and P-gp levels was determined with Chi-square test. Results: Twenty-one had high and 55 had low P-gp expression. On analyzing P-gp expression with response by World Health Organization (WHO) criteria, statistical significance was obtained (P = 0.038). Similarly, assessment of P-gp expression with response by Response Evaluation in Solid Tumors (RECIST) criteria in 48 patients showed statistical significance (P = 0.0005). Conclusion: This study proves that P-gp expression is a determinant factor in predicting response to NACT. Finally, detection of P-gp expression status before initiation of chemotherapy can be used as a predictive marker for NACT response and will also aid in avoiding the toxic side effects of NACT in non-responders.
Keywords: Breast cancer, neoadjuvant chemotherapy, P-glycoprotein
|How to cite this article:|
Vishnukumar S, Umamaheswaran G, Anichavezhi D, Indumathy S, Adithan C, Srinivasan K, Kadambari D. P-glycoprotein expression as a predictor of response to neoadjuvant chemotherapy in breast cancer. Indian J Cancer 2013;50:195-9
|How to cite this URL:|
Vishnukumar S, Umamaheswaran G, Anichavezhi D, Indumathy S, Adithan C, Srinivasan K, Kadambari D. P-glycoprotein expression as a predictor of response to neoadjuvant chemotherapy in breast cancer. Indian J Cancer [serial online] 2013 [cited 2019 Jun 17];50:195-9. Available from: http://www.indianjcancer.com/text.asp?2013/50/3/195/118726
| » Introduction|| |
Breast cancer is one of the leading causes of mortality worldwide, affecting the life and productivity of women. The trend in the management of locally advanced breast cancer (LABC) has changed from radical surgery to multimodality approach involving surgery, radiotherapy, and chemotherapy. Breast cancer accounts for 33% of all female cancers and is responsible for 20% of the cancer-related deaths in women.  The incidence of breast cancer in India is on the rise and is rapidly becoming the number one cancer in females pushing the cervical cancer to the second spot. It is reported that 1 out of 22 women in India is likely to suffer from breast cancer during her lifetime.  Neoadjuvant chemotherapy (NACT) is an integral part in the management of LABC. All tumors do not respond to chemotherapy in the same manner. This variability in response may be due to several factors like absorption, distribution, metabolism, and elimination of the drugs mediated by drug transporters and drug metabolizing enzymes that contribute to resistance at the cellular level.  One of the factors that have been studied very often is the MDR1 gene also known as ABCB1 mapped on chromosome 7 at q21.1 and consists of 29 exons ranging in size from 49 to 209 bp.  This gene codes for P-glycoprotein (P-gp), an ATP- dependent drug efflux pump which transports various hydrophobic substrates including anti-cancer drugs such as anthracyclines (doxorubicin), epipodophyllotoxins (etoposide), vinca alkaloids (vincristine), dactinomycin, and paclitaxel. P-gp is normally expressed in tissues with excretory function, including the jejunum, kidney, liver, and adrenal gland.  Over-expression of MDR1 will result in increased drug efflux activity of P-gp leading to multi-drug resistance (MDR).
Chemoresistance is an important factor which determines the response of a tumor to a particular chemotherapy regime. The degree of expression, regulation, and activity of P-gp influenced by MDR1 gene polymorphisms can directly affect the therapeutic efficacy of P-gp substrates.  With this background, this study was aimed to find out the baseline P-gp expression in patients with breast cancer and to determine its role in predicting the response to NACT in LABC.
| » Materials and Methods|| |
After approval by the Institute Ethics Committee, 81 patients with LABC according to AJCC (American Joint Committee on Cancer) classification were included in the study. Core needle biopsy was done to confirm the diagnosis in all patients and P-gp expression was assessed from the tissue sample. Also, estrogen receptor (ER), progesterone receptor, and human epidermal growth factor receptor 2 (Her2/neu) status was assessed from the core biopsy samples. Routine investigations included complete blood counts, renal and liver function tests. Metastatic workup was done with chest x-ray and ultrasound of the abdomen. Echocardiography was done to assess left ventricular function as all patients received adriamycin-based chemotherapy. Detailed history and clinical examination were performed.
Clinical assessment of the tumor was done and measurements of the two longest diameters were taken. Patients underwent contrast-enhanced computed tomography (CECT) thorax before starting chemotherapy. Thus, the tumor size and axillary lymph nodal status were determined both clinically and by using CECT thorax before chemotherapy. Patients received three cycles of chemotherapy [CAF regime - Cyclophosphamide, Adriamycin, and 5-Fluorouracil]. Two weeks after the completion of the third cycle of chemotherapy, the response was assessed clinically and by CECT thorax using RECIST (Response Evaluation Criteria in Solid Tumors) criteria and was taken up for surgery. The pathological response (gold standard) from the specimen was compared with the RECIST response. The tissue bits were taken before the treatment and stored in RNA later solution(TM) at -80°C and assessed for P-gp expression.
Objective response was defined as patients with complete response (CR) or partial response (PR) [CR: Complete disappearance of tumor in CECT, PR: at least 30% decrease in the sum of the longest diameters (LD) of the target lesions, taking reference the baseline sum LD] after three cycles of NACT. Non-responders were patients with less than 30% response, no change or local progression. Pathological CR (pCR) was defined as the absence of any gross or microscopic evidence of tumor in the specimen.
| » mRNA expression analysis using RT-PCR|| |
Total RNA was extracted from breast cancer tissue using Trizol reagent (Medox). The complementary DNA (cDNA) was synthesized from total RNA according to manufacturer's protocol (Strategene, Agilent Technologies). After reverse transcription, standard cDNA was serially diluted to five standard solutions (200 to 0.02 ng) to prepare the standard reference curve. MDR1 mRNA expression levels was measured by real-time quantitative polymerase chain reaction (qRT-PCR) using 7300 RT - PCR system (Applied Biosystems, Foster City, CA, USA) - with brilliant SYBR Green quantitative PCR assay (Strategene, Agilent Technologies). Glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) was used as endogenous reference. The reactions were carried out in 25 μl final volume in that contained of 12.5 μl of SYBR Green mastermix, 0.75 μl of each primer, 0.375 μl of reference dye, 5.625 μl of deionized water and 5 μl of cDNA as template. The assays were performed in triplicates in order to confirm the efficiency and reliability of the method and the mean Ct values were taken for each sample derived from duplicate reactions.
The thermocycler conditions included 1 cycle at 50°C for 2 min; 1 cycle at 95°C for 10 min to activate the AmpliTaq Gold polymerase followed by 40 cycles of denaturation at 92°C for 15 s and annealing/extension at 60°C for 1 min. The melting curve program consisted of 1 cycle at 95°C for 15 s, 60°C for 30 min, and 95°C for 15 s with a linear temperature transition rate of 0.1°C/s with continuous fluorescence acquisition. The relative standard curve method was used for quantification and the normalized Ct values of MDR1 to that GAPDH were used to plot the standard curve.
The resulting melting curves allowed for discrimination between primer-dimers and specific products. PCR amplification was carried out using forward primer 5'-GACAGGCATCTCCAAGCATT-3' and reverse primer 5'-ATCTGGTTTGTGCCCACTCT-3' for MDR1 gene and forward primer 5'-ACCACAGTCCATGCCATCAC-3' and reverse primer 5'-TCCACCACCCTGTTGCTGTA-3' for GAPDH gene.
| » Statistical analysis|| |
Statistical analysis was done using Statistical Package for Social Software (SPSS, Windows version release 16). The association between P-gp expression, immunohistochemical markers, and response to NACT was analyzed by calculating odds ratio (OR) and 95% confidence intervals (CIs) using the chi-square test. P < 0.05 was considered significant.
| » Results|| |
Eighty-one patients were recruited initially. Five patients did not complete the treatment protocol and were excluded from the final analysis. Both CECT scans could be done only in 48 patients and so the response as per RECIST criteria could be assessed only in them.
The mean age at presentation in this study was 49.7 years (range 31-70 years) with majority of patients in the age group of 40-49. The mean tumor size before chemotherapy was 7 cm. The mean duration of symptoms was 8.4 months. Fifty patients (61.7%) had T3 tumor and 31 (38.3%) had T4 tumor at presentation. Out of 76 patients, 14 had complete response, 20 had partial response, and 42 had no response. Of the 76 patients only 48 patients could be assessed by using RECIST criteria. The expression was found to be high in 21 patients (27.6%) and low in 55 (72.3%) patients.
| » WHO response|| |
the 21 patients in the high P-gp expression group, 1 (4.8%) showed complete response, 4 (19%) had partial response, and 16 (76.2%) did not respond. The objective response was in 5/21 (23.8%). Of the 55 in the low expression group, 13 (23.6%) had complete clinical response, 16 (29.1%) had partial response, and 26 (47.3%) showed no response. The objective response was in 29/55 (52.7%). On analyzing P-gp expression with WHO response criteria, statistical significance was found with a P-value of 0.038. The OR was 3.6 with 95% CI of 1.15-11.1 [Table 1].
| » Response evaluation criteria in solid tumors response|| |
Of the 48 patients assessed using Response evaluation criteria in solid tumors (RECIST) criteria, 14 (29.1%) had high expression and 34 (70.8%) had low expression of P-gp. In the high expression group, no patients had complete response. Two out of 14 (14.3%) had partial response and 12/14 (85.7%) had no response. Thus, objective response was observed in 2/14 (14.3%). In the low expression group, 7/34 (20.6%) had complete response, 17/34 (50%) had partial response, and 10/34 (29.4%) had no response. The objective response was found in 24/34 (70.6%). On analyzing P-gp expression with RECIST response criteria, statistical significance was found with a P-value of 0.0005. The OR was 14.4 with 95% CI of 2.71-76.43 [Table 2].
|Table 2: Association of P-gp expression with response by RECIST criteria|
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Of the 14 patients with complete clinical response, 1 (7.1%) had a high P-gp expression as compared to 13 (92.9%) with low P-gp expression. Of 7 (20.6%) patients in whom CECT scan showed complete response, all 7 (100%) had low P-gp expression. Histopathological examination revealed complete pathological response in 6 patients all of whom (100%) had low P-gp expression in their tumor.
Statistical analysis failed to show any significant association between ER, PR and Her2neu status, and response criteria.
| » Discussion|| |
NACT has become the standard of care and has changed the management of LABC. P-gp plays a role as an efflux pump in these tissues thereby providing a protective mechanism against chemicals in diet. This capacity of P-gp leads to strong resistance to chemotherapy. 
P-gp expression was detected in a broad range of human tumors (leukemia, lymphoma, cancers of ovary, breast, soft tissues tumors, gastric and colorectal cancers).  From various studies it was found that P-gp was more highly expressed in HCC (hepatocellular carcinoma) than in cirrhotic liver, which is consistent with the known resistance of HCC to chemotherapy. 
RT-PCR can be one of the most sensitive, efficient, fast, and reproducible methods of measuring gene expression. RT-PCR can be used to quantify mRNA levels from much smaller samples. In fact, this technique is sensitive enough to enable quantitation of RNA from a single cell. ,
Of the 81 enrolled in the study, 76 were finally included and their data was used for analysis. The mean age at presentation in this study was 49.7 years with majority of patients in the age group of 40-49. This was comparable with similar studies done earlier in this institute and elsewhere. The mean initial size of tumor in this study was 7.02 cm. The mean tumor size was 4.5 cm in the National Surgical Adjuvant Breast and Bowel Project (NSABP) trial-B27).  In a study done by Chintamani et al, the mean tumor size was 8 cm. Previous studies in this institute had shown a mean tumor size of 6.8 cm. This may be because most of the patients from Indian population present late to the hospital. The mean duration of symptoms at the time of presentation was 8.5 months in this study.
In the present study, P-gp expression was found to be high in 21 patients (27.6%) and low in 55 patients (72.3%). Chintamani et al found P-gp expression to be positive in 26/50 (52%) and negative in 24/50 (48%).  Li En-Xiao et al found that P-gp was positive in 48.3% of cases and they had a significantly lower 5-year survival rate. Distant metastasis occurred more frequently in the P-gp-positive cases than in the P-gp-negative cases receiving adjuvant chemotherapy.  Another study conducted by Tsukamoto et al showed 35/94 (37.2%) patients were positive for P-gp expression and had a significantly shorter disease-free survival period than P-gp-negative patients.  Gregorcyk et al found 24/55 (44%) P-gp positivity and concluded that P-gp-positive patients were at significantly greater risk for disease recurrence. 
Statistical analysis with Fisher's exact test showed significant association between tumor response and P-gp expression levels. Patients with high expression levels 16/21 (76.2%) had poor response and those with low expression levels 29/55 (52.7%) had good response.
Using WHO response criteria patients with high P-gp expression had 3.6 times more chance of having poor response to NACT when compared with those having low expression levels. On using RECIST criteria patients with high P-gp expression had 14.4 times more chance of having poor response to NACT when compared with those having low expression levels. All six patients with pathological complete response had low P-gp levels.
Burger et al found 17% response rate with high MDR1 expression compared with 68% with low expression levels.  Trock et al conducted a critical review and meta-analysis of 31 studies and found that patients with tumors expressing P-gp were three times more likely to fail to respond to chemotherapy than patients whose tumors were P-gp negative. P-gp expression in breast tumors is associated with a poor response to chemotherapy. 
In a study done by Chintamani et al, they found that with an increase in P-gp expression, the response rate was found to drop significantly. The P-gp positivity correlated inversely with clinical response to NACT in their study.  Gasparini et al, in their study, found that the presence of P-gp was significantly predictive of poor response with anthracycline based chemotherapy (P = 0.03).  As shown by other studies, our study also showed a poor response in the patients with high P-gp expression.
Molecular markers like ER and PR status, Her2neu oncogene have been studied in the recent past as predictive factors for chemotherapy. Although their role as prognostic factor is being evaluated, the data about these markers as a predictive factor for chemotherapy remains controversial and inconclusive.  . The present study failed to show any significant association between any of the markers and clinical response. Drug efficacy can be modulated by several genetic and non-genetic factors. Nevertheless, a large amount of the inter-patient variability in treatment response is attributable to the presence of molecular variations in the genes encoding drug metabolism and transporter proteins. Recently, George et al. has shown that patients with homozygous T allele had 2.3 times greater chance of achieving response to NACT when compared to carriers of CC genotype of MDR1 (3435C>T) gene polymorphism in South Indian breast cancer patients.  Since, the P-gp activity is haplotype-dependent linkage disequilibrium analysis, gene-environment interactions are required to elucidate the precise role of MDR1 gene polymorphism in influencing gene expression as well as in predicting the response to NACT.
| » Conclusions|| |
Patients with high P-gp expression had 3.6 times more chance of having poor response to NACT. By RECIST criteria they had 14.4 times more chance of having poor response to NACT. All patients with pathological complete response had low P-gp expression. This study proves that P-gp expression is a determinant factor in predicting response to NACT in breast cancer patients. Thus, detection of P-gp expression status before initiation of chemotherapy can be used as a predictor for response and also avoid its toxic side effects in non-responders.
| » Acknowledgments|| |
The authors would like to thank all the patients who participated in the study. A special thanks to Dr. Ghorpade Arun for his assistance in statistical analysis. This work was financially supported by Intramural Research Grant, JIPMER and partly by ICMR Centre for Advanced Research in Pharmacogenomics.
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[Table 1], [Table 2]