Effect of Neoadjuvant Chemotherapy on in-vivo MRS determined tCho and Membranous and Cytoplasmic b-catenin Expression in Breast Cancer Patients
Naranamangalam R Jagannathan1, Khushbu Agarwal1, Uma Sharma1, Sandeep Mathur2, Vurthaluru Seenu3, and Rajinder Parshad3

1Department of NMR and MRI Facility, All India Institute of Medical Sciences, New Delhi, India, 2Department of Pathology, All India Institute of Medical Sciences, New Delhi, India, 3Department of Surgical Disciplines, All India Institute of Medical Sciences, New Delhi, India

Synopsis

We evaluated the changes in tCho levels and β-catenin expression (membrane and cytoplasm) after III neoadjuvant chemotherapy in breast cancer patients. Significant reduction in β-catenin expression (membranous and cytoplasmic) was observed after therapy. Post-therapy, tCho reduced significantly in tumors with Grades 1 and 2 membranous β-catenin expression and also in tumors with IRS 0 and Grade 1 cytoplasmic β-catenin. Prior to therapy, tCho was positively associated with cytoplasmic β-catenin while negatively with membranous protein. However post-therapy tCho was negatively associated with both cytoplasmic and membranous β-catenin. This signifies antiproliferative and apoptosis induction effects of chemotherapy drugs on breast cancer patients.

Purpose

To determine the effect of chemotherapy on the expression of membranous and cytoplasmic β-catenin and tCho concentration in breast cancer patients

Methodology

The present study was undertaken to explore the changes associated with neoadjuvant chemotherapy (NACT) treatment on levels of tCho and expression of β-catenin (membrane and cytoplasm) in breast cancer patients. Twenty patients with locally advanced breast cancer (LABC) were recruited for in vivo 1H magnetic resonance spectroscopy (MRS) at 1.5 T (Avanto, Siemens). Table 1 presents the clinical details of the patients. tCho concentration was calculated using the integral values1 of the acquired MR spectrum from each patient both prior to and after III NACT. Tissues (biopsy before NACT and surgical specimen after NACT) obtained from the same set of patients were immediately fixed in formalin and paraffin embedded blocks were prepared. Immunohistochemistry (IHC) was carried out using anti-β-catenin antibodies. The intensity was scored as follows: 0 = none, 1+ = weak, 2+ = moderate, and 3+ = strong/intense. The percentage of β-catenin positive cells was scored from 1 to 7 (1 = 0%; 2 = 1–4%; 3 = 5–19%; 4 = 20–39%; 5 = 40–59%; 6 = 60–79%; 7 = 80–100%). Immunoreactive scores (IRS) were calculated by multiplying the scores for percentage and intensity2. Patients with IRS of 3-8 were categorized as Grade 1 and IRS of 9-21 as Grade 2. Wilcoxon matched pairs two tailed test and Spearman’s rank analysis were used to calculate the significance and correlations. A p-value of ˂0.05 was considered significant. Institutional ethical committee approved the study and written informed consent were obtained from all patients.

Results and discussion

In the present study, the effect of chemotherapy on the expression of β-catenin in both membrane and cytoplasm as well as on tCho concentration was investigated in breast cancer patients. Table 2 presents the grades based on IRS for expression of β-catenin (membrane and cytoplasm) and the tCho concentration both prior to and after III NACT. Figure 1a shows the positive control (Split end, SPEN) and Figs. 1b-e are the representative slides showing β-catenin expression in cytoplasm and membrane both pre- and post-therapy. Prior to therapy, membranous immunoreactivity to β-catenin was seen in 18/20 (90%) patients; of these 15% showed Grade 1 while 75% patients had Grade 2 staining. Cytoplasmic immunoreactivity to β-catenin was seen in 10/20 (50%) patients prior to therapy; of these, 15% showed Grade 1 while 35% patients showed Grade 2 staining. After III NACT, both membranous and cytoplasmic expression of β-catenin reduced significantly (Figure 2; Table 2). Further, our data showed that the concentration of tCho significantly reduced following chemotherapy compared to its pre-therapy value in tumors with membranous expression of β-catenin of Grade 1 and Grade 2 (Table 2). The tumors with cytoplasmic β-catenin expression in IRS 0 and Grade 1 also showed a significant reduction in tCho concentration after therapy (Table 2). The decrease in tCho concentration observed may be due to the cytotoxic activity of chemotherapy drugs. This may be related to decrease in the activity of major enzymes involved in choline synthesis pathway like choline kinase, phospholipase D (PLD) and phospholipase A23. Prior to therapy, a positive association between tCho concentration and cytoplasmic β-catenin while a negative correlation with membranous β-catenin was observed which may be attributed to the translocation of β-catenin from membrane to cytoplasm. The increased levels of β-catenin in cytoplasm increases the activity of PLD, which in turn enhances the tCho concentration as observed prior to therapy. However, post-therapy a negative association of tCho with both cytoplasmic and membranous β-catenin was observed. The antiproliferative and apoptosis induction effects of chemotherapy drugs mediate the suppression of Wnt/β-catenin signaling, and these actions might contribute to a decrease in β-catenin expression in membrane and cytoplasm of the cells. This leads to decreased activity of PLD thereby decreasing the tCho concentration after NACT in breast cancer patients.

Conclusion

In the present study the association between tCho concentration and the cytoplasmic and membranous β-catenin emphasizes the possibility that both tCho and β-catenin influence breast cancer progression. While their decrease followed by NACT treatment reflects the cytotoxic effect of chemotherapy drugs on these molecular pathways in these LABC patients. The results of our present study call for additional studies to determine the mechanism of NACT effect on different molecular pathways involved in breast cancer.

Acknowledgements

The authors thank the Department of Science and Technology, Government of India for the financial assistance.

References

(1) Sah RG, Sharma U, Parshad R et al. Association of Estrogen Receptor, Progesterone Receptor, and Human Epidermal Growth Factor Receptor 2 Status with Total Choline Concentration and Tumor Volume in Breast Cancer Patients: An MRI and In Vivo Proton MRS Study. MRM 2012;68(4):1039-47; (2) Lpoez Knowles E, Zardawi SJ, McNeil CM et al. Cytoplasmic localization of beta-catenin is a marker of poor outcome in breast cancer patients. Cancer Epidemiol Biomakers Prev 2010:19(1);301-9; (3) Glunde K, Jie C, Bhujwalla Z et al. Molecular cause of aberrant choline phospholipid metabolism in breast cancer. Cancer Res 2004;64(12):4270-4276.

Figures

Table 1: Detailed clinical characteristics including menopausal status, histological findings, staging, and chemotherapy regimen used in breast cancer patients

Table 2: Pre- and Post-therapy immunoreactive scores (IRS) for β-catenin expression (membrane and cytoplasm) and Grade wise distribution of tCho concentration in breast cancer patients (n=20)

Figure 1: Representative example of IHC slides showing (a) SPEN: ß-catenin positive control; (b) Pre-therapy; ß-catenin with 70% cells showing moderate cytoplasmic staining; (c) Post-therapy; Reduced ß-catenin expression with only 5% cells showing weak cytoplasmic staining; (d) Pre-therapy; ß-catenin with 75% of cells showing strong membranous staining; (e) Post-therapy; Reduced ß-catenin expression with only 10% cells showing moderate membranous staining

Figure 2: Box plots showing the change in tCho concentration (a); cytoplasmic β-catenin (b) and membranous β-catenin (c) as a result of therapy

Figure 3: Scatter plots showing (a) tCho concentration and cytoplasmic β-catenin expression; (b) tCho concentration and membranous β-catenin expression prior to therapy in breast cancer patients



Proc. Intl. Soc. Mag. Reson. Med. 24 (2016)
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