Choline as a biomarker for cell proliferation: Do the results from proton MR spectroscopy show difference between HER2/neu positive and negative breast cancers?

Dear Sir,

HER2/neu gene is a member of family of genes encoding trans-membrane receptors for four growth factors, including the epidermal growth factor receptor, HER2/neu, HER-3 and HER-4. The intracellular domain of HER2/neu has tyrosine kinase activity which regulates cell growth and proliferation.1–5 Overexpression of HER2/neu can transform cultured cells into more aggressive phenotype and accelerate tumorigenesis.1, 6 HER2/neu is overexpressed in 20%–25% of invasive breast cancers and associated with an aggressive tumor, an early relapse and reduced survival rate.7–9

It has been found that a tumor cell line overexpressing HER2/neu resulted in an increase in levels of choline-containing compounds (tCho) measured by in vitro proton MR spectroscopy (MRS), including phosphocholine (PCho), glycerophosphocholine and choline.10 It was postulated that growth factor-mediated activation of the tyrosine kinase cascade can lead to an increase in PCho levels.10 Although recent ¹H-MRS studies conducted at clinical MR scanners have shown that invivo proton MRS can be used to distinguish between malignant and benign tissues based on the detection of choline (tCho),11–15 further investigation in a large population is needed to determine whether the additional information measured by ¹H-MRS (tCho) may improve the specificity of DCE-magnetic resonance imaging (MRI) (morphological and kinetic enhancement characteristics) in differentiating benign from malignant tumors.

Choline measured by ¹H-MRS may provide an imaging marker for cell proliferation. Our recently published article16 analyzing the MR imaging features with respect to HER2/neu overexpression in invasive breast cancer demonstrated a higher choline detection rate in HER2/neu positive compared to negative cancer. The number of patients in that study was, however, very small and conclusion could not be drawn. Here, we reported a larger series study to further investigate the choline expression between HER2/neu +/− cancers.

Sixty-six breast cancer patients (range 32–76 years old, mean 51 year) enrolled from March 2005 to October 2006, who were scanned with the MRI/MRS protocol were included in this study. The inclusion criteria were patients with biopsy confirmed diagnosis of malignant lesions that measured 1.5 cm or larger on MR images. Of the 66 malignant lesions, 41 (77%) were invasive ductal carcinomas, 7 (11%) were invasive lobular carcinomas, and the other 8 (12%) were mixed invasive ductal and lobular carcinomas. HER2/neu status was determined initially using the immunohistochemical staining (IHC), positive in tumors with 3+ staining score, and negative for score of 0 and 1+. For those with IHC 2+ staining, fluorescent in situ hybridization (FISH) was conducted to determine the status.

The MRI study was performed using a 1.5 T Phillips Eclipse MR scanner with a standard bilateral breast coil (Philips Medical Systems, Cleveland, OH). The imaging protocol consisted of high-resolution precontrast imaging from the concerned breast, bilateral dynamic contrast-enhanced imaging, and ¹H-MRS. For dynamic acquisition, the MR contrast agent gadodiamide (Omniscan®, GE Healthcare, Princeton, NJ, 0.1 mmol/kg body weight) was manually injected. After the MRI study was completed, single-voxel ¹H-MRS was performed using a point-resolved spin-echo sequence. The spectroscopic voxel was carefully positioned to maximize the coverage of the contrast-enhanced lesions while minimizing the inclusion of adipose tissue. The voxel size was from 2.4 to 8.0 mL. The absolute tCho concentration was analyzed using as an internal reference method.17 The tumor size was measured by a radiologist based on the maximum intensity projection of the contrast subtraction images.

Of 66 cancers, 45 (68%) were HER2/neu negative and 21 (32%) were HER2/neu positive. The mean size of 66 malignant tumors was 3.4 cm (range, 1.5–8.6 cm). The ¹H-MRS result was positive for tCho in 53 (80%) of 66 patients. The measured Cho levels ranged from 0 to 8.5 mmol/kg (mean ± SD, 1.9 ± 1.9 mmol/kg), which were consistent with the previously published value by Bolan et al.17 Table I summarizes in vivo breast ¹H-MRS results in HER2/neu positive and negative groups. The choline detection rate was higher in HER2/neu positive group (91%) than in HER2/neu negative group (76%), but not reaching significant level (p = 0.26, χ² test). The comparison between two groups in tCho concentration and tumor size is also shown in Figure 1, but no significant difference was observed. Figures 2 and 3 are two represented cases, one shows positive choline detection in HER-2 positive breast cancer, and the other showing negative choline in HER-2 negative breast cancer.

Figure 1. Comparison of tumor size and total choline-containing compounds (tCho) in HER2/neu-positive and negative groups. There was no significant difference in tumor size (a) and tCho level (b) between these two groups.

Figure 2. MRI and ¹H-MRS measurement in a 40-year-old patient with HER-2/neu overexpression in invasive breast cancer. The spectroscopic voxel (size, 2 × 2 × 2 cm³) is superimposed on the contrast-enhanced lesion in the subtraction axial image (a). The MR spectrum was measured from the selected lesion. A Cho peak at 3.23 ppm is clearly visible in the water-fat suppressed spectrum (b). The Gaussian model fitting of the Cho peak produces a measurement of [Cho] = 2.33 ± 0.54 mmol/kg.

Figure 3. MRI and ¹H-MRS measurement in a 50-year-old patient without HER-2/neu overexpression in invasive breast cancer. The spectroscopic voxel (size, 2 × 2 × 2 × 3 cm⁴) is superimposed on the contrast-enhanced lesion in the subtraction axial image (a). The MR spectrum was measured from the selected lesion. A Cho peak at 3.21 ppm is not visible in the water-fat suppressed spectrum (b).

Table I. Sensitivity and tCHO Concentration in HER/neu Positive and Negative Breast Cancers Using In Vivo Proton MR Spectroscopy

	No. of true positives	No. of false negatives	Sensitivity	tCho level (mmol/kg)
Note: tCho = total choline-containing compounds. No significant difference was found between both groups in tCho level and sensitivity of proton MR spectroscopy in detecting choline.
HER2/neu
Positive (N = 21)	19	2	91%	1.50
Negative (N = 45)	34	11	76%	2.03
Overall	53	13	80%	1.87

HER2 receptor mediates signaling to cancer cells and stimulates proliferation.3–5In vitro cell line study by overexpressing HER2/neu in MCF7 cells showed higher proliferation rate.18 Overexpression/amplification of HER2 is associated with tumor aggressiveness and a poor prognosis in breast cancer. Limited literature is available correlating MR imaging features with HER-2 biomarkers expressed in invasive breast cancer. Tse et al.19 reported 17 out of 19 breast cancer patients showing positive choline detection. The two false-negative cases were negative for HER-2/neu oncogene expression, suggesting that a false-negative spectroscopic result may be related to an absence of Her-2 overexpression in carcinoma of the breast. Analyzing a subset of patients in year 2005 included this article, Agrawal et al.16 showed more choline detection rate in HER-2/neu positive patients compared to Her-2/neu negative cohort in small number of patients (4/7 vs. 0/8, p < 0.05). The case number was too small in the two aforementioned studies. In this much larger series study, the sensitivity of in vivo¹H-MRS in HER2/neu negative group, although lower, was not significantly different from that of HER2/neu positive group, and also the absolute tCho levels did not appear to be related to HER2/neu overexpression. The reason why our finding was not consistent with the result reported in the aforementioned invitro cell line study10 might be because of the heterogeneity of the breast cancer tissue, especially when considering the fact that current determination of HER-2 status, whether using IHC or FISH, is based on staining score which only requires a certain level of breast cancer cells to be positive for HER-2.

From our present study, it was therefore suggested that in vivo¹H-MRS can not provide useful information for characterizing HER2/neu overexpression in carcinoma of the breast.

Yours sincerely,

Hyeon-Man Baek, Jeon-Hor Chen, Orhan Nalcioglu, Min-Ying Su