PURPOSE:

Transverse rectus abdominis myocutaneous (TRAM) flaps are commonly used for breast reconstruction following mastectomy for breast cancer. Recurrence of breast cancer in reconstructed breast with autologous tissue is reported to be approximately 7%. Imaging for occult or recurrent breast cancer in these patients is unreliable at best.  In this study, our primary aim is to test whether NM404, a phospholipid ether analogue, would identify occult breast cancer in a breast reconstructed by a TRAM flap and complicated by fat necrosis.  Secondarily we sought to compare NM404 to MRI, the gold standard in breast imaging, and elucidate a mechanism of action for NM404.

METHOD:

Twenty female Sprague-Dawley rats underwent elevation of unilateral TRAM flaps based on inferior epigastric vessels.  The flap was folded over the mammary gland located in the flank.  In order to simulate fat necrosis, a 300mg sample of fat was removed from the contralateral axillary fat pad and placed within the TRAM.  4T1 breast cancer cells were xenografted into the mammary gland with the overlying TRAM flap.  I¹²⁴-NM404 was then administered through a tail vein injection and at 2,4, 8 and 12 days later, rats were imaged utilizing dual modality microCT/microPET.  Images were acquired, reconstructed, visualized, and analyzed utilizing commercially available software.  Animals were sacrificed, tumors explanted, and sent for pathology and histology.  Laser capture microdissection along with QPCR, western blot and amplex red assays were used to selectively investigate the molecular mechanism of NM404.

RESULTS:

In all 20 animals, NM404 was able to selectively visualize established breast cancer as early as four days and as late as 12 days post-injection.  NM404 did not enhance fat necrosis occurring within the TRAM flap.  The signal activity correlated with tumor presence.  Activity images were converted to gradient profiles and a series of gating protocols to delineate tumor margins.  Estimated tumor volumes by dual-modality imaging and NM404 correlated well with actual histological volumes (R² =0.91) versus MRI (R²=0.70).  Molecular assays confirmed dysregulation of PLD1 and 2 as a mechanism of action. Gene expression, protein expression and enzymatic activity all demonstrated statistically significant alterations at the molecular level.

CONCLUSION: This study is the first to demonstrate a tumor selective biomarker for breast cancer.  This biomarker may assist with early detection of breast cancer in patients who have undergone autologous breast reconstruction.  Furthermore, a tumor selective biomarker may allow for therapy to be targeted or longitudinal evaluation of on-going therapy.  In the future, NM404 may also be a beneficial adjunct to detecting cancer in stem cell regenerated tissues.