A Babsky, Ju Shenghong - Apparent diffusion coefficient of waterin evaluation of treatment response in animal body tumors - страница 1
ЕКСПЕРИМЕНТАЛЬНІ РОБОТИ / EXPERIMENTAL WORKS
APPARENT DIFFUSION COEFFICIENT OF WATER
IN EVALUATION OF TREATMENT RESPONSE IN ANIMAL BODY TUMORS
Andriy M. Babsky 12, Shenghong Ju 13, Navin Bansal 1
department of Radiology, Indiana University 950, West Walnut St., R2 E124, Indianapolis, IN 46202, USA 2Ivan Franko National University of Lviv, 4, Hrushevskyi St., Lviv, 79005, Ukraine
3Zhongda Hospital, Southeast University 87, Dingjiaqiao Rd, Nanjing, 210009, China e-mail: firstname.lastname@example.org
The review summarizes the author's results and literature data on the evaluation of diffusion-weighted magnetic resonance imaging (DWI) as a cancer biomarkers that reflects structural, cellular, apoptotic, and necrotic changes in tumor tissue. Diffusion measurements reflect the effective displacement of water molecules allowed to migrate for a given time. It was demonstrated that apparent diffusion coefficient (ADC) of water estimated from 1H DWI is important tool for the detection and characterization of neoplastic transformation as well as monitoring response to therapy. The possible mechanisms of pre- and post-therapy changes in water ADC in animal body tumors are discussed.
Key words: tumor, chemotherapy, ADC, sodium, MRI.
Abbreviations: 5FU, 5-fluorouracil; ADC, apparent diffusion coefficient; BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea; Cp, cyclophosphamide; DWI, diffusion weighted imaging; ECS, extracellular space; ICS, intracellular space; MRI, magnetic resonance imaging; [Na+]e, extracellular Na+; [Na+],, intracellular Na+; [Na+]t, total tissue Na+; PET, positron-emission tomography; RIF-1, radiation induced fibrosar-coma-1; sc, subcutaneous; SI, signal intensity; SQ, single-quantum 23Na; TQF, triple quantum-filtered 23Na; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.
The prediction and detection of the therapeutic response, as well as characterization of residual disease, are very important for effective cancer therapy. Current assessment of tumor treatment response relies on evaluating changes in the maximal cross-sectional area or the diameter of the tumor [1, 2], weeks to months after the conclusion
of a therapeutic protocol [3, 4]. Several noninvasive imaging methods, such as computed tomography, positron-emission tomography, single-photon emission computerized tomography, magnetic resonance spectroscopy and diffusion-weighted magnetic resonance imaging (DW MRI or DWI) are being evaluated for assessing early therapeutic response that are independent of late changes in tumor volume [5-10].
The diffusion of tissue water in vivo can be accurately and noninvasively estimated as an apparent diffusion coefficient (ADC) by using 1H DWI. It is a well known diagnostic tool to evaluate central nervous system pathologies. The first reported evaluation of mean tumor ADC following chemotherapy of an animal tumor model was performed by Ross et al. in 1994 , who studied effect of BCNU treatment on orthotopic rat 9L glioma. For many years the use of DWI was limited to the brain because the image quality of DWI of non-central nervous system tissue was inferior. Physiologic motion and the challenging magnetic environment outside the brain made it difficult to achieve DWI with sufficient image quality within a reasonable acquisition time. Within the last years advances in MRI software and hardware-sector of MRI improved image quality considerably and led to several reports describing the potential of DWI in the evaluation of extracranial diseases.
DW MRI has a number of advantages over other imaging techniques (e.g., computed (CT) and positron-emission tomographies (PET)). DW MRI is noninvasive, and does not require ionizing radiation exposure or the administration of contrast medium. The short examination time, especially when using parallel imaging, is an additional advantage, as is the ability to assess the tumor completely. In addition, parallel imaging offers the benefit of reduced artifacts, which becomes very important in DW MRI. Prediction of response to treatment or even early detection of non-responders would allow changes of therapy in order to minimize treatment-related toxicity. Furthermore, both conventional morphologic and physiologic assessments can be made during the same examination . DWI provides both quantitative and qualitative information that can be useful for tumor treatment.
In preclinical models, there is abundant evidence that the ADC in tumors increases early in response to successful treatment. This has been shown in sarcoma, glioma, and breast carcinoma xenografts treated with cytotoxic chemotherapies, cytostatic chemotherapies, radiation therapy, and gene therapies [13, 14]. Treatments that caused cells to shrink led to early increases in ADC that were predictive of the ultimate tumor response. The observations have generated increasing interest in the clinical application of DW MRI for assessing tumor response during follow-up with various treatment modalities (e.g., chemotherapy and radiation therapy) [15-19].
Changes detected in mean tumor ADC values after treatment in rodent tumor models revealed that this approach has merit for preclinical drug development studies as a sensitive and early predictor of therapeutic efficacy [8,14,18,20-24]. Furthermore, some preliminary studies have shown correlations between pre-treatment ADC value and tumor response to therapy. However, because of tumor heterogeneity and suboptimal methods of digital image analysis [15, 25-28], clinical utility of this approach is more complicated and therefore the application was delayed comparing with preclinical utility in rodent models. Many of the clinical studies evaluating DWI for assessing treatment response have been performed in relatively small numbers of patients. Nevertheless, the body of evidence suggests that ADC measurement is a potentially useful tool that provides unique prognostic information and should be more widely investigated in large clinical studies in the future .
The common points of view on ADC changes in tumor tissue are summarized in Fig. 1. Neoplastic transformation can lead to an increase in water ADC due to less
■<- ADC -
Fig. 1. Schematic diagram of possible mechanisms of the water apparent diffusion coefficient (ADC) increase (+) or decrease (—) in tumor tissue (Adapted from Moffat et al.  and Koh and Collins )
efficient cell packing in tumors compared to the healthy tissue. Ross et al. hypothesized that the changes in tumor ADC that occur after effective therapy can be related to changes in cell density [11, 30-34] as result of necrotic and/or apoptotic processes. Both of these processes should lead to an increase in extracellular space (ECS) and an increase in water ADC. From other side, the loss of tumor membrane integrity causes water inflow, cell swelling, and a decrease in both ECS and water ADC especially in the beginning of the post-treatment period. Heterogeneous tumor tissues usually contain regions ofintratumoral edema, necrosis, and cyst with high extracellularwatercontent. Post-treatment dynamic reorganization of these regions involving tissue water drainage can also decrease water ADC. Furthermore, intensive fibrosis in tumor tissue can also contribute to restriction of water diffusion. Moffat et al. stated that „the changes in cell density due to cell killing along with tissue reorganization may lead to heterogeneous changes in the underlying tissue morphology (e.g., ratio of intra- to extracellular water), resulting in spatially varying changes in tumor ADC values" . Koh and Collins consider that the explanation of ADC changes has to include not only the ECS changes but also the contribution of intravascular perfusion to the diffusion measurement, especially when the DWIs are acquired using low b-values, which are sensitive to vascular perfusion effects . The diagram in Figure 1 also does not include the contribution of intracellular water ADC to total tissue value. The direct measurements of ADC of small molecules like sodium, gadolinium, mannitol, glucose and others show that intra- and extracellular ADCs are rather similar than different [36, 37]. The use of ADC to estimate chemotherapy efficiency has been assessed in a number of animal studies and summarized in Table and described below.
1. Variability in Animal Models and MRI Techniques in Water ADC Studies
The extensive application of water ADC as a marker of the animal body tumor chemotherapy efficiency began about 13 years ago using mainly rats (Fisher, Wistar, Lud-wig, etc.) and mice (C3H, BALB nude, SCID, GBNIH nude, etc.). For that purpose both clinical (1.5-4.7 T) and small animal (4.7-14 T) MRI systems were used. Different type of MR coils such as birdcage- [38, 39], loop-gap- [40-42], wrist-  and surface-coils  were used accordingly to the tumors location and volume. Majority of the experiments were performed using subcutaneous (sc) tumors. Sc tumor models are very convenient for NMR studies because they avoid the effects of motion present in the abdominal area and provide high quality MR images due to close location of the MR coil to the tumor tissue. The values of water ADC before treatment for many sarcomas [40, 42, 45, 46] and human breast cancer xenografts [47, 48] were reported in the range 0.4 - 0.7x10-3 mm2/s. However, Thoeny et al. [43, 49] show the higher water ADC value for rhabdomyosarcoma (1.26x10-3 mm2/s). Water ADC in sc gliomas (gliosarcomas) [38, 39, 41] and mammary tumor xenografts  were found before treatment in the range 0.9 - 1.1x10-3 mm2/s. Pre-treated sc tumor volumes used for the water ADC estimation varied from 0.1-0.6 cm3 (mostly for carcinomas) to 1-2 cm3 (mostly for gliomas). Absolute ADC values, however, depend on the b-values applied for DW MRI. Unfortunately, the number of b-values in the different post-treatment studies of ADC varied very much - from two (0 and 300 s/mm2) [38, 39, 50] to fourteen (15-2741 s/mm2) . Only in few cases b-values higher than 1,000 mm2/s have been used [38-42, 51, 52]. Some investigators use b-value = 0 s/mm2 [40-43, 46, 48], some of them do not [39, 44, 45, 47, 53]. These variations in b-values make the comparison of the ADC data complicated. As a quantitative parameter calculated from DWI, ADC reflects not only diffusion but also perfusion in microvessels . Previous studies show that for low b-values (< 100 s/mm2) perfusion dominate diffusion by a factor of 10 [54, 55]. However, by using high b-values (> 500 s/mm2), the influence of perfusion is largely attenuated. Thoeny et al.  divided b-values on low (0, 50, and 100 s/mm2) and high (500, 750, and 1,000 s/mm2). They propose to use the difference between ADClow and ADChi has a perfusion component of the tissue ADC. However, Zhao et al.  assume that the vascular contribution to tumor tissue water diffusion is minimal despite the greater water ADC in blood. This conclusion is based on the Braunschveiger study  showing that blood occupied less than 5% of sc RIF-1 volume. In addition, the effect of perfusion is diminished when wide ranges of b-values are used in a study. The challenges and recommendations concerning the application of DWI as a cancer biomarker are widely discussed in the recent review by Padhani et al.  published in Neoplasia.
In our paper, studies of water ADC in different animal body tumor models, such as a tumor of connective tissue, muscle, colon, breast, liver, prostate, and other tissues will be discussed.
2. Fibrosarcomas and Myosarcomas
The first report on the application of water ADC measurement for monitoring response to the body cancer therapy was published by Zhao et al. in 1996 . This study was performed using a diffusion-weighted spectroscopy pulse sequence. The effects of cyclophosphamide (Cp), an alkyliting agent, on sc-implanted radiation induced fibrosar-coma-1 (RIF-1) in C3H mice were investigated using a 4.7 T MR system. Cp itself is a pro-drug, which is oxidized in the liver to 4-hydroxycyclophosphamide and subsequently
converted to nitrogen mustard and other metabolites. In tumors, Cp metabolites do not directly disrupt cell metabolism, but rather these metabolites alkylate DNAand proteins. WaterADC of RIF-1 tumors increased at day 2, 3 and 4 after Cp treatment in the absence or before a decrease in tumor volume . The magnitude and duration of the changes in ADC were dose dependent. A 300 mg/kg Cp dose caused a larger and more sustained increase in the ADC compared to a 150 mg/kg dose. Because water ADC was increased substantially at a time when there was no change in tumor volume for a dose which produces minimal cell kill, authors suggest that ADC measurement could provide a novel means for early detection of response to anti-cancer therapy.
More recently the increase in water ADC in sc-implanted RIF-1 tumors after chemo-therapywith Cp and 5-fluorouracil (5-FU) has been confirmed using DW H MRI [40, 42]. A single injection of Cp (300 mg/kg) or 5FU (25 mg/kg) significantly increased the water ADC of RIF-1 tumors two and three days post-treatment while tumor volume was significantly decreased. Unlike Cp, the action of 5FU involves its incorporation into RNA and metabolic activation to 5-fluoro-2'-deoxyuridine-5'-monophosphate, which inhibits thymidilate synthetase, a key enzyme in DNAsynthesis and repair. In control (untreated) groups tumor ADC remained unchanged throughout the experiment. 5FU is most commonly used for treatment of breast and gastrointestinal cancer 5FU shows anti-hepatocellular carcinoma activity, both clinically [58, 59] and in animals . Examples of water ADC images for a control and Cp-treated tumor before and one, two, and three days after Cp injection are shown in Fig. 2. After Cp injection, water ADC increased progressively during the first three days post treatment. The water ADC increase was observed not only in the tumor regions with low cell density (bright region in the lower right quadrant of the tumor), but throughout the whole tumor. In the Cp group, the average water ADC increased from 0.49 + 0.02x10-3 mm2/s (before treatment) to 0.58 + 0.03x10-3 mm2/s (day 2, p < 0.05) and
Fig. 2. Water apparent diffusion coefficient (ADC) maps and 23Na MR images of representative control and cyclophosphamide (Cp) treated RIF-1 tumors. WaterADC and 23Na signal intensity increased with time after Cp treatment. A vial filled with a NaCl solution was placed near the tumor as a reference
0 1 2
Days after Cp treatment
0.73 + 0.04x10-3 mm2/s (day 3, p < 0.01). In 5FU group, the average water ADC increased from 0.52 + 0.02x10-3 mm2/s (before treatment) to 0.61 + 0.02x10-3 mm2/s (day 2, p < 0.05) and 0.65 + 0.03x10-3 mm2/s (day 3, p < 0.05). The average water ADC of treated tumors was also significantly higher (p < 0.01) compared to the unchanged ADC of untreated control tumors two and three days post-treatment.
Braunschweiger  has shown that Cp treatment reduces cell proliferation and increases extracellular, interstitial and plasma water volumes during the initial five days after the treatment. The increase in extracellular water, cell death, and/or reduction in cell volume may increase the overall mobility of water in the damaged tissue and lead to an increase in waterADC. Destructive chemical analysis and histological results of RIF-1 tumors support this hypothesis. Measurment of relative ECS by destructive chemical analysis show 46 + 8% ECS in treated tumors compared to 26 + 4% in untreated tumors. Similarly, histological data shows a decrease in the number of cells and an increase in extracellular space [40, 42]. It has been shown previously that the increase in waterADC correlates with both the increase in tumor necroticfraction in RIF-
3. Colon Tumor
The earliest significant response of water ADC to chemotherapy was reported for the treatment of the sc-implanted human colon H29 carcinoma xenografts in GBNIH nude mice with inhibition of HIF-1a by PX-478 . PX-478 is a novel agent that suppresses both constitutive and hypoxia-induced levels of HIF-1a in cancer cells . PX-478-in-duced inhibition of tumor growth is associated with HIF-1a levels in different human tumor xenografts. At 24 and 36 hrs after the treatment with this drug waterADC increased by 94.5% and 38.4% (p < 0.01), respectively, before returning to the pretreatment ADC level. However, PX-278 had no effect on the ADC of a drug-resistant tumor system.
Roth et al. treated sc-implanted C26 colon carcinomas with doxorubicin and with aminolevulenic acid-based photodynamic therapy (PDT) . In malignant cells, doxoru-bicin-induced intercalation inhibits nucleotide replication and action of DNA and RNA polymerases. The interaction of doxorubicin with topoisomerase II to form DNA-clea-vable complexes appears to be an important mechanism of doxorubicin cytocidal activity. The photodynamic therapy is a regional therapy that induces early destruction oftissue, whereas the effect of doxorubicin chemotherapy is systemic and seen much later. To quantify the diffusion characteristics of the tumor tissue, the authors defined a diffusion index, RD. They stated that ARD refect changes in tumor viability, intracellular water fraction, and integrity of cell membranes that may affect permeability. In doxorubicin-treated carcinomas, a significant correlation was found between RD measured prior to treatment, and changes in tumor volume after therapy. An average negative change in tumor RD was observed after chemotherapy (ARD = -3.1 + 0.5) and PDT (ARD = -4.4 + 1.0) 24 or 48 hr post-treatment. In general, a negative change in RD corresponds to an increase in water ADC. No substantial changes in diffusion were observed in control tumors. Tumors with high pretreatment viability responded better to chemotherapy with doxorubicin than more necrotic tumors. In tumors treated with photodynamic therapy, no such correlation was detected. Changes observed in water diffusion 24-48 hrs after treatment correlated with later carcinoma growth rate for both therapies.
The most durable increase in waterADC was reported by Seierstad et al.  when sc-implanted human HT29 colon adenocarcinoma xenografts were treated weekly with the 5FU prodrug capecitabine, daily oxaliplatin, and fractional irradiation. Oxaliplatin is a platinum-based chemotherapy drug in the same family as cisplatin and carboplatin. It is typically administered in combination with fluorouracil and leucovorin in a combination known as FOLFOXforthe treatment of colorectal cancer. Compared to cisplatin the two amine groups are replaced by cyclohexyldiamine for improved antitumor activity. The chlorine ligands are replaced by the oxalato bidentate derived from oxalic acid in order to improve water solubility. Seierstad et al. found an increase in waterADC by -19% with combinations of the capecitabine and oxaliplatin on 11 days after the drug applications . The combination of these drugs with fractionated irradiation showed a significant increase in tumor doubling growth delay compared to the tumors that received radiation only. Histological examination showed that both treated and control tumors had necrotic centers, ranging from 12 to 84%, and that there was a much higher ADC value compared to the surrounding viable regions. However, the statistical analysis revealed no differences in necrotic fraction between the six treatment groups including control one. The same authors have studied the effect of 15 Gy irradiation alone on the same tumor model, and have found that 24 hr post-radiation the waterADC was increase by 5.0% (p < 0.002) . This increase was followed by a significant decrease (-6.9%,
p < 0.001) three days post-radiation and a renewed increase of the waterADC on day 7 (+ 6.2%, p< 0.03) and 11 (+ 11.1%, p < 0.001). The decrease in waterADC three days post-radiation was accompanied with increased fibrosis in the treated tumors.
4. Breast Tumor
Galons et al. monitored the chemotherapy response of human breast MCF-7 cancer tumor xenografts to paclitaxel (27 mg/kg after the first MRI experiment and 18 mg/kg every other day) was monitored . Paclitaxel binds to the p subunit of tubulin interfering with normal microtubule breakdown during cell division. This drug is related to taxa-nes, which also induce apoptosis in cancer cells by binding to an apoptosis stopping protein called Bcl-2 and thus arresting its function. Water ADC increased from 0.5 -0.7x10-3to 0.7 - 1.5x10-3mm2/s 48 hr after successful therapy in parental drug-sensitive MCF-7/S tumors, but there was no change in the waterADC in p-glycoprotein-positive tumors MCF-7/D40, which are resistant to paclitaxel. The authors concluded that the mechanism underlying these changes is consistent with apoptotic cell shrinkage and a concomitant increase in the extracellularwaterfraction.