Molecular Imaging of Cancer: Prediction and Early Detection of Response by NMR Spectroscopy and Imaging

Systemic therapy (chemotherapy, immunotherapy and radio-immunotherapy) of cancer is currently administered according to statistically based protocols developed through standardized random clinical trials with the choice of treatment being determined by consultation between the oncologist and the patient. The physicians base their recommendations on clinical trials and their previous experience. This strategy has led to the development of certain standard first-line and second-line approaches to treatment of various diseases, such as antiestrogen, paclitaxel or doxorubicin-based treatment of breast cancer, anti-androgen treatment of prostate cancer, platinum-based therapy of ovarian cancer, temozololide treatment of brain tumors, fluorouracil treatment of colon cancer and rituximab (R) plus cyclophosphamide, doxorubicin, vincristine, prednisone (CHOP) (also called RCHOP) treatment of lymphomas. Patients who fail firstline therapy are then directed to second-line treatments. While this traditional approach has led to beneficial results for many patients, it has also led to exposure of many patients to agents that have little or no beneficial effect for management of their cancer. Treatment with these ineffective agents invariably leads to toxicity, adds unnecessary cost, and most unfortunately, leads to unnecessary delays in treatment with more effective agents. Hence, there is a need to tailor-fit the therapy to the patient’s cancer, which can only be accomplished with reliable methods for prediction of therapeutic response or response failure before treatment is initiated or by the use of methods that accurately detect response at an early stage of treatment.

Here we describe the development of phosphorus-31 (31P) Magnetic Resonance Spectroscopy (MRS)-based methods for non Here we describe the development of phosphorus-31 (31P) Magnetic Resonance Spectroscopy (MRS)-based methods for noninvasively predicting response failure in non-Hodgkin’s lymphoma (NHL) patients prior to initiation of treatment. Early results from a multi-institutional NCI funded collaborative program involving Columbia University, Memorial Sloan-Kettering Cancer Institute, The Royal Marsden Hospital in London, the CR UK Cambridge Research Institute, Cambridge, UK, Radboud University Nijmegen Medical Center, The Netherlands, and the University of Pennsylvania indicate that these methods appear to be capable of identifying about 2/3 of the patients who will fail to exhibit a complete clinical response (i.e., complete disappearance of the local lesion). We will also describe 1H MRS and MRI methods that can detect response very soon after the initiation of therapy (e.g., within 48 hr of initiation of RCHOP treatment). The 1H MRS and MRI methods are still under development, in many cases on mouse models of NHL, and are just being translated into the clinic, whereas the 31P MRS methods have been under development for over a decade and have been applied to about 250 human patients. The initial 31P MRS studies were conducted on patients with various forms of this disease who were treated with a variety of therapeutic methods. Currently, the studies are being conducted on patients with specific forms of the disease treated with specific regimens. The most conclusive results have been obtained with patients with diffuse large B-cell lymphoma (DLBCL), the most common form of NHL that afflicts about 1/3 of the NHL patients, who have been treated with RCHOP, the most common therapeutic regimen. Therefore, the conclusions being drawn here must be considered tentative and subject to revision as research and clinical translation progresses.

Development of methods for predicting or detecting therapeutic response is best performed on malignancies that exhibit about a 50% response rate, preferably with a substantial proportion of complete responses. In addition, preliminary animal and clinical data suggested that the relative levels of phosphomonoester (PME) intermediates, phosphocholine (PCh) and phosphoethanolamine (PEt), decreased significantly following effective cancer therapy in a variety of animal and human cancers [1]. Hence, the initial hypothesis was that 31P MRS measurements of the sum of these metabolites normalized to total nucleoside triphosphates (NTP), which remain approximately constant at 5 mM within most tumor cells, would be a suitable response indicator, i.e., that the (PCho + PEt)/NTPß or PME/NTP ratio, where the metabolite abbreviations refer to integrated resonance intensities measured under conditions of 1H decoupling to eliminate heteronuclear spin-spin coupling and induce a maximal nuclear Overhauser enhancement, would be a suitable parameter for prediction and early detection of therapeutic response. Because of the relatively low sensitivity of 31P MRS, this required that the studies be performed on relatively large superficial tumors (≥ 27 cm3). These requirements led to the choice of four malignancies for initial investigation in a multiinstitutional preliminary trial – NHL, squamous cell carcinoma of the head and neck, softtissue sarcomas and locally advanced breast cancer. However, despite the participation of multiple institutions, adequate accrual was only achieved with NHL patients; therefore, the study became limited to this single malignancy. Also, the requirement that the patients remain in the magnet for approximately an hour discouraged most patients from returning for a post-treatment follow-up examination; hence, the acquired data were limited to pretreatment examinations. Despite these limitations and the fact that data were initially acquired from previously untreated or recently relapsed patients with all forms of NHL treated with a variety of different therapeutic agents, the results proved remarkably useful [2-6]. Figure 1 shows that when patients were stratified by tumor grade based on the International Prognositic Index and the PME/NTP ratio was plotted for each tumor grade, a line drawn through the medians of the low and low-to-high grade tumors and extended to the other columns provided a very useful basis for predicting CR. With one exception, all the points above the line originated from tumors that did not exhibit a CR, whereas data from below the line were approximately evenly split into CRs and NCRs. Thus, with 96% accuracy one could predict response failure (NCR) for patients whose tumor PME/NTP ratios fell above this arbitrary line. An even better prediction has recently been achieved by defining an optimum cut-off value of PME/NTP for each tumor grade (Cooperative Group in NMR Spectroscopy of Cancer, submitted for publication). Similar results have also been obtained by the Cooperative Group in preliminary studies of squamous cell carcinoma of the head and neck [7].

Correlation of the International Prognostic Index (IPI) and the pretreatment PME/NTP per patient in the whole NHL cohort. The long-term response to treatment outcome of each patient was also plotted as CR, complete response (filled circles) and noncomplete responders (open circles). Tumors were stratified on the basis of the IPI as low grade (L), low to intermediate (LI), high to intermediate (HI) and high grade (H) tumors. A line was drawn between the medians of the L and LI points and extended through the HI and H columns. This line was a useful threshold for predicting failure to exhibit a CR.

The key limitation of the 31P MRS technique was its low sensitivity, which necessitated about 1 hour examination times, limited observations to large superficial tumors and predicted response failure rather than response success. To overcome these limitations, the Penn component of the Cooperative Group undertook preclinical 1H MRS and MRI as well as 31P MRS studies of human DLBCL xenografts in nude mice utilizing the DLCL2 tumor model introduced by Mohammed et al [8,9]. Treatment of these xenografts with CHOP, rituximab (R), RCHOP, CHOP plus bryostatin (CHOPB) or radiation therapy (RTX) were evaluated using protocols similar to the clinical protocols except that the drug doses were slightly modified, the time per cycle of drug therapy was decreased from three weeks to one week because of the shorter doubling time of the tumor in the mouse, and the RTX was administered as a single 15 Gy bolus instead of multiple 2Gy fractions [10-13]. Tumor volume, measured with calipers, was the response endpoint. Relative response followed the order RTX>CHOPB>CHOP=RCHOP>R, with R producing only a slight growth delay. The 1H MRS studies indicated that decreases in steady state lactic acid (Lac) were statistically significant following one cycle of therapy with CHOP, CHOPB or within 24 hour following RTX, whereas treatment with R alone had no effect on Lac but decreased total choline (tCho), which was also decreased by RCHOP, CHOPB or RTX. CHOPB and RTX also decreased the PME/NTP ratio of the tumor, but none of the other treatments had a significant effect on metabolites detected by 31P MRS. Of potentially greatest clinical significance was the observation that, with respect to treatment of the DLCL2 xenograft model with CHOP or RCHOP, which are the two most common therapies for DLBCL patients, Lac was selectively responsive to CHOP, whereas tCho was selectively responsive to R; hence, a decrease of Lac but an increase in tCho following RCHOP therapy could indicate rituximab resistance, which could be treated in the clinic with lenalidomide, an agent that restores immunological response of NHL tumors. This ability to identify rituximab refractory patients remains to be confirmed in the clinic. This principle is demonstrated in Figure 2, which shows data from a DLBCL patient who was examined on a Monday, started RCHOP therapy on Wednesday and was re-examined on Friday. His tumor exhibited a 70% decrease in Lac and a 15% increase in tCho suggesting that this patient may have been rituximab refractive, but no confirmatory data of this were obtained. The patient went on to exhibit a CR and is still in remission several months after treatment.

63 year-old male with inguinal node diffuse large B-cell lymphoma. (A) T2-weighted image, TE=15 ms, TR=3000ms (B) Lactate image measured with Had-SelMQC-CSI sequence.

The utility of two forms of MRI (diffusionweighted (DWI) and T2-weighted (T2WI)) was evaluated on DLCL2 xenografts treated with CHOPB [14]. These imaging methods produce tumor images showing sub-millimeter in-plane resolution at acquisition times on the order of ten minutes. Bryostatin produces a more robust response of this tumor model to CHOP chemotherapy by inhibiting multidrug resistance or Bcl-2 expression [8]. A significant increase in the apparent diffusion constant (ADC) was detected by DWI following only one cycle of CHOPB, whereas T2WI required two therapy cycles to detect a statistically significant but anomalous decrease in the average T2 [14]. However, the most interesting observation was that in three of the five tumors examined, the changes in ADC or T2 were not uniform over the entire tumor but were limited to distinct regions of the tumor. This regional response pattern could be due to various causes including heterogeneity in tumor perfusion, oxygenation or apoptotic ability or drug resistance (which are energy and hence perfusion dependent). The exact mechanism producing this heterogeneity is under examination, but it is apparent that if, for example, it proves to be related to perfusion, then there are various interventions that could be used to produce a more homogeneous and, therefore, extensive response of the tumor. Thus, image-guided therapy is a distinct possibility, if not a probability for the future.

About 30% of all the new cancer drugs under development by pharmaceutical companies target signal-transduction pathways. These drugs usually act by phosphorylating or acetylating key kinases that then modify critical cell properties such as proliferation, apoptosis, angiogenesis, bioenergetics, gene expression, protein expression, etc. However, there are no non-invasive imaging methods for monitoring the actual labeling events on the level of the affected proteins. There is, however, a great need to monitor these processes within the individual patient. To develop such a method, we proceeded on the assumption that in order to modify vital cellular functions, signal-transduction had to modify cellular metabolism; cellular metabolism can be monitored by NMR and PET methods. The goal was, therefore, to identify specific metabolic pathways that were modified when specific signaling pathways were inhibited. As proof of principle, we chose the mTOR pathway, which is selectively inhibited by rapamycin and a number of other agents. Addition of seven doses of rapamycin (10 mg/kg x 2 doses/day) to the DLCL2 tumor model model produced a 90% decrease in tumor Lac detected by 1H MRS (Seung-Cheol Lee, Michal Marzec, Xiaobin Liu, Suzanne Wehrli, Kanchan Kantekure, Puthiyaveettil N Ragunath, E. James Delikatny, Jerry D. Glickson, Mariusz A. Wasik, submitted for publication). There was no significant change in tCho. Gene chip and Western blot analysis indicated an approximate 35% decrease in the expression of hexokinase-2, the key enzyme involved in regulating tumor glycolysis. A number of other glycolytic enzymes exhibited smaller decreases in expression following rapamycin treatment including phosphofructokinase (15-20%), enolase 1 (18%) and pyruvate kinase (5%). There was a small decrease in choline kinase and a small increase in phospholipase A2. Therefore, the glycolytic pathway appears to be selectively inhibited. This was confirmed in the Ramos line (derived from human Burkitt’s lymphoma) of NHL as well as in a variety of different human NHL cell lines and with a variety of different mTOR inhibitors. Thus, it appears that inhibition of mTOR can be monitored selectively by 1H MRS using lactate imaging methods [15-17] and probably also by FDG PET.

In summary, we have shown that pretreatment ³¹P MRS can predict about 2/3 of the response failures among human NHL patients. These patients could be directed to more vigorous therapeutic regimens followed by bone marrow transplantation or to experimental new therapeutic agents. Phosphorus-31 NMR is limited to large superficial tumors and only provides a predictor of response failure rather than successful response. Proton NMR data can be used to monitor much smaller tumors in any site in the human body. Proton spectroscopy has two response markers, lactate and choline, that can selectively detect response to CHOP chemotherapy and rituximab immunotherapy, re-spectively. This may provide a non-invasive method for detecting patients refractory to rituximab therapy who can be treated with thalidomiderelated agents that restore rituximab response. Finally, we have demonstrated a general strategy for non-invasively monitoring response to inhibitors of specific signal transduction pathways by monitoring the corresponding metabolic pathway that is modified by signaltransduction inhibition. We have demonstrated that in the case of mTOR, inhibition of this signaling pathway can be detected by inhibition of glycolysis, which can be detected by 1H MRS lactate imaging or FDG PET.

Acknowledgements

This work has been supported by NIH grants CA101700, CA41078 and CA118559. Animal studies were performed at the Small Animal Imaging Facility of the University of Pennsylvania that is operated with partial support from an NCI Small Animal Resource grant. Much of this research has been conducted by members of the Cooperative Group on 5U24CA08315-07 and as a core facility of the Abramson Comprehensive Cancer Center that is supported by 5P30CA016520-34. Clinical studies have been performed by the NMR Spectroscopy in Cancer Cooperative Group that includes the following participants: Columbia University (Truman R. Brown and Fernando Arias-Mendoza), Memorial Sloan-Kettering Cancer Center (Jason A. Koutcher, Kirsten Zakian, Amita Shukla-Dave), The Royal Marsden Hospital, London, UK (Martin O. Leach, Geoffrey S. Payne, Adam J. Schwarz, David Cunningham), CR UK Cambridge Research Institute, Cambridge, United Kingdom (John R. Griffiths, Marion Stubbs), Radboud University Nijmegen Medical Center, The Netherlands ( Arend Heerschap), The University of Pennsylvania (Harish Poptani, Seung-Cheol Lee and Jerry D. Glickson).

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