Combined Diffusion and Perfusion MR Imaging as Biomarkers of Prognosis in Immunocompetent Patients with Primary Central Nervous System Lymphoma

Here, the authors sought to determine if ADC values and perfusion measurements provided prognostic information in 25 treatment-naïve immunocompetent patients with brain lymphoma. The studied parameters were obtained from the enhancing tumor regions. The results of this investigation reinforce the validity of ADC values as a prognostic biomarker and provide evidence of low tumor CBV as a novel risk factor for adverse prognosis in immunocompetent patients with primary brain lymphoma. BACKGROUND AND PURPOSE: ADC derived from DWI has been shown to correlate with PFS and OS in immunocompetent patients with PCNSL. The purpose of our study was to confirm the validity of ADC measurements as a prognostic biomarker and to determine whether rCBV measurements derived from DSC perfusion MR imaging provide prognostic information. MATERIALS AND METHODS: Pretherapy baseline DWI and DSC perfusion MR imaging in 25 patients with PCNSL was analyzed before methotrexate-based induction chemotherapy. Contrast-enhancing tumor was segmented and coregistered with ADC and rCBV maps, and mean and minimum values were measured. Patients were separated into high or low ADC groups on the basis of previously published threshold values of ADCmin < 384 × 10−6 mm2/s. High and low rCBV groups were defined on the basis of receiver operating curve analysis. High and low ADC and rCBV groups were analyzed independently and in combination. Multivariate Cox survival analysis was performed. RESULTS: Patients with ADCmin values < 384 × 10−6 mm2/s or rCBVmean values < 1.43 had worse PFS and OS. The patient cohort with combined low ADCmin–low rCBVmean had the worst prognosis. No other variables besides ADC and rCBV significantly affected survival. CONCLUSIONS: Our study reinforces the validity of ADC values as a prognostic biomarker and provides the first evidence of low tumor rCBV as a novel risk factor for adverse prognosis in immunocompetent patients with PCNSL.

P CNSL, an extra-nodal variant of non-Hodgkin lymphoma confined to the CNS, 1 is an aggressive primary brain cancer with rising incidence and variable response to therapy and clinical outcome. [2][3][4] Unlike systemic lymphomas, relatively little is known about the pathobiology of PCNSL. 5 Immune deficiency is the only established risk factor for developing PCNSL with uniformly devastating clinical outcome. 6 In immunocompetent individuals, however, the disease etiology remains unknown and prognosis can be highly variable. 7 Several markers of adverse prognosis have been suggested such as age older than 60 years, low performance scores, high lactate dehydrogenase level, high CSF protein level, enhancing lesions involving the brain stem, 8,9 and no initial response to steroids. 10 However, noninvasive biomarkers of prognosis and tumor biology that can be measured serially and quantitatively are needed to identify high-risk subgroups at initial diagnosis for formulation of a personalized therapeutic strategy, to assess response to therapy, and to detect tumor recurrence without delay.
Physiology-based MR imaging modalities, such as DWI and DSC perfusion MR imaging, are used clinically to characterize tumor biology beyond structural abnormalities. 11 A recently published report suggests a significant correlation between DWI-derived ADC values and clinical outcomes of immunocompetent patients with PCNSL treated with high-dose methotrexate. Specifically, pretherapeutic minimum ADC values Ͻ384 ϫ 10 Ϫ6 mm 2 /s within enhancing tumor were predictive of shorter progression-free and overall survival. 12 Although there are no published data on DSC perfusion MR imaging and prognosis in patients with PCNSL, there are several studies that have demonstrated the value of rCBV as a predictor of grade and prognosis in gliomas. [13][14][15][16] The purpose of our study was to confirm the validity of ADC measurement as a prognostic biomarker and to determine whether rCBV measurements derived from DSC perfusion MR imaging provide prognostic information.

Patient Population
Twenty-five patients treated at our institution between June 2001 and July 2009 were selected for this retrospective study on the basis of the following criteria: histopathologic diagnosis of B-cell PCNSL as defined by the World Health Organization; negative immunodeficiency virus status; and absence of disease outside the CNS based on CT scans of the chest, abdomen, and pelvis. All patients had a pathologic diagnosis of large B-cell CNS lymphoma and received identical methotrexate-based induction chemotherapy treatment. Of 121 patients who met the inclusion criteria for this investigation, 25 had both diffusion and perfusion MR imaging available for analysis before their diagnostic biopsy. Of the 25, 15 were women and 10 were men. The average performance score at pretherapy baseline was 69.2 with a range of 50 -100.
The induction chemotherapy (cycle ϭ 28 days, 4 cycles) comprised a 3-drug regimen: methotrexate, 8 g/m 2 , days 1 and 15; temozolomide, 150 -200 mg/m 2 /day, days 7-11; and rituximab, 375 mg/m 2 , every week. In patients who achieved a complete response, defined as complete resolution of contrast-enhancing lesions on follow-up MR imaging, to induction therapy, 2-3 additional cycles of methotrexate (8 g/m 2 ) were administered every 14 -21 days as consolidation therapy. Patients who achieved a partial response, defined as an interval decrease in the contrastenhancing lesion, or who exhibited disease progression, defined by an increase in contrast-enhancing lesion volume or development of new enhancing lesions on follow-up MR imaging, were offered high-dose chemotherapy including methotrexate or whole-brain irradiation as salvage therapy. All patients with PCNSL included in this study underwent restaging with follow-up MR imaging within 5 cycles of methotrexate chemotherapy. 2 The clinical performance score, the Karnofsky Performance Score, was assessed at pretherapy baseline evaluation.
The standard DSC perfusion MR imaging protocol (TR/TE, 1250/54; flip angle 35°) at our institution was used to acquire a series of gradient-echo echo-planar images immediately before, during, and after a bolus injection of gadolinium diethylene triamine pentaacetic acid (Magnevist; Bayer Healthcare, Wayne, New Jersey). Eight axial 4-mm-thick sections were used to cover the entire tumor volume, as determined with T2-weighted FLAIR images. The first 10 echo-planar acquisitions were performed before gadolinium diethylene triamine pentaacetic acid (0.1 mmol/Kg body weight) was injected intravenously by using an MR imaging-compatible power injector (Spectris Solaris; MedRad, Indianola, Pennsylvania) at a rate of 4 -5 mL/s through a 18-or 20-ga angiocatherer and followed immediately by a 20-mL continuous saline flush. A multisection image set was obtained every 1.25 seconds before, during, and after the first pass of contrast agent until images were obtained at 60 time points.

MR Image Processing
The contrast-enhanced 3D spoiled gradient-recalled images and raw diffusion data were transferred to a commercially available workstation, aligned to the same axial location, and processed by using FuncTool software and the Advantage workstation (GE Healthcare). ADC maps were calculated on a voxel-by-voxel basis from the diffusion imaging sets. ADC measurements of the contrast-enhancing tumor volume were measured. The average ADC min values in units of 10 Ϫ6 mm 2 /s for all ROIs were calculated. The investigator responsible for production of the region of interest was blinded to pathologic and clinical outcome (F.E.V.). All ROIs were approved by an attending neuroradiologist certified by the American Board of Radiology with a Certificate of Added Qualification in neuroradiology (S.C.).
The raw data of T2*-weighted DSC perfusion MR images were transferred to the same workstation as mentioned above. rCBV maps were derived by using the methods published previously. 17 Contrast-enhanced 3D spoiled gradient-recalled images with ROIs of enhancing tumor were aligned with rCBV maps by using the aforementioned image-processing software. In addition, one 50-mm 2 region of interest was manually drawn around the enhancing region with the higher CBV for each transaxial section. A 50-mm 2 region of interest within the contralateral normal-appearing white matter for each transaxial section was used to standardize the CBV measurements (rCBV mean and rCBV min ).

Statistical Analysis
The ADC min group was stratified into low ADC min (Ͻ 384) and high ADC min (Ն384) subgroups. The rCBV mean group was stratified into low rCBV mean (Ͻ1.43) and high rCBV mean (Ն1.43) subgroups by using a receiver operating characteristic analysis. Similar analysis for the rCBV min yielded a low rCBV min subgroup (Ͻ0.56) and a high rCBV min subgroup (Ն0.56).
Using the Wilcoxon rank sum test, we performed multiplecomparison analysis among the 4 groups (high ADC/high rCBV, high ADC/low rCBV, low ADC/high rCBV, and low ADC/low rCBV). Univariate analyses were performed comparing patient age at diagnosis, CEL volume, edema volume, ADC values, and rCBV values.
Two clinical end points were measured in months: OS and PFS, defined as the time from initiation of therapy to death and to the first recurrence, respectively. A single covariate survival analysis was performed by using a logrank test. A Cox proportional hazards model was used for multivariable survival analysis. A P value Ͻ.05 was considered to indicate a significant difference between groups.

Anatomic Tumor Burden at Pretherapy Baseline Does Not Predict Clinical Outcome
The CEL volume varied from 5.0 to 39.45 cm 3

Lower Pretherapy Baseline ADC min Measurements Predict Shorter Survival
Within the patient cohort, we identified a wide range (133-1010 ϫ 10 Ϫ6 mm 2 /s) of intertumoral variability in pretherapy ADC min values. Seven of the 25 patients included in this study had an ADC min Ͻ 384 within enhancing regions and were assigned to the low ADC min subgroup. The remaining 18 patients (ADC min Ն 384) were designated as the high ADC min subgroup. As shown in Fig 1, patients in the low ADC min subgroup had a greatly increased risk of both progression (P Ͻ .01 logrank test, hazard ratio ϭ 0.24; Fig 1D) and death (P ϭ 0.02, hazard ratio ϭ 0.29; Fig  1A) compared with the high ADC min group. Mean PFS and mean OS were significantly shorter in the low ADC min group versus the high ADC min group (P ϭ .04 and P ϭ .05, respectively).

Lower Pretherapy Baseline rCBV mean and rCBV min Measurements Predict Adverse Outcome
Ten of the 25 patients in the study with rCBV mean values Ͻ1.43 were assigned to the low rCBV mean subgroup, and the remaining 15 patients with rCBV mean Ն 1.43 were designated as the high rCBV mean subgroup. Patients in the low rCBV mean subgroup had increased risk for both rapid progression (P ϭ .03 logrank test, hazard ratio ϭ 0.28; Fig 1E) and death (P ϭ .03 logrank test, hazard ratio ϭ 0.30; Fig 1B). Eighteen of the 25 patients had rCBV min values Ͻ 0.56 and were designated the low rCBV min subgroup, and the remaining 7 patients (rCBV min Ն 0.56) were assigned to the high rCBV min subgroup. When patient outcome was stratified by rCBV min , the low subgroup had increased risk for rapid progression (P Ͻ .01, logrank test; Fig 1F) and death (P ϭ .01, logrank test; Fig 1C).

Combined ADC-rCBV Groups Are Predictive of Outcomes in Which the Low ADC-low rCBV Group Has the Worst Prognosis
To evaluate the potential additive effects of ADC and rCBV stratification methods, we grouped patient cohorts by using a combined stratification method. Figure 2 illustrates survival difference among different combinations of the ADC-rCBV cohort, in which patients in the low ADC-low rCBV group had the greatest risk for both rapid disease progression (P Ͻ .01, logrank test) and death (P Ͻ .01, logrank test).
The PFS outcomes (Fig 2C, -D) were observed to cluster into nonbimodal cohorts, while the overall survival outcomes (Fig 2A, -B) were observed to cluster into bimodal cohorts for both categorization strategies.
When we compared the combined variables with each individual variable, the overall survival is better stratified into high and low survival groups by using both ADC and rCBV than by using only 1 individual variable as shown in Fig 3. The high ADChigh rCBV group and the high ADC group have statistically different survival rates (P ϭ .01); similarly the low ADC-low rCBV group and the low rCBV group have different survival rates (P ϭ .03).

DISCUSSION
Here, we confirm the validity of ADC measurement as a prognostic biomarker and present the first evidence of rCBV values as an additional imaging biomarker of clinical outcome in patients with PCNSL.
In this study, we reproduced the previously reported finding of tumor ADC values derived from DWI as a powerful predictor of clinical outcomes in immunocompetent patients with PCNSL who are treated uniformly with methotrexatebased chemotherapy. We found that the lower the pretherapy baseline tumor ADC values, the shorter the PFS and OS in this group of patients with lymphoma. Both mean and minimum ADC values of tumor at pretherapy show a strong statistical correlation with clinical end points of PFS and OS. When the patients were separated into high-risk (low ADC) versus low-risk (high ADC) subgroups based on the previously published cutoff value of ADC min Ͻ 384 ϫ 10 Ϫ6 mm 2 /s, the difference in clinical outcomes between the 2 groups was highly significant, in that the low-risk group had a greater than 2-fold increase in OS and an almost 3-fold increase in PFS compared with the high-risk group.
The exact biologic or molecular correlate of ADC measurement in PCNSL remains unclear. Previous studies have shown significant inverse correlation between tumor cell attenuation and ADC values in PCNSL, suggesting ADC as a surrogate marker of tumor proliferation. Summarizing from numerous published reports on DWI of gliomas in which ADC values have been closely linked to tumor cell attenuation and nuclear-to-cytoplasmic ratio, [18][19][20][21] one can postulate that ADC values in PCNSL are imaging surrogates of tumor cell attenuation and that lower ADC values reflect higher cellular proliferation in PCNSL, hence correlating with adverse clinical outcome.
We found those patients with low tumor   First, rCBV values measure bulk vessel attenuation and thus reflect tumor angiogenesis. Second, rCBV values depend on patent vessels for delivery of intravenous gadolinium contrast agent. Therefore, low tumor rCBV values in PCNSL may signify a relative lack of tumor angiogenesis or a hypoxic microenvironment and decrease in patent vessels that are able to deliver intravenous methotrexate to the tumor bed. Our results are in agreement with the literature, in which PCNSL has been found to have hypoxic environments due to low perfusion 22 and these hypoxic environments lead to hypoxic tumors that are resistant to treatment. [23][24][25] From further stratification based on a combination of ADC-rCBV values, we found the strongest imaging prediction of clinical outcomes in those patients with combined low tumor ADC and low rCBV values. The high ADC-high rCBV group had very low mortality (no patients died within 5 years), while the low ADC-low rCBV group had 100% mortality within 2 years (Fig 2A, -B). This new stratification suggests that the prognostic value of each imaging variable is additive and synergistic, in that the combination of both variables best predicted the worst prognostic subgroup of patients with PCNSL.
The results of our study have potentially high clinical impact in the treatment and management of patients with PCNSL. Noninvasive pretherapeutic imaging biomarkers that can accurately categorize patients into different risk groups of methotrexate-based chemotherapy could greatly facilitate clinical decision-making, allowing the initiation of individualized second-line salvage therapies, which may result in improved clinical outcome. [26][27][28][29] While ADC may signal a subtype of PCNSL that is more aggressive as demonstrated by cellularity, 12 our rCBV measure may help predict which tumors would be less treatable by chemotherapy due to lower perfusion.
Our study had several limitations. The results of our study may be influenced by its modest sample size and retrospective nature. Second, due to the paucity of available tumor-tissue samples, we were unable to correlate the rCBV measurements observed to histopathologic standards of tumor angiogenesis and hypoxia.

CONCLUSIONS
Our study further supports the important role of ADC measurement as a prognostic biomarker in immunocompetent patients with PCNSL in which the lower ADC measurement at pretherapy baseline indicates shorter overall survival. In addition, we present the first evidence of rCBV measurement as a potential prognostic biomarker in patients with PCNSL, in which lower value is associated with adverse prognosis. The predictive power of adverse outcome is even greater when risk stratification is based on combined low ADC and low rCBV values, suggesting the additive and complementary role of diffusion and perfusion MR imaging as prognostic biomarkers in patients with PCNSL.
Disclosures: Francisco Valles-RELATED: Grant: (Doris Duke Charitable Foundation Clinical Research Fellowships, Comments: The DDCF Clinical Research Fellowship program for medical students at UCSF is a 1-year mentored clinical research opportunity designed to span a broad range of research ranging from bench laboratory science to clinical and translational science, epidemiology, and outcomes research. We define "clinical and translational research" as research designed to address a question of clinical importance. The program is designed to be flexible and will be tailored to meet the needs of each student. James Rubenstein-RELATED: Grant: Dr Rubenstein is a Clinical Scholar of the Leukemia and Lymphoma Society and is a recipient of National Cancer Institute R01 CA139083-01A1.