Monitoring the effect of first line treatment in RAS/RAF mutated metastatic colorectal cancer by serial analysis of tumor specific DNA in plasma

C B Thomsen
T F Hansen
R F Andersen
J Lindebjerg
L H Jensen
A Jakobsen
Monitoring the effect of first line treatment in RAS/RAF mutated metastatic colorectal cancer by serial analysis of tumor specific DNA in plasma
10.1186/s13046-018-0723-5R E S E A R C H Open Access
Background: Precision medicine calls for an early indicator of treatment efficiency. Circulating tumor DNA (ctDNA) is a promising marker in this setting. Our prospective study explored the association between disease development and change of ctDNA during first line chemotherapy in patients with RAS/RAF mutated metastatic colorectal cancer (mCRC). Methods: The study included 138 patients with mCRC receiving standard first line treatment. In patients with RAS/ RAF mutated tumor DNA the same mutation was quantified in the plasma using droplet digital PCR. The fractional abundance of ctDNA was assessed in plasma before treatment start and at every treatment cycle until radiologically defined progressive disease.Results: RAS/RAF mutations were detected in the plasma from 77 patients. Twenty patients progressed on treatment and 57 stopped treatment without progression. The presence of mutated DNA in plasma was correlated with poor overall survival. A low level of ctDNA after the first cycle of chemotherapy was associated with a low risk of progression. On the other hand, a significant increase of ctDNA at any time during the treatment course was associated with a high risk of progression on continuous treatment. The first increase in ctDNA level occurred at a median of 51 days before radiologically confirmed progression.Conclusions:The results indicate that the ctDNA level holds potential as a clinically valuable marker in first line treatment of mCRC. A rapid decrease was associated with a prolonged progression free interval, whereas a significant increase gave notice of early progression with a relevant lead time.

Treatment efficiency

Background Precision medicine is an important subject with growing clinical interest in cancer treatment. The concept may be complicated, but its core is as simple as "the right treatment to the right patient at the right time". The issue has two aspects. Giving the right treatment is essential, but discontinuing ineffective and potentially toxic treatment holds the same importance and places monitoring in a central position.

Easily accessible biomarkers validated for clinical application are warranted. Blood samples have several advantages over tissue biopsies and consequently, the "liquid biopsy" concept has seen increased interest [1].

Tumor specific mutations in tissue rank high as tumor markers of clinical relevance and are detectable by Next Generation Sequencing (NGS). However a complete characterization takes considerable resources and the sensitivity is low, around 2% [2].

In blood a promising approach is analysis of circulating tumor DNA (ctDNA) by droplet digital PCR (ddPCR) with a high sensitivity (> 0.01%).

RAS/RAF mutations occur in 50-60% of colorectal cancer (CRC) patients [3,4] and predict resistance to EGFRinhibiting antibodies. In the clinic the mutation analysis thus serves as a tool for selection of targeted treatment. New data show that ctDNA may replace tumor tissue analysis [5][6][7][8], and serial analysis may provide information as to the relation of ctDNA with treatment effect and progression [9]. There are few studies in solid tumors dealing with this issue. A proof of concept study has indicated that fluctuations in the ctDNA level reflect changes of tumor size in metastatic breast cancer [10]. A recent study has looked at mutational dynamics during anti-EGFR therapy [11], but there are no studies dealing with prediction of progression in CRC during a treatment course.

The aim of the present study was to investigate the possible value of ctDNA monitoring during chemotherapy with respect to efficiency of first line treatment of metastatic CRC (mCRC).

Methods

Study population

Patients with mCRC receiving first line treatment were offered inclusion in a prospective biomarker study at Danish Colorectal Cancer Center South, Vejle Hospital, Denmark. The inclusion criteria are listed in the supplementary material. Plasma samples from all patients were collected at baseline and before every treatment cycle throughout the treatment course. Treatment effect was evaluated at every third cycle according to the RECIST criteria blinded of mutational status.

The study was approved by The Regional Committees on Health Research Ethics for Southern Denmark (S-20100005) and the investigation was conducted in accordance with the REMARK criteria [12].

Tumor analysis

DdPCR (Bio-Rad®, Hercules, CA, USA) was used for the analysis of all mutations in both tissue and plasma as described below. The mutations were selected by combining the results from two previous studies [13,14]. The analyses were performed in three consecutive rounds (Additional file 1: Table S1) as previously described [15] and are further described in Additional file 2: Supplementary material.

Blood sample analysis

Patients with RAS/RAF mutated DNA in the tumor were selected for plasma analysis. We investigated the 26 most frequent KRAS and NRAS mutations in CRC and the most frequent RAF mutation, BRAF V600E, as further described in the supplementary material (Additional file 3: Table S2) [13,14]. The blood sampled at each cycle of treatment was analyzed for the specific mutation detected in the tumor tissue. A detailed description of the blood sample analysis and the ddPCR can be found in Additional file 2: Supplementary material.

We have recently published a method allowing for screening with subsequent quantitative analysis of 27 RAS/RAF mutations in the blood [16]. This method was applied in the screening of RAS/RAF mutations at time of progression in patients with a wild-type tumor.

The specific mutations and the distribution found in the plasma at baseline are listed in Additional file 4: Table S3.

Data management

The ctDNA level was defined as the fraction of ctDNA and expressed as the proportion of mutant alleles in the total circulating cell-free DNA (mutated DNA/(mutated DNA + wild-type DNA) and calculated by the Quantasoft software. The variation of the ctDNA level is reported as a 95% confidence interval (95% CI) based on Poisson statistics.

A substantial increase of the ctDNA level (HctDNA) during the treatment course was defined as a value with no overlap between the 95% CI of the current and immediately preceding measurements.

Low ctDNA (LctDNA) after the first treatment cycle was defined as a level with the lower 95% CI overlapping zero.

Statistical analysis

Full description is given in the Additional file 2: Supplementary material.

Results

Between March 2010 and November 2015, 152 patients were enrolled in the study. The patient flow is shown in Fig. 1. One patient withdrew his consent and one suffered from another malignant disease and was mistakenly enrolled leaving 150 patients for investigation. Analysis of mutational status was not possible in 12 patients (8%) due to limited access to tumor tissue or insufficient material in the specimens. Thus data were available from 138 patients (92%). Eighty-two patients (59%) had a tumor RAS/RAF mutation and in 77 patients (94%) the mutation was found in the plasma. Baseline patient characteristics of patients with mutated DNA in plasma are shown in Table 1. There was no statistically significant difference in terms of age, gender, and tumor location between the group of patients with wild-type DNA in the plasma and patients with mutated DNA (data not shown). The 77 patients had at least a baseline blood sample drawn and contributed with a total of 571 analyzed blood samples. Twenty patients progressed on treatment (26%), one of which only had a baseline blood sample. Fifty-seven patients stopped treatment without progressive disease (PD) (74%).

The time-of-progression plasma sample from patients with a wild-type tumor was screened for RAS/RAF mutations. All patients were plasma wild-type except one (1/21), whose blood samples were not included in the analysis.

Effect of plasma mutational status

The median follow-up time was 35.3 months as estimated by inverse Kaplan-Meier analysis. The overall survival (OS) analysis was based on 114 events in the whole cohort of 138 patients of which 124 had at least one response evaluation. Based on tissue samples the best overall response rate (partial response or complete response) was 29%, 18% and 59% in RAS mutated patients, BRAF mutated patients, and wild type patients, respectively. In plasma the same rates were 33%, 20% and 52%, respectively. The difference in response rate between patients with mutated DNA and wild type in plasma translated into a significant difference in progression free survival (PFS). The median PFS was 5.7 months (range 4.2-7.0 months) and 7.8 months (range 6.9-9.9 months), respectively, hazard ratio (HR) = 2.02, 95%CI = 1.38-2.95, P < 0.001. A significant difference also applied to OS. The median values were 12.7 months (range 10.

Effect of ctDNA level at baseline

Among the 77 patients with tumor and plasma mutation, the ctDNA level at baseline ranged between 0.0026 and 80.43% of the total cell free DNA. The results indicated a clear correlation between the ctDNA level as divided according to the median value and PFS (HR = 3.95, P = 0.017). Further calculation on the quantitative importance of the ctDNA level was analyzed by logistic regression. Increased ctDNA at baseline was clearly associated with an increased risk of progression on treatment (relative risk (RR) = 1.02, 95%CI = 1.01-1.03, P < 0.001). Figure 3 shows fluctuation of ctDNA level during the treatment course up to nine cycles. It only includes patients on treatment censored at discontinuation irrespective of reason. It is obvious that the ctDNA level decreases rapidly during the first three cycles of treatment from around 20% at baseline to nadir values of approximately 3% before cycle four. The confidence intervals during these and the few subsequent cycles were rather narrow, but they widened towards the third evaluation (pre-cycle nine). The reason was mainly a reduced number of patients for observation but also an increasing The figure also shows an abrupt decrease of ctDNA after start of treatment. LctDNA was found in 40% (27/ 65) after the first cycle of chemotherapy (before cycle 2). There was a strong correlation between LctDNA and risk of progression during treatment. Only two patients out of 27 (7%) progressed in the LctDNA group compared to 17/38 (45%) in the other group (non-LctDNA) (p = 0.001). This relationship is further underlined in the analysis of PFS during treatment (Fig. 4). The difference according to ctDNA status was statistically significant (HR = 0.16, P = 0.017).

Treatment related dynamics of ctDNA level

HctDNA at any point during the treatment course was highly related to progression on treatment (RR = 4.58, 95%CI = 1.99-10.51, P < 0.001). A consecutive HctDNA resulted in a similar high risk of progression (RR = 4.03, 95%CI = 2.11-7.70, P < 0.001). The increased level appearing before progression was found by CT scan with a median lead time of 51 days (range 14-133).

Discussion

In this prospective study of mCRC patients receiving first line chemotherapy we observed a strong correlation between the ctDNA level and clinical endpoints.

Chemotherapy is the corner stone in mCRC treatment. Modern combinations of two or three drugs have improved survival but the treatment still has severe limitations. Firstly, it is only beneficial to two thirds of the patients and secondly, it has serious toxicity. The current methods for monitoring mCRC treatment are far from ideal. The only  recommended evaluation modality is imaging [17]. However, CT scanning has drawbacks as to tumor size and volume and is not applicable for small metastases (< 10 mm). Furthermore, progression according to the RECIST criteria calls for at least a 20% size increase. Therefore, the identification of markers able to guide the treatment holds high priority, especially biomarkers allowing for discontinuation of ineffective treatment at an early time. An indicator in the context of treatment effect should be easily accessible for repeated measurement, which in practice excludes tissue markers. Also, it requires high specificity and sensitivity. Somatic tumor mutations seem to be adequate for this purpose with ctDNA being highly specific. The literature, however, is still sparse.

An approach based on ddPCR has high sensitivity and seems to be in accordance with other techniques described in the literature [18,19]. It is also highly reproducible as shown in our recent paper [20]. We here present results of serial measurements over a whole treatment course in patients with RAS/RAF mutated tumors. Mutated DNA was detected in the blood stream in > 90% of the patients. This is in agreement with several other studies [21,22].  We chose to investigate the clinical value of the fractional abundance of ctDNA, since previous studies have focused on this quantity as a central measure in the prognostic and predictive setting [21,23]. The interpretation of ctDNA data in a clinical context calls for careful consideration and at present there is no general agreement as to quantitative classification related to clinical parameters. Relative reduction of ctDNA level has limitations, since it does not account for low baseline levels. An absolute value in ng/ml or copies/ml seems more attractive. We chose the 95% CI, which is a simple and unique measure. It is easily calculated and seems relevant to account for different values at different time points over a long treatment course.

The prognostic value of ctDNA in mCRC patients has previously been evaluated. Recent studies found a marked prognostic value of ctDNA in OS estimates [24,25]. Our result supports their conclusion, as patients with a plasma mutation were shown to have a significantly worse survival compared to the rest of the cohort (Fig. 2). We also found that patients presenting with mutated DNA in plasma had a lower response rate than wild-type patients. This is in line with a recently conducted study that found the same relation but only based on the first radiologic assessment [21].

The present results indicate an influence of a high baseline ctDNA level on treatment effect. With each increase in baseline ctDNA level of 1%, the risk of treatment failure increased by 2%. This suggests patients with a high ctDNA level to have a poor prognosis, but a more detailed quantification of the threshold for effect is needed.

LctDNA correlated with a low risk of progression under treatment (Fig. 4). Our findings confirm the results in a recent paper by Garlan et al. [24]. They reported a low ctDNA level at baseline, or a level decreasing below 0.1 ng/ml before cycle two, to indicate long PFS and OS. Tie et al. reported that a reduction in ctDNA level before cycle two correlated with response at the first radiological evaluation and a trend for increased PFS [21]. Our results showed agreement between reduction of ctDNA level and improved PFS, but we did not find an association with response. The difference may be explained by different response rates. It was 52% in the study by Tie et al. and only 31% in the present one. The lower rate may be due to RAS/RAF mutations in all our patients.

The present results are the first to show the relationship between increasing level of ctDNA and progression over the whole treatment course. HctDNA at any time implied a high risk of progression (RR = 4.58). The importance of just one such increase was accentuated by the fact that two consecutive HctDNA measurements had a similar impact on the risk of progression (RR = 4.03). Hence, there does not seem to be evidence as to postpone the change in treatment strategy until a second increase is observed. On the other hand, a stable ctDNA level encourages treatment continuation. The increase appears with a lead time of approximately 2 months, which is relevant in the clinical setting.

The present study has several limitations. First of all, it only comprised patients with RAS/RAF mutated tumors, which, however, applied to around 60% of the patients. Additionally, the sample size of patients ending treatment due to PD was small. The reasons for treatment discontinuation, listed in the flow chart, are believed to be in agreement with clinical practice. The correlations of LctDNA with PFS and HctDNA with progression suggest a predictive value of changes in ctDNA level, but a randomized trial is needed for final proof.

Previous studies conclude that ctDNA is a more reliable marker than carcinoembryonic antigen (CEA) which may even be misleading [21,26,27]. Further, CEA is not part of the ESMO guidelines [17] and consequently not included in the present study. We only analyzed the plasma samples from tumor wild-type patients at the time of progression, but it is not likely that patients without mutated DNA at progression would have presented with mutated DNA before start of treatment.

Conclusions

The present study adds preliminary evidence as to the clinical relevance of ctDNA during treatment. An early decrease holds promise as to a long progression free interval. On the other hand a significant increase gives notice of progression. Thus, ctDNA monitoring may be a step towards precision medicine.

Additional files

2-16.0 months) and 24.2 months (range 18.4-34.3 months), respectively, HR = 2.50, 95%CI = 1.69-3.71, P < 0.001 (Fig. 2). Median PFS was 6.2 months (range 4.4-7.2 months) and median OS was 14.8 months (range 10.2-16.8 months) for plasmatic RAS mutated patients alone (n = 67).

Fig. 1
1Flow chart fraction of progressing patients with a broad range of ctDNA values.

Fig. 2
2Overall survival according to mutational statusThe difference is statistically significant, HR = 2.50, 95%CI = 1.69-3.71, P < 0.001.Tick marks at censor points.

Fig. 4
4Progression free survivalPFS during treatment according to ctDNA at end of cycle one until end of treatment. The difference is statistically significant, HR = 0.16, P = 0.017. Tick marks at censor points.

Fig. 3
3Observed variation in fractional abundance (ctDNA level)ctDNA level is shown over the whole treatment course in all 77 patients with plasma ctDNA.The figure gives mean value and 95%CI.

.
Mutations tested in three consecutive rounds of tumor analyses. (JPEG 44 kb) Additional file 2: Suplementary material. (DOC 35 kb) Additional file 3: Table S2. BioRad PrimePCR ddPCR assays for specific mutations. (JPEG 118 kb) Additional file 4: Table S3. Distribution of mutations in plasma DNA found at baseline. (JPEG 29 kb) Abbreviations CEA: Carcinoembryonic antigen; CI: Confidence interval; CRC: Colorectal cancer; ctDNA: Circulating tumor DNA; ddPCR: Droplet digital polymerase chain reaction; HctDNA: a substantial increase in the ctDNA; HR: Hazard ratio; LctDNA: Low ctDNA; mCRC: Metastatic CRC; NGS: New Generation Sequencing; OS: Overall survival; PD: Progressive disease; PFS: Progression free survival; RR: Relative risk

Table 1
1Baseline characteristics of patients with mutated DNA in plasma (N = 77)All patients with mutated
DNA in plasma

Parameter
N (%)

Age, years

Median (range)
66 (35-79)

Gender

Female
29 (38)

Male
48 (62)

PS

0-1
73 (95)

2
4 (5)

Location of primary tumor

Right/transverse colon
25 (32)

Left colon
21 (27)

Rectum
31 (40)

Primary disseminated disease
63 (81)

Number of metastatic sites

≤ 2
53 (69)

> 2
24 (31)

Mutations in plasma DNA

KRAS
64 (83)

NRAS
3 (4)

BRAF
10 (13)

Abbreviations: N, number; PS, performance status. Some percentages do not
add up to 100% due to rounding

AcknowledgementsWe would like to thank the study participants and the staff at Vejle Hospital who collected samples for this study. Also, we gratefully acknowledge the technicians Lone Hartman Hansen, Pia Nielsen, Tina Brandt Christensen, and Birgit Roed Sørensen for technical assistance, Flemming Brandt Sørensen for his expertise in laser microdissection, and study nurse Margit Søgaard Jakobsen. The authors have declared no conflicts of interest.Publisher's NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.We gratefully thank René Depont for his statistical support. Furthermore, we express gratitude to Karin Larsen for linguistic editing of the manuscript.FundingNo financial support was given to any of the authors in relation to this work.Availability of data and materialsThe dataset supporting the conclusions of this article is not available due to Danish legislation.Authors' contributions CBT collected patient data, interpreted the data regarding the influence of ctDNA and was a major contributor in writing the manuscript. TFH designed and initiated the trial and collected patient data. RF performed the ddPCR analyses. AJ designed the trial, contributed to write the manuscript and to critically review the content. All authors read and approved the final manuscript.Ethics approval and consent to participate All patients gave oral and written consent to participate in the study. The study was approved by The Regional Committees on Health Research Ethics for Southern Denmark (S-20100005) and the investigation was conducted in accordance with the REMARK criteria[12].Consent for publicationNot applicable.Competing interests• We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal Submit your next manuscript to BioMed Central and we will help you at every step:
Circulating DNA and survival in solid tumors. A Ocaña, L Díez-González, D C García-Olmo, Cancer Epidemiol Biomark Prev. 252Ocaña A, Díez-González L, García-Olmo DC, et al. Circulating DNA and survival in solid tumors. Cancer Epidemiol Biomark Prev. 2016;25(2):399-406.

Liquid biopsies: genotyping circulating tumor DNA. L A Diaz, A Bardelli, J Clin Oncol. 326Diaz LA, Bardelli A. Liquid biopsies: genotyping circulating tumor DNA. J Clin Oncol. 2014;32(6):579-86.

Prognostic and predictive value of common mutations for treatment response and survival in patients with metastatic colorectal cancer. J Souglakos, J Philips, R Wang, Br J Cancer. 1013Souglakos J, Philips J, Wang R, et al. Prognostic and predictive value of common mutations for treatment response and survival in patients with metastatic colorectal cancer. Br J Cancer. 2009;101(3):465-72.

Addition of cetuximab to oxaliplatin-based first-line combination chemotherapy for treatment of advanced colorectal cancer: results of the randomised phase 3 MRC COIN trial. T S Maughan, R A Adams, C G Smith, Lancet. 3779783Maughan TS, Adams RA, Smith CG, et al. Addition of cetuximab to oxaliplatin-based first-line combination chemotherapy for treatment of advanced colorectal cancer: results of the randomised phase 3 MRC COIN trial. Lancet. 2011;377(9783):2103-14.

Clinical utility of circulating DNA analysis for rapid detection of actionable mutations to select metastatic colorectal patients for anti-EGFR treatment. A R Thierry, S El Messaoudi, C Mollevi, Ann Oncol. 28Thierry AR, El Messaoudi S, Mollevi C, et al. Clinical utility of circulating DNA analysis for rapid detection of actionable mutations to select metastatic colorectal patients for anti-EGFR treatment. Ann Oncol. 2017;28:2149-59.

Blood-based detection of RAS mutations to guide anti-EGFR th2erapy in colorectal cancer patients: concordance of results from circulating tumor DNA and tissue-based RAS testing. W Schmiegel, R J Scott, S Dooley, Mol Oncol. 112Schmiegel W, Scott RJ, Dooley S, et al. Blood-based detection of RAS mutations to guide anti-EGFR th2erapy in colorectal cancer patients: concordance of results from circulating tumor DNA and tissue-based RAS testing. Mol Oncol. 2017;11(2):208-19.

Concordance of blood-and tumor-based detection of RAS mutations to guide anti-EGFR therapy in metastatic colorectal cancer. J Grasselli, E Elez, G Caratù, Ann Oncol. 286Grasselli J, Elez E, Caratù G, et al. Concordance of blood-and tumor-based detection of RAS mutations to guide anti-EGFR therapy in metastatic colorectal cancer. Ann Oncol. 2017;28(6):1294-301.

RAS testing of liquid biopsy correlates with the outcome of metastatic colorectal cancer patients treated with first-line FOLFIRI plus cetuximab in the CAPRI-GOIM trial. N Normanno, R Esposito Abate, M Lambiase, Ann Oncol. 29Normanno N, Esposito Abate R, Lambiase M, et al. RAS testing of liquid biopsy correlates with the outcome of metastatic colorectal cancer patients treated with first-line FOLFIRI plus cetuximab in the CAPRI- GOIM trial. Ann Oncol. 2018;29:112-18.

Plasma ctDNA RAS mutation analysis for the diagnosis and treatment monitoring of metastatic colorectal cancer patients. J Vidal, L Muinelo, A Dalmases, Ann Oncol. 286Vidal J, Muinelo L, Dalmases A, et al. Plasma ctDNA RAS mutation analysis for the diagnosis and treatment monitoring of metastatic colorectal cancer patients. Ann Oncol. 2017;28(6):1325-32.

Mutation tracking in circulating tumor DNA predicts relapse in early breast cancer. I Garcia-Murillas, G Schiavon, B Weigelt, Sci Transl Med. 7302Garcia-Murillas I, Schiavon G, Weigelt B, et al. Mutation tracking in circulating tumor DNA predicts relapse in early breast cancer. Sci Transl Med. 2015;7(302):302ra133.

Clonal evolution and resistance to EGFR blockade in the blood of colorectal cancer patients. G Siravegna, B Mussolin, M Buscarino, Nat Med. 21Siravegna G, Mussolin B, Buscarino M, et al. Clonal evolution and resistance to EGFR blockade in the blood of colorectal cancer patients. Nat Med. 2015;21:795-801.

Reporting recommendations for tumor marker prognostic studies. L M Mcshane, D G Altman, W Sauerbrei, S E Taube, J Clin Oncol. 2336McShane LM, Altman DG, Sauerbrei W, Taube SE. Reporting recommendations for tumor marker prognostic studies. J Clin Oncol. 2005; 23(36):9067-72.

Frequency of KRAS, BRAF, and NRAS mutations in colorectal cancer. C P Vaughn, S D Zobell, L V Furtado, Genes Chromosom Cancer. 505Vaughn CP, Zobell SD, Furtado LV, et al. Frequency of KRAS, BRAF, and NRAS mutations in colorectal cancer. Genes Chromosom Cancer. 2011;50(5):307-12.

Panitumumab-FOLFOX4 treatment and RAS mutations in colorectal cancer. J-Y Douillard, K S Oliner, S Siena, N Engl J Med. 36911Douillard J-Y, Oliner KS, Siena S, et al. Panitumumab-FOLFOX4 treatment and RAS mutations in colorectal cancer. N Engl J Med. 2013;369(11):1023-34.

The prognostic value of simultaneous tumor and serum RAS/RAF mutations in localized colon cancer. Ceb Thomsen, A L Appelt, R F Andersen, Cancer Med. 65Thomsen CEB, Appelt AL, Andersen RF, et al. The prognostic value of simultaneous tumor and serum RAS/RAF mutations in localized colon cancer. Cancer Med. 2017;6(5):928-36.

Screening for circulating RAS/RAF mutations by multiplex digital PCR. R F Andersen, A Jakobsen, Clin Chim Acta. 458Andersen RF, Jakobsen A. Screening for circulating RAS/RAF mutations by multiplex digital PCR. Clin Chim Acta. 2016;458:138-43.

ESMO consensus guidelines for the management of patients with metastatic colorectal cancer. E Van Cutsem, A Cervantes, R Adam, Ann Oncol. 0Van Cutsem E, Cervantes A, Adam R, et al. ESMO consensus guidelines for the management of patients with metastatic colorectal cancer. Ann Oncol. 2016;0:1-38.

Analysis of circulation nucleic acids. I Hudecova, Pcr Digital, Clin Biochem. 48Hudecova I, Digital PCR. Analysis of circulation nucleic acids. Clin Biochem. 2015;48:948-56.

Detecting biomarkers with microdroplet technology. V Taly, D Pekin, A A El, P Laurent-Puig, Trends Mol Med. 187Taly V, Pekin D, El AA, Laurent-Puig P. Detecting biomarkers with microdroplet technology. Trends Mol Med. 2012;18(7):405-16.

International Interlaboratory digital PCR study demonstrating high reproducibility for the measurement of a rare sequence variant. A S Whale, A S Devonshire, G Karlin-Neumann, Anal Chem. 893Whale AS, Devonshire AS, Karlin-Neumann G, et al. International Interlaboratory digital PCR study demonstrating high reproducibility for the measurement of a rare sequence variant. Anal Chem. 2017;89(3):1724-33.

Circulating tumor DNA as an early marker of therapeutic response in patients with metastatic colorectal cancer. J Tie, I Kinde, Y Wang, Ann Oncol. 26Tie J, Kinde I, Wang Y, et al. Circulating tumor DNA as an early marker of therapeutic response in patients with metastatic colorectal cancer. Ann Oncol. 2015;26:1715-22.

Clinical validation of the detection of KRAS and BRAF mutations from circulating tumor DNA. A R Thierry, F Mouliere, El Messaoudi, S , Nat Med. 204Thierry AR, Mouliere F, El Messaoudi S, et al. Clinical validation of the detection of KRAS and BRAF mutations from circulating tumor DNA. Nat Med. 2014;20(4):430-5.

Analysis of circulating tumour DNA to monitor disease burden following colorectal cancer surgery. T Reinert, L V Schøler, R Thomsen, Gut. 65Reinert T, Schøler LV, Thomsen R, et al. Analysis of circulating tumour DNA to monitor disease burden following colorectal cancer surgery. Gut. 2016;65:625-34.

Early evaluation of circulating tumor DNA as marker of therapeutic efficacy in metastatic colorectal cancer patients (PLACOL study). F Garlan, P Laurent-Puig, D Sefrioui, Clin Cancer Res. 2310Garlan F, Laurent-Puig P, Sefrioui D, et al. Early evaluation of circulating tumor DNA as marker of therapeutic efficacy in metastatic colorectal cancer patients (PLACOL study). Clin Cancer Res. 2017;23(10):5416-26.

KRAS mutations in tumor tissue and plasma by different assays predict survival of patients with metastatic colorectal cancer. J-M Xu, X-J Liu, F-J Ge, J Exp Clin Cancer Res. 331104Xu J-M, Liu X-J, Ge F-J, et al. KRAS mutations in tumor tissue and plasma by different assays predict survival of patients with metastatic colorectal cancer. J Exp Clin Cancer Res. 2014;33(1):104.

Circulating mutant DNA to assess tumor dynamics. F Diehl, K Schmidt, Choti M A, Nat Med. 149Diehl F, Schmidt K, Choti M a, et al. Circulating mutant DNA to assess tumor dynamics. Nat Med. 2008;14(9):985-90.

Circulating DNA as a strong multimarker prognostic tool for metastatic colorectal cancer patient management care. S El Messaoudi, F Mouliere, Du Manoir, S , Clin Cancer Res. 2212El Messaoudi S, Mouliere F, Du Manoir S, et al. Circulating DNA as a strong multimarker prognostic tool for metastatic colorectal cancer patient management care. Clin Cancer Res. 2016;22(12):3067-77.


Hyperdynamic CSF motion profiles found in idiopathic normal pressure hydrocephalus and Alzheimer's disease assessed by fluid mechanics derived from magnetic resonance images
2017

Ken Takizawa
Department of Neurosurgery
Tokai University School of Medicine
143 Shimokasuya2591193IseharaKanagawaJapan

Mitsunori Matsumae
Department of Neurosurgery
Tokai University School of Medicine
143 Shimokasuya2591193IseharaKanagawaJapan

Naokazu Hayashi
Department of Neurosurgery
Tokai University School of Medicine
143 Shimokasuya2591193IseharaKanagawaJapan

Akihiro Hirayama
Department of Neurosurgery
Tokai University School of Medicine
143 Shimokasuya2591193IseharaKanagawaJapan

Satoshi Yatsushiro
Department of Neurosurgery
Tokai University School of Medicine
143 Shimokasuya2591193IseharaKanagawaJapan

Kagayaki Kuroda
Department of Neurosurgery
Tokai University School of Medicine
143 Shimokasuya2591193IseharaKanagawaJapan

Hyperdynamic CSF motion profiles found in idiopathic normal pressure hydrocephalus and Alzheimer's disease assessed by fluid mechanics derived from magnetic resonance images

Fluids Barriers CNS
1429201710.1186/s12987-017-0077-yRESEARCHIdiopathic normal pressure hydrocephalusAlzheimer's diseaseCerebrospinal fluidMagnetic resonance imagingFluid dynamics
Background: Magnetic resonance imaging (MRI) does not only ascertain morphological features, but also measures physiological properties such as fluid velocity or pressure gradient. The purpose of this study was to investigate cerebrospinal fluid (CSF) dynamics in patients with morphological abnormalities such as enlarged brain ventricles and subarachnoid spaces. We used a time-resolved three dimensional phase contrast (3D-PC) MRI technique to quantitatively evaluate CSF dynamics in the Sylvian aqueduct of healthy elderly individuals and patients with either idiopathic normal pressure hydrocephalus (iNPH) or Alzheimer's disease (AD) presenting with ventricular enlargement.Methods: Nineteen healthy elderly individuals, ten iNPH patients, and seven AD patients (all subjects ≥ 60 years old) were retrospectively evaluated 3D-PC MRI. The CSF velocity, pressure gradient, and rotation in the Sylvian aqueduct were quantified and compared between the three groups using Kolmogorov-Smirnov and Mann-Whitney U tests.Results:There was no statistically significant difference in velocity among the three groups. The pressure gradient was not significantly different between the iNPH and AD groups, but was significantly different between the iNPH group and the healthy controls (p < 0.001), and similarly, between the AD group and the healthy controls (p < 0.001). Rotation was not significantly different between the iNPH and AD groups, but was significantly different between the iNPH group and healthy controls (p < 0.001), and similarly, between the AD group and the healthy controls (p < 0.001).Conclusions: Quantitative analysis of CSF dynamics with time resolved 3D-PC MRI revealed differences and similarities in the Sylvian aqueduct between healthy elderly individuals, iNPH patients, and AD patients. The results showed that CSF motion is in a hyperdynamic state in both iNPH and AD patient groups compared to healthy elderly individuals, and that iNPH patients and AD patients display similar CSF motion profiles.

Background

The Cerebrospinal fluid (CSF) removes unnecessary substances and heat produced by metabolic activity from the brain parenchyma [1]. Therefore, understanding CSF dynamics is important for evaluating intracranial changes, pathological analysis, and treatment management of idiopathic normal pressure hydrocephalus (iNPH) and Alzheimer's disease (AD) patients. Lately, noninvasive techniques using magnetic resonance imaging (MRI) have become prevalent in the analysis of CSF motion dynamics, and researchers commonly use phase contrast (PC) MR images to understand CSF physiology [2][3][4][5][6]. In the present study, we analyzed CSF motion using PC. Specifically, we used a time-resolved three-dimensional PC (3D-PC) MRI acquisition where the time dimension is added to the anterior-posterior, right-left, and head-foot dimensions in order to quantify and analyze CSF motion in elderly healthy individuals, idiopathic normal pressure hydrocephalus (iNPH) patients, and Alzheimer's disease (AD) patients. In two-dimensional imaging, CSF velocity can only be calculated in-plane. Using 3D-PC, CSF motion can be calculated in three dimensions, and therefore the pressure gradient and rotation of the CSF motion can be measured in addition to velocity. Understanding fluid dynamics is important to determine the pressure gradient and rotation. CSF motion in elderly groups has only been investigated in a small number of studies and there is no consensus on the characteristics of CSF motion in AD patients [3,7,8]. In the iNPH study, CSF motion appears to be hyper dynamic [3,9,10]. The aim of this study was to compare the CSF dynamics of healthy elderly volunteers with those of AD and iNPH patients.

Methods

This study was conducted with informed consent from volunteers and patients after explaining the purpose of the study in accordance with the ethics regulations of the authors' affiliated institution.

Patients

The patient's characteristics are shown in Table 1. The subjects included seven AD patients (age range 66-89 years), ten iNPH patients (age range 67-87 years), and nineteen healthy elderly volunteers (age range 67-80 years). The iNPH group was selected in accordance with the guidelines outlined by the Japanese Society of Normal Pressure Hydrocephalus [11] and consisted of patients who displayed at least one symptom of the classical medical triad (gait disturbance, cognitive dysfunction, or urinary incontinence) and presented an enlargement of the anterior lateral ventricle horn with an Evans ratio ≥ 0.3, narrowing of high convexity subarachnoid space, and enlargement of the Sylvian fissure [12]. Eight out of ten iNPH patients received a shunting procedure: six received a ventriculo-peritoneal shunt and two patients a lumbo-peritoneal shunt. Gait disturbance improved in all patients, cognitive function improved in four patients, and urinary dysfunction improved in six patients after the shunting procedure. The AD group included individuals with probable AD according to the criteria delineated by the National Institute of Neurological and Communicative Diseases and Stroke/ Alzheimer's Disease and Related Disorders Association [13]. The healthy elderly control group was selected from volunteers recruited at our institution that did not have a history of central nervous system disorders, did not exhibit neurological abnormalities upon examination by neurologists or neurosurgeons, and did not present abnormalities on neurological and standard MRI.

MRI acquisition

A 1.5 Tesla MRI scanner with an eight-channel phased array head and neck surface coil (Gyroscan; Philips, Best, Netherlands) was used. A time resolved 3D-PC MRI sequence was used, providing a 4D velocity field. Flow-encoding directions were head-foot, right-left, and anterior-posterior. A dynamic cine image of one cardiac cycle was created by imaging 32 phases per heartbeat (without interpolation), synchronized with the heartbeat. The imaging parameters were: repetition time, 9.8-16.4 ms; echo time, 6.6-6.7 ms; flip angle, 20°; field of view, 22 × 22 for females and 32 × 32 cm 2 for males; velocity encoding, 5 cm/s for volunteers, 30 cm/s for AD and iNPH patients; acquisition matrix, 1.96 × 1.96 × 1.96 mm 3 (isotropic); sensitivity encoding factor of 2. Ten slices per volume were acquired, using 4-8 pixels to calculate the velocity and pressure gradient, and 12-20 pixels for the rotation. The acquisition time for this sequence was on average 32 min, depending on heart rate. The trigger for timing the 3D-PC was the peripheral pulse, measured from a finger.

Image processing

Pressure gradients were calculated from the velocity data using Navier-Stokes equations. Color-coded CSF velocity vectors, pressure gradients, and rotations were overlaid on T 2 -weighted images. All processing was performed on a workstation (Power Mac Pro, Quad-Core Intel Xeon; Apple Inc., Cupertino, CA, USA) with our in-house software, written in Matlab (R2012b; Mathworks, Natick, MA, USA). Regions of interest (ROIs) at both the entry point and outlet point of the Sylvian aqueduct were drawn on sagittal images by the researchers, including a neurosurgery specialist, using an in-house mouse-operated graphical user interface. Partial volume effects caused by the relatively large voxel size (approximately 2 mm) used can lead to segmentation errors on T 2 -weighted images. Therefore, a "spatial-based fuzzy clustering" segmentation method [14] was used to reduce partial volume effects.

The principle of this study was based on obtaining velocity and rotation in three-dimensional space (anterior-posterior, right-left, and head-foot directions) using the PC technique. The pressure gradient was then calculated from these velocity data using Navier-Stokes equations. Further details of this method can be found elsewhere [4].

Statistical analysis

CSF velocity and pressure gradient data calculated from the ROIs were represented as box plots. CSF velocities and pressure gradients were compared between groups as nonparametric data using Kolmogorov-Smirnov and Fig. 1 Velocity mapping of the volunteer by 3D-PC at 4 different stages of the cardiac cycle. In-plane velocities were visualized using vectors and through-plane velocities were visualized using colors. The color-coded CSF velocity field vector was then superimposed onto T 2 -weighted images of the stationary tissues. Red circle indicates Sylvian aqueduct Mann-Whitney U tests. All statistical analyses were conducted using SPSS software version 13 (SPSS Japan Inc., Tokyo).  Figure 2 shows the calculated velocity in the Sylvian aqueduct of AD patients, iNPH patients, and healthy elderly volunteers. There was no statistically significant difference (p = 0.380) between the AD (median = 0.868 cm/s) and iNPH groups (median = 1.452 cm/s). Similarly, there were no significant differences (p = 0.912) between the AD and healthy controls (median = 0.801 cm/s) or between the iNPH and healthy controls (p = 0.271).  Figure 4 shows the pressure gradient in the Sylvian aqueduct of AD patients, iNPH patients, and healthy elderly volunteers. The median pressure gradients for each group were: 426.6 Pa/m for the AD group, 473.8 Pa/m for iNPH group, and 117.8 Pa/m for the healthy elderly group. Both the AD and iNPH groups had significantly greater pressure gradients in the Sylvian aqueduct compared to the healthy controls (p < 0.001 for both comparisons). There was no significant difference between the AD and iNPH groups (p = 0.696). Figure 5 shows rotation images for a 67-year-old male volunteer. Figure 6 shows the rotation in the Sylvian aqueduct of AD patients, iNPH patients, and healthy elderly volunteers. There was no significant difference (p = 0.845) between the AD (median = 10.243 cycle/s cm 2 ) and iNPH groups (median = 9.159 cycle/s cm 2 ). Both the AD and iNPH groups had greater rotation compared to the healthy elderly group (median = 3.447 cycle/s cm 2 , p < 0.001 for both comparisons).

Results

Comparison of velocity between AD patients, iNPH patients, and healthy elderly volunteers

Comparison of pressure gradient between AD patients, iNPH patients, and healthy elderly volunteers

Comparison of rotation between AD patients, iNPH patients, and healthy elderly volunteers

Discussion

In the present study, we assessed three elements that describe CSF motion (CSF velocity, pressure gradient, and rotation) in the Sylvian aqueduct, using a time resolved 3D-PC MRI sequence and compared these between AD patients, iNPH patients, and healthy elderly volunteers. Although previous studies have investigated the physiology and pathology of AD and iNPH patients, few reports have analyzed CSF dynamics using MR, particularly in AD patients. Moreover, apart from one preliminary study [3], no other studies have shown that the pressure gradient is elevated in iNPH patients compared to healthy elderly controls, and our present report is the Fig. 2 Box plots of velocity in the Sylvian aqueduct for AD patients, iNPH patients, and healthy elderly volunteers. There were no statistically significant differences between the patient groups and volunteer group. AD Alzheimer's disease, iNPH idiopathic normal pressure hydrocephalus outside values are indicated by a small "o" and far-out values are indicated by an asterisk first to investigate the pressure gradient in AD patients. Therefore, the present study analyzed the components of fluid dynamics such as velocity, pressure gradient, and rotation, in order to better understand the pathology of a disease that also presents as a similar enlargement of CSF spaces.

The results showed that CSF velocity was not statistically significantly different between the AD and iNPH patient groups and healthy elderly volunteers. However, pressure gradient and rotation were significantly greater in AD and iNPH patient groups compared to the healthy elderly group. Pressure gradient was determined using the Navier-Stokes equations, consisting of acceleration, convection, and viscosity terms. We hypothesized that CSF viscosity and convection, as well as the anatomical morphology of the Sylvian aqueduct are not significantly different between the AD and iNPH patient groups and the healthy elderly group. This would mean that only acceleration has a significant effect on the pressure gradients of CSF, with acceleration increases resulting in pressure gradient increases. In other words, the AD and iNPH patient groups had greater CSF flow acceleration compared to the healthy controls, and this consequently influenced the pressure gradient. Furthermore, Fig. 3 Pressure gradient color mapping of the volunteer by 3D-PC at 4 stages of the cardiac cycle. The intensity of the pressure gradient was indicated using a color scale. The color-coded CSF pressure gradient were then superimposed onto T 2 -weighted images of the stationary tissues. Red circle indicates Sylvian aqueduct large velocity changes may lead to a disturbance in CSF motion, inducing turbulence in the Sylvian aqueduct, and this may have also been reflected in the large differences in rotation. Thus, it appears that CSF dynamics are in a hyper-dynamic state in both the AD and iNPH patient groups. The details of the relationship between pressure gradient and or velocity compared with rotation is described elsewhere [15]. To the best of our knowledge, this is the first study to report CSF rotation in AD and iNPH patients. Moreover, apart from one preliminary study [3], no other studies have shown that the pressure gradient is elevated in iNPH patients compared to healthy elderly controls, and our present report is the first to investigate the pressure gradient in AD patients. On the other hand, there are various PC MRI studies that have shown that the CSF flow in iNPH patients is in a hyperdynamic state, which is in accordance with our results. Specifically, it has been shown that mean CSF flow is greater in iNPH but not AD patients compared to healthy controls [7, 10], CSF motion increases in iNPH patients [16], and stroke volume increases in iNPH patients [9, 17,18]. However, it is not currently possible to use markers such as stroke volume [8] to distinguish between iNPH and AD. Also, we have compared the result of preoperative CSF dynamics and postoperative surgical results in iNPH group. Due to the limited number of patients, we currently could not differentiate between shunt responders and shunt non-responders.

These results pose the question as to why CSF is in a hyper-dynamic state in our patients compared to the healthy elderly group. A decrease in brain volume due to aging or AD leads to a relative increase in ventricular volume and subarachnoid space leading to a general increase in CSF volume, and this consequently increases the space available for free CSF motion. It is unlikely that this increased freedom leads to pressure gradient elevation, rotation increase, or turbulence. On the contrary, it would most likely decrease pressure gradient and rotation. If so, the most probable factor inducing elevated CSF pressure gradients and increased rotation would be the restriction in CSF motion due to decreased compliance in the cerebrospinal cavity that surrounds the subarachnoid space and ventricles. Mase and colleagues reported that iNPH patients had lower compliance compared to patients with ventricular dilatation or asymptomatic ventricular dilatation and healthy volunteers [19][20][21]. On the other hand, Edwards et al. described that the compliance of spinal subarachnoid space (especially dural sac) has a large effect on the compliance of overall CSF, indicating that degenerative diseases of the spinal cord induce decreased compliance [22]. Studies using mathematical models showed that decreased compliance is a significant element in ventricle enlargement [23,24], and simulations using these models are extremely easy to understand. These results all indicated that decreased compliance in the CSF environment is important in Fig. 4 Box plots of pressure gradients in the Sylvian aqueduct between AD patients, iNPH patients, and healthy elderly volunteers. Both the AD and iNPH groups had significantly greater pressure gradients compared to the volunteer group. AD Alzheimer's disease, iNPH idiopathic normal pressure hydrocephalus outside values are indicated by a small "o" diseases such as iNPH that are associated with enlarged ventricles. Furthermore, Bateman et al. investigated compliance in iNPH and AD groups and found that iNPH patients had significantly lower compliance compared to healthy volunteers and that the AD group had a compliance that was midway between the healthy volunteers and the iNPH group [25], corroborating the presence of decreased compliance in iNPH patients and suggesting that further investigations are required for AD patients. Based on these reports, it seems likely that the increased CSF pressure gradient and rotation in the Sylvian aqueduct of iNPH patients are due to decreased compliance in the cerebrospinal cavity. This is presumably the case in AD patients who also exhibit increased CSF pressure gradients and rotation in the Sylvian aqueduct.

In the above section, we discussed the common pathological state between iNPH and AD based on the fluid dynamics analysis of CSF. Regarding the discrimination of iNPH and AD, there are some contradictory findings in brain tissue biopsy [26,27] and examinations using CSF biomarkers may not be able to differentiate between these groups [28]. This indicated that there are overlaps between iNPH and AD in addition to the pathological condition assessed based on the fluid dynamics analysis Fig. 5 Rotation mapping of the volunteer by 3D-PC at 4 stages of the cardiac cycle. In-plane rotations were visualized using vectors and throughplane rotations were visualized using colors. The color-coded CSF rotation field vector was then superimposed onto T 2 -weighted images of the stationary tissues. Red circle indicates Sylvian aqueduct of CSF we reported in the present study. Therefore, it can be concluded that there is not currently a suitable method to distinctly classify iNPH and AD.

A limitation of the present technique is that this quantitative measurements based on the PC technique can only measure fluid velocity with 10% accuracy [29], especially for small voxel size.

Conclusions

In the present study, we quantitatively analyzed timeresolved 3D-PC MRI data of the Sylvian aqueduct in iNPH and AD patients, and healthy elderly volunteers. The 3D-PC method is useful to understand CSF dynamics in AD, iNPH, and healthy elderly volunteers. As iNPH and AD patients show a different CSF motion profile from that of healthy elderly individuals, it suggests that there is a difference in compliance between the patient groups and healthy controls. Fig. 6 Box plots of rotation in the Sylvian aqueduct between AD patients, iNPH patients, and healthy elderly volunteers. Both the AD and iNPH groups had greater rotation compared to the volunteer group. AD: Alzheimer's disease, iNPH idiopathic normal pressure hydrocephalus outside values are indicated by a small "o"

Figure 1
1shows velocity images for a 67 year old male volunteer.

Figure 3
3shows pressure gradient images for a 67-yearold male volunteer.

Table 1
1Characteristics of the groupiNPH
AD
Control

Number of subjects
10
7
19

Mean age
73
77
74

Age range
67-87
66-89
67-80

Female
5
5
12

AcknowledgementsThe authors would like to thank the patients with AD referred from the Department of Neurology in Tokai University Hospital, as well as the volunteers that participated in the study.Competing interestsKagayaki Kuroda is an employ of the Bioview Inc. Other authors have no financial conflicts or any interest with any commercial product used in this study or any substantial relationship with an entity that may impact or benefit from the conclusions of this research.Availability of data and materialsThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.Consent for publicationAll of authors agree with publication of this manuscript.Ethics approval and consent to participateThis research was approved by our institution's Institutional Review Board for Clinical Research, Tokai University Hospital (http://irb.med.u-tokai.ac.jp/) IRB No. 13R-066 (Flow dynamic study of cerebrospinal fluid using MRI). All volunteers were examined after providing appropriate informed consent, consistent with the terms of approval from the internal review board of Tokai University Hospital, Isehara, Kanagawa, Japan. Written informed consent concerning diagnostic procedures and image analysis was obtained from all volunteers and patients.FundingThis study was supported in part by the Research and Study Project of Tokai University Educational System General Research Organization, and Health and Labor Sciences Research grants from the Japanese government for research on rare and intractable disease.Publisher's NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Guidelines for management of idiopathic normal pressure hydrocephalus: second edition. E Mori, M Ishikawa, T Kato, H Kazui, H Miyake, M Miyajima, M Nakajima, M Hashimoto, N Kuriyama, T Tokuda, Neurol Med Chir (Tokyo). 52Mori E, Ishikawa M, Kato T, Kazui H, Miyake H, Miyajima M, Nakajima M, Hashimoto M, Kuriyama N, Tokuda T, et al. Guidelines for management of idiopathic normal pressure hydrocephalus: second edition. Neurol Med Chir (Tokyo). 2012;52:775-809.

Diagnosis of idiopathic normal pressure hydrocephalus is supported by MRI-based scheme: a prospective cohort study. M Hashimoto, M Ishikawa, E Mori, N Kuwana, Cerebrospinal Fluid Res. 718Hashimoto M, Ishikawa M, Mori E, Kuwana N. Diagnosis of idiopathic normal pressure hydrocephalus is supported by MRI-based scheme: a prospective cohort study. Cerebrospinal Fluid Res. 2010;7:18.

Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA work group under the auspices of Department of Health and Human Services Task Force on Alzheimer's Disease. G Mckhann, D Drachman, M Folstein, R Katzman, D Price, E M Stadlan, Neurology. 34McKhann G, Drachman D, Folstein M, Katzman R, Price D, Stadlan EM. Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA work group under the auspices of Department of Health and Human Services Task Force on Alzheimer's Disease. Neurology. 1984;34:939-44.

Cerebrospinal fluid image segmentation using spatial fuzzy clustering method with improved evolutionary expectation maximization. A Abdullah, A Hirayama, S Yatsushiro, M Matsumae, K Kuroda, Conf Proc IEEE Eng Med Biol Soc. 2013Abdullah A, Hirayama A, Yatsushiro S, Matsumae M, Kuroda K. Cerebrospi- nal fluid image segmentation using spatial fuzzy clustering method with improved evolutionary expectation maximization. Conf Proc IEEE Eng Med Biol Soc. 2013;2013:3359-62.

R Feynman, R Leighton, M Sands, The Feynman lectures on physics. Addison Wesley Longman2Feynman R. Leighton R. Sands M: The Feynman lectures on physics. Addison Wesley Longman; 1970. p. 2.

MRI of CSF flow in normal pressure hydrocephalus. G Schroth, U Klose, Psychiatry Res. 29Schroth G, Klose U. MRI of CSF flow in normal pressure hydrocephalus. Psychiatry Res. 1989;29:289-90.

Changes in aqueductal CSF stroke volume and progression of symptoms in patients with unshunted idiopathic normal pressure hydrocephalus. A Scollato, R Tenenbaum, G Bahl, M Celerini, B Salani, Di Lorenzo, N , AJNR Am J Neuroradiol. 29Scollato A, Tenenbaum R, Bahl G, Celerini M, Salani B, Di Lorenzo N. Changes in aqueductal CSF stroke volume and progression of symptoms in patients with unshunted idiopathic normal pressure hydrocephalus. AJNR Am J Neuroradiol. 2008;29:192-7.

Relationship between cerebrospinal fluid flow, ventricles morphology, and DTI properties in internal capsules: differences between Alzheimer's disease and normal-pressure hydrocephalus. J Daouk, B Chaarani, J Zmudka, C Capel, A Fichten, R Bouzerar, Gondry-Jouet , C Jouanny, P Baledent, O , Acta Radiol. 55Daouk J, Chaarani B, Zmudka J, Capel C, Fichten A, Bouzerar R, Gondry- Jouet C, Jouanny P, Baledent O. Relationship between cerebrospinal fluid flow, ventricles morphology, and DTI properties in internal capsules: dif- ferences between Alzheimer's disease and normal-pressure hydrocepha- lus. Acta Radiol. 2014;55:992-9.

Noninvasive estimation of intracranial compliance in idiopathic NPH using MRI. M Mase, T Miyati, H Kasai, K Demura, T Osawa, M Hara, Y Shibamoto, K Yamada, Acta Neurochir Suppl. 102Mase M, Miyati T, Kasai H, Demura K, Osawa T, Hara M, Shibamoto Y, Yamada K. Noninvasive estimation of intracranial compliance in idi- opathic NPH using MRI. Acta Neurochir Suppl. 2008;102:115-8.

Noninvasive MRI assessment of intracranial compliance in idiopathic normal pressure hydrocephalus. T Miyati, M Mase, H Kasai, M Hara, K Yamada, Y Shibamoto, M Soellinger, C Baltes, R Luechinger, J Magn Reson Imaging. 26Miyati T, Mase M, Kasai H, Hara M, Yamada K, Shibamoto Y, Soellinger M, Baltes C, Luechinger R. Noninvasive MRI assessment of intracranial compliance in idiopathic normal pressure hydrocephalus. J Magn Reson Imaging. 2007;26:274-8.

Dynamic state of water molecular displacement of the brain during the cardiac cycle in idiopathic normal pressure hydrocephalus. H Kan, T Miyati, M Mase, T Osawa, N Ohno, H Kasai, N Arai, M Kawano, Y Shibamoto, Comput Med Imaging Graph. 40Kan H, Miyati T, Mase M, Osawa T, Ohno N, Kasai H, Arai N, Kawano M, Shi- bamoto Y. Dynamic state of water molecular displacement of the brain during the cardiac cycle in idiopathic normal pressure hydrocephalus. Comput Med Imaging Graph. 2015;40:88-93.

Chronic hydrocephalus in adults. R J Edwards, S M Dombrowski, M G Luciano, I K Pople, Brain Pathol. 14Edwards RJ, Dombrowski SM, Luciano MG, Pople IK. Chronic hydrocepha- lus in adults. Brain Pathol. 2004;14:325-36.

A model of pulsations in communicating hydrocephalus. M Egnor, L Zheng, A Rosiello, F Gutman, R Davis, Pediatr Neurosurg. 36Egnor M, Zheng L, Rosiello A, Gutman F, Davis R. A model of pulsations in communicating hydrocephalus. Pediatr Neurosurg. 2002;36:281-303.

A mathematical analysis of physiological and molecular mechanisms that modulate pressure gradients and facilitate ventricular expansion in hydrocephalus. K P Wilkie, G Nagra, M Johnston, Int J Numer Anal Model B. 316Wilkie KP, Nagra G, Johnston M. A mathematical analysis of physiologi- cal and molecular mechanisms that modulate pressure gradients and facilitate ventricular expansion in hydrocephalus. Int J Numer Anal Model B. 2012;316:65-81.

Can MR measurement of intracranial hydrodynamics and compliance differentiate which patient with idiopathic normal pressure hydrocephalus will improve following shunt insertion?. G A Bateman, A M Loiselle, Acta Neurochir (Wien). 149discussion 462Bateman GA, Loiselle AM. Can MR measurement of intracranial hydro- dynamics and compliance differentiate which patient with idiopathic normal pressure hydrocephalus will improve following shunt insertion? Acta Neurochir (Wien). 2007;149:455-62 (discussion 462).

Prevalence of Alzheimer's disease in patients investigated for presumed normal pressure hydrocephalus: a clinical and neuropathological study. S Savolainen, L Paljarvi, M Vapalahti, Acta Neurochir (Wien). 141Savolainen S, Paljarvi L, Vapalahti M. Prevalence of Alzheimer's disease in patients investigated for presumed normal pressure hydrocepha- lus: a clinical and neuropathological study. Acta Neurochir (Wien). 1999;141:849-53.

Alzheimer's disease comorbidity in normal pressure hydrocephalus: prevalence and shunt response. J Golomb, J Wisoff, D C Miller, I Boksay, A Kluger, H Weiner, J Salton, W Graves, J Neurol Neurosurg Psychiatry. 68Golomb J, Wisoff J, Miller DC, Boksay I, Kluger A, Weiner H, Salton J, Graves W. Alzheimer's disease comorbidity in normal pressure hydrocepha- lus: prevalence and shunt response. J Neurol Neurosurg Psychiatry. 2000;68:778-81.

Alzheimer CSF biomarkers may be misleading in normal-pressure hydrocephalus. N R Graff-Radford, Neurology. 83Graff-Radford NR. Alzheimer CSF biomarkers may be misleading in normal-pressure hydrocephalus. Neurology. 2014;83:1573-5.

Accuracy of phase-contrast flow measurements in the presence of partial-volume effects. C Tang, D D Blatter, D L Parker, J Magn Reson Imaging. 3Tang C, Blatter DD, Parker DL. Accuracy of phase-contrast flow measure- ments in the presence of partial-volume effects. J Magn Reson Imaging. 1993;3:377-85.