Introduction The predictive value of serum myeloma protein in solitary plasmacytoma
2020

Won Ick Chang
Department of Radiation Oncology
Seoul National University College of Medicine
SeoulKorea

Hyeon Kang Koh
Department of Radiation Oncology
Konkuk University Medical Center
SeoulKorea

Sung-Soo Yoon
Department of Internal Medicine
Seoul National University College of Medicine
SeoulKorea

Han-Soo Kim
Department of Orthopedic Surgery
Seoul National University College of Medicine
SeoulKorea

Keun-Yong Eom
Department of Radiation Oncology
Department of Radiation Oncology
Seoul National University Bundang Hospital
SeongnamKorea

Seoul National University College of Medicine
101 Daehak-ro, Jongno-gu03080SeoulKorea

IlHan Kim
Department of Radiation Oncology
Seoul National University College of Medicine
SeoulKorea

Cancer Research Institute
Seoul National University
SeoulKorea

Il Han Kim
Introduction The predictive value of serum myeloma protein in solitary plasmacytoma

Radiat Oncol J
382202010.3857/roj.2019.00570Received: October 28, 2019 Revised: April 29, 2020 Accepted: May 26, 2020Original Article pISSN Correspondence:PlasmacytomaMyeloma proteinRisk factorRadiotherapy
Solitary plasmacytoma is a rare disease which accounts for less than 10% of plasma cell neoplasm[1]. It is diagnosed at a median age of 60 and is a male-dominant disease with a male to female ratio of approximately 2:1 [2,3]. Solitary plasmacytoma is subdivided into two disease entities. Solitary plasmacytoma originating from bones are classified as solitary plasmacytoma of the bone (SPB), whereas soft tissue origin plasmacytoma is classified as soli-Purpose: To identify the clinical usefulness of serum M protein and to establish a rationale for regular follow-up with serum protein electrophoresis in solitary plasmacytoma. Materials and Methods: Sixty-nine patients with solitary plasmacytoma and solitary plasmacytoma with minimal marrow involvement according to the International Myeloma Working Group criteria were retrospectively reviewed. Results: At a median follow-up of 6.2 years, 5-year local control (LC), 5-year multiple myeloma-free survival (MMFS), 5-year failure-free survival (FFS), and 5-year overall survival (OS) were 82.6%, 44.1%, 41.8%, and 85.1%, respectively. Among the patients whose initial serum M protein was present or not evaluated, 37.3% of patients showed disappearance of serum M protein after various treatment. MMFS of these patients were comparable to non-secretory plasmacytoma with undetectable levels of M protein, and significantly better than patients with persistent M protein. Increase of serum M protein ≥0.1 g/dL was most predictive of treatment failure with area under the curve of 0.731. Conclusion: Patients who eventually showed persistence of serum M protein after treatment showed worse MMFS and FFS compared to those whose serum M protein disappeared or who had initially non-secretory disease. The increase of serum M protein level ≥0.1 g/dL from current nadir was predictive of treatment failure. Therefore, regular follow-up with serum M protein is highly recommended especially unless the patient had initially non-secretory disease.

ter local radiation therapy (RT) [4][5][6]. Although there is no consensus regarding the optimal dose of RT, but RT dose ≥ 40 Gy was reported to improve LC [4,7]. Nevertheless, more than half of the patients after local or systemic treatment eventually progress to MM with long-term follow-up [3,4,8,9].

Although there is a consensus criteria for treatment response assessment for multiple myeloma [10], there is no widely used criteria for solitary plasmacytoma. Also, the prediction of treatment failure in solitary plasmacytoma patients is not available although early predictions of treatment failure is important considering the high rates of progressions to MM in order to make early salvage treatment possible.

Myeloma protein, which is also called M protein, is an abnormal protein produced in excess by an abnormal monoclonal proliferation of plasma cells and is typically detected in serum or urine of patients with MM or plasma cell tumors. Although only 3% of MM patients are non-secretory [11], i.e., no detectable serum and urine M protein at diagnosis, M protein is present in only 33% to 64% of solitary plasmacytoma patients at diagnosis [4,5,12,13]. Also in patients with M protein, the median level of serum M protein at diagnosis of solitary plasmacytoma patients is less than 1 g/dL [5,13], which is lower than that of MM ( > 3 g/dL) [11]. Nevertheless, the measurements of serum M protein level for solitary plasmacytoma is important in that the persistence of serum M protein after treatment was associated with poor prognosis [12][13][14] and therefore, the European Expert Panel recommended serum and urine electrophoresis and immunofixation to be performed during follow-up [15].

However, the usefulness of serum M protein as a biomarker to assess treatment response and to predict treatment failures in solitary plasmacytoma is yet to be studied. In MM, randomized trials implemented the increase in serum M protein more than 0.5 g/dL from nadir to define disease progression [16,17]. Considering the high incidence of non-secretory plasmacytoma and low level of serum M protein level at diagnosis, this criterion seems not to be appropriate for solitary plasmacytoma. Therefore, we tried to identify the clinical usefulness of serum M protein for predicting treatment failures and to establish a rationale for regular follow-up with serum protein electrophoresis to evaluate serum M protein level.

Materials and Methods

Patients and diagnostic work-up

Medical records of patients with solitary plasmacytoma and solitary plasmacytoma with minimal marrow involvement according to the International Myeloma Working Group (IMWG) criteria [18] were retrospectively reviewed. A total of 69 solitary plasmacytoma patients who were taken care of between 1986 and 2019 at Seoul National University Hospital and Seoul National University Bundang Hospital were identified. At initial diagnostic work-up, all patients were evaluated with at least one radiological imaging workups (100.0%) including simple X-ray, CT, MRI, bone scan, or FDG-PET scan and laboratory work-ups including serum protein electrophoresis (62.3%), urine protein electrophoresis (55.1%), and serum free light chain ratio (31.9%).

The characteristics of all patients are shown in Table 1. The median age at the diagnosis of solitary plasmacytoma was 60.5 years (range, 29.7 to 79.8 years) and male was predominant (59.4%). The most common initial presenting symptom was pain at the involved site (59.4%). Vertebra was the most common primary site of the 51 SPB patients. Head and neck was the most common primary site of the 18 SEP patients. At initial diagnostic work-up, serum M protein, Bence Jones proteinuria, and abnormal free light chain ratio was present in 58.1%, 15.8%, and 50.0% of patients whose pre-treatment data was available, respectively. Patients treated with surgery alone or surgery plus adjuvant RT included more patients with insufficient initial work-up studies including serum and urine protein electrophoresis (p < 0.05) (Supplementary Table S1).

Treatment and follow-up

All patients received a curative treatment; definitive RT alone (46.4%), surgical resection (24.6%), and surgical resection plus adjuvant RT (17.4%). The median total radiation dose was 45.0 Gy (range, 23.4 to 70.0 Gy). Curative surgical resection was performed in 33 patients (47.8%) and 90.9% of them underwent complete resection. Chemotherapy was administered to 8 patients (11.6%). After the completion of treatment, patients were followed up with work-ups including radiological imaging studies, serum protein electrophoresis, and serum free light chain ratio. The median time from the initial serum protein electrophoresis at the time of diagnosis to first post-treatment serum protein electrophoresis was 3.5 months (range, 1.3 to 36.5 months). In this study, we defined the "disappearance of serum M protein" in order to assess treatment response. When post-treatment serum M protein of a patient whose initial serum M protein was present or not evaluated was measured and showed no detectable serum M protein during follow-up, it was regarded as disappearance of serum M protein.

Patients were defined as progression to MM, whenever meeting the criteria of IMWG for the diagnosis of MM during follow-up [18]. The diagnosis of MM requires clonal bone marrow plasma cells ≥ 10% or a biopsy-proven plasmacytoma plus the presence of one or more myeloma-defining events. Myeloma defining events include the presence of hypercalcemia, renal insufficiency, anemia, and bone lesion. Also, clonal bone marrow plasma cells ≥ 60%, sehttps://doi.org/10.3857/roj.2019.00570 rum free light chain ratio ≥ 100, or more than one focal lesion on MRI are diagnosed as MM regardless of the presence of myeloma defining event.

Statistical analysis

The characteristics according to various treatments were compared using Fisher's exact test. The actuarial LC, multiple myeloma-free survival (MMFS), failure-free survival (FFS), and overall survival (OS) were calculated with Kaplan-Meier analysis. All survivals were calculated from the first day of initial treatment. The events were local failure before progression to MM for LC, progression to MM or death from any cause for MMFS, any failure or death from any cause for FFS, and death from any cause for OS. Log-rank test was used for univariate analysis of the prognostic factors. Based on prognostic factors with p < 0.05, multivariate analysis was conducted. To identify a set of independent predictive factors a multivariate analysis with Cox proportional hazards model or Cox regression with Firth's penalized likelihood was performed as appropriate. In all analyses p < 0.05 was considered statistically significant. All statistical analyses were conducted in R version 3.5.0.

Results

Survival outcome and cause of death

At a median follow-up of 6.8 years (range, 0.1 to 29.3 years), 9 patients (13.0%) experienced a local failure; 8 within 3 years and one at 9 years after treatment (Fig. 1A). The 5-and 10-year LC rate were 82.6% and 68.9%. The LC rates did not differ significantly across treatment modalities. Anaplastic plasmacytoma was the only prognostic factor for LC ( Table 2, 3). In the meanwhile, RT improved LC for extramedullary lesions (100.0% vs. 53.6%; p = 0.028).

Overall, 32 patients (46.8%) progressed to MM. Most of the progressions to MM (96.9%) occurred within the first 5 years after treatment. The 5-and 10-year MMFS were 44.1% and 36.7%. In univariate analysis and multivariate analysis, SPB (hazard ratio [HR] = 8.63, p = 0.036), tumor size ≥ 5 cm (HR = 2.84, p = 0.012), and anaplastic histology (HR = 50.9, p = 0.002) were adverse prognostic factors for MMFS (Tables 2, 3). The 5-and 10-year FFS were 41.8% and 34.9%. As they were for MMFS, SPB (HR = 10.3, p = 0.023), tumor size ≥ 5 cm (HR = 2.18, p = 0.043), and anaplastic histology (HR = 66.6, p = 0.001) were significant adverse prognostic factors for FFS in the univariate and multivariate analysis (Tables 2, 3). The median OS was 14.0 years (95% confidence interval [CI], 11.8 years to upper limit not reached). The 5-and 10year OS were 85.1% and 70.6%. There was no significant prognostic factor for OS (Table 4). One patient with unknown dose excluded. At the time of the analysis, 22 deaths (31.9%) were reported, and 6 patients died from the progression of MM. There were two deaths related to treatments; one from complications after surgical resection and another from side effects of salvage chemotherapy. Five deaths were unrelated to plasmacytoma or treatment and the cause of death of remaining 9 patients was unknown.

Prognostic factors in patients treated with RT

In the subgroup of patients treated with any treatment that includes RT, tumor location, tumor size, and serum free light chain ratio were significant prognostic factors for MMFS and FFS in the univariate analysis. In the multivariate analysis, SPB (HR = 22.1, p < 0.001), tumor size ≥ 5 cm (HR = 2.64, p = 0.045), and abnormal serum free light chain ratio (HR = 6.29, p = 0.008) were associated with poor MMFS. For FFS, SPB (HR = 22.7, p < 0.001) and abnormal serum free light chain ratio (HR = 7.38, p = 0.003) were statistically significant adverse prognostic factors. However, there were no significant prognostic factors identified for LC and OS for patients treated with RT. The addition of surgical resection, chemotherapy, or RT dose ≥ 45 GyEQD2 did not result in significantly improved prognosis.     (Fig. 1B). The MMFS of non-secretory plasmacytoma patients and patients whose serum M protein disappeared were comparable (p > 0.05). Both groups of patients showed significantly superior 5-year MMFS compared to patients who showed persistence of serum M protein and patients whose serum M protein level was not evaluated during follow-up (p < 0.05).

Disappearance of serum M protein after treatment as a prognostic factor

Correlation of disappearance of serum M protein and RT

Among patients who initially had serum M protein, the post-treatment serum M protein level was available from 20 patients. Two patients who did not receive RT showed persistence of serum M protein after treatment, whereas disappearance of serum M protein was observed in 7 patients (38.9%) after RT (  (range, 0.02 to 2.1 g/dL). Considering the high incidence of non-secretory M protein and low level of serum M protein in solitary plasmacytoma at diagnosis, we evaluated the increase of serum M protein level ≥ 0.1 g/dL from current nadir instead of 0.5 g/dL, which is implemented in MM, to predict treatment failure. The area under the curve (AUC) of the prediction model with this criterion was 0.731 (Table 5, Fig. 1C). Specificity and sensitivity of this model were 84.2% and 61.9%, respectively. The median time to treatment failure from the day serum M protein level increased 0.1 g/dL or more from current nadir was 16.9 months (95% CI, 6.9 months to upper limit not reached).

The increase of serum M protein level as a predictive marker for treatment failure

In addition, three other criteria were evaluated, which are two increases of serum M protein level, two consecutive increases of serum M protein level, and increase of serum M protein above double the value of nadir. The AUC of the prediction models with these criteria were 0.690, 0.662, and 0.614, respectively ( Table 5). As the increase of serum M protein level ≥ 0.1 g/dL from current nadir showed highest AUC amongst the four criteria compared, we adopted this criterion to predict treatment failure.

Discussion and Conclusion

In this study, we evaluated the prognostic value of serum M protein level at diagnosis and its level change or conversion during follow-up to assess treatment response and predict treatment failures of solitary plasmacytoma. Prognostic factors with contradictory prognostic values for solitary plasmacytoma were demonstrated in previous studies due to different treatment profiles and small number of patients included in each study. Although local control was excellent in this study and was also consistent with that of previous studies, progression to MM remained to be the main obstacle for failure-free survival as observed in Fig. 1A. In this study, SPB and tumor size ≥ 5 cm were associated with poor MMFS and FFS, which is consistent with the results of previous studies [3,5,8,9,19,20]. Older age [3,5,[19][20][21], the abnormal post-treatment free light chain ratio [12], the pres- ence of serum M protein at diagnosis [5] and after treatment [12] were also suggested to be associated with poor prognosis. Warsame et al. [22] reported clonal plasma cells in BM, presence of urine Bence Jones protein, and higher RT dose as adverse prognostic factors for progression-free survival.

In addition to SPB and tumor size ≥ 5 cm, anaplastic plasmacytoma was associated with poor LC, MMFS, and FFS. Anaplastic plasmacytoma is an extremely rare type of plasmacytoma which can develop in patients with immunosuppression and combined Epstein-Barr virus infection [23]. However, because of its rarity the clinical course of anaplastic plasmacytoma is unknown. In this study, two patients of anaplastic plasmacytoma were included and they showed rapid local failure and progression to MM. However, both patients survived more than 10 years. It seems that anaplastic plasmacytoma shows rapid progression but does not result in poor overall survival, but the clinical course of anaplastic plasmacytoma needs to be investigated with larger number of patients.

In this study, patients with non-secretory plasmacytoma and patients whose serum M protein disappeared after treatment showed superior MMFS compared to patients who had persistent serum M protein after treatment. However, the prognostic value of serum M protein level at diagnosis is controversial. Non-secretory plasmacytoma showed better MMFS in the study by Reed et al. [5], whereas the opposite result showing that non-secretory plasmacytoma is associated with worse MMFS and cause-specific survival was also reported [13,14]. In contrast, the persistence of M protein after treatment is uniformly reported to be associated with poor MMFS [12][13][14], which was also observed in this current study. Moreover, we showed the usefulness of the increase of serum M protein level ≥ 0.1 g/dL from current nadir for predicting treatment failures. Therefore, it would be reasonable to follow-up patients, who initially had serum M protein or whose pre-treatment serum M protein level was not evaluated, with a regular serum protein electrophoresis test along with radiologic examinations until the serum M protein disappears. In addition, even if patients had non-secretory disease or their serum M protein disappeared, a regular follow-up with serum protein electrophoresis would be needed in order to make earlier detection and salvage treatment possible.

Immunofixation and free light chain ratio are another tests to evaluate serum M protein. It has been reported that 9.7% of normal serum protein electrophoresis showed positive immunofixation result [24] and therefore, immunofixation could be a good complementary test when combined with serum protein electrophoresis during follow-up. Abnormal serum free light chain ratio was a significant prognostic factor in patients who were treated with RT in our study and in a previous study [12]. Considering this, serum free light chain ratio could be another potential predictive biomarker to be investigated and utilized.

RT has been known as the treatment of choice for solitary plasmacytoma [15,25,26] with evidences from retrospective studies showing improved LC [3], disease-free survival [3], and OS [2] with RT. Regarding the dose of RT, higher doses were recommended for SEP [27] and larger tumors [19], although no definite dose-response relationship for RT > 30 Gy was observed in another study [3]. In this study, we also could not find a definite dose-response relationship. However, RT ≥ 45 GyEQD2 seemed to increase the rates of disappearance of serum M protein, which can be interpreted as good treatment response, although the number of patients were insufficient to show definite relationship.

In this study, since solitary plasmacytoma without evidence of MM is very rare in nature, we could find only a small number of patients. Patients who were incidentally diagnosed as solitary plasmacytoma after surgical resection lacked the pre-treatment workup for solitary plasmacytoma as shown in Supplementary Table S2. Therefore, our definition of disappearance of serum M protein included patients whose initial serum M protein level was not evaluated when predicting the prognosis with respect to serum M protein level. However, considering that it is often the case that solitary plasmacytoma is incidentally diagnosed after surgery, as it was in this study, it would be still meaningful to include patients whose initial serum M protein was not evaluated and predict their prognosis. Also, the data of proportion of clonal plasma cells in the bone marrow was not available in 46.4% of the patients and therefore, we could not show any prognostic difference between solitary plasmacytoma and solitary plasmacytoma with minimal marrow involvement according to the IMWG criteria. However, with long-term follow-up data, we could derive a meaningful conclusion regarding the clinical value of serum M protein level, although further studies with larger number of patients are needed for validation.

In conclusion, we found that patients who eventually showed persistent serum M protein after treatment had worse prognosis compared to those whose serum M protein disappeared or initially had non-secretory disease. Also, the increase of serum M protein level ≥ 0.1 g/dL from current nadir was predictive of treatment failure. Therefore, we recommend a regular follow-up with serum protein electrophoresis after the treatment of solitary plasmacytoma to assess treatment response and predict treatment failure. Also, closer follow-up with serum protein electrophoresis is needed for patients who initially had serum M protein or whose serum M protein level was not evaluated.

Conflict of Interest

No potential conflict of interest relevant to this article was reported.

Supplementary Materials

Supplementary materials can be found via https://doi.org/10.3857/ roj.2019.00570 Table S1. Risk factors associated with treatment outcome of solitary plasmacytoma treated with RT (univariate analysis) Table S2. Characteristic of the patients according to their treatments

Fig. 1 .
1(A) Treatment outcomes of solitary plasmacytoma after various treatments. (B) Multiple myeloma-free survival of solitary plasmacytoma after various treatment with respect to serum M protein. (C) Receiver operating characteristic (ROC) curve and area under the curve (AUC) of increase of serum M protein level ≥0.1 g/dL from current nadir to predict treatment failure. LC, local control; MMFS, multiple myeloma-free survival; FFS, failure-free survival; OS, overall survival. *p < 0.05.

At the time of diagnosis, 18 patients were non-secretory. Among the remaining 51 patients who had serum M protein or whose pre-treatment serum M protein level was not evaluated, serum M protein disappeared in 19 patients after a median period of 2.4 months (range, 0.0 to 51.1 months) following treatment. The 5-year MMFS of patients with non-secretory plasmacytoma, disap-

Next, we tried to predict treatment failures with the level change of serum M protein. Overall, 40 patients were followed up with at least two serum protein electrophoresis tests during follow-up before any treatment failure, defined as local failure and progression to MM. The median value of post-treatment nadir value of serum M protein was 0.0 g/dL (range, 0.0 to 3.2 g/dL). During follow-up, 17 patients experienced an increase of serum M protein level at least once before clinically detected treatment failure. The median of maximum increase of serum M protein from nadir was 0.7

Table 4 .
4Correlation of disappearance of serum M protein and radiaradiation therapy; EQD2, equivalent dose in 2-Gy fractions at α/β of 10. a) One patient with unknown dose excluded. https://doi.org/10.3857/roj.2019.00570

Table 1 .
1Patient characteristics of solitary plasmacytoma SPB, solitary plasmacytoma of bone; SEP, solitary extramedullary plasmacytoma; RT, radiation therapy; EQD2, equivalent dose in 2-Gy fractions at α/β of 10.One patient received additional radiosurgery.Characteristic
Number of patients (%)
Age (yr)
< 60
32 (46.4)
≥ 60
37 (53.6)
Sex
Male
41 (59.4)
Female
28 (40.6)
Site of lesion
SPB
51 (73.9)
Craniofacial bone
11 (15.9)
Vertebra
21 (30.4)
Pelvic bone
9 (23.1)
Extremity
4 (5.8)
Others
6 (8.7)
SEP
18 (26.1)
Head and neck
14 (20.3)
Others
4 (5.8)
Anaplastic histology
No
67 (97.1)
Yes
2 (2.9)
Serum M protein at diagnosis (g/dL)
0
18 (26.1)
0.1-1.0
11 (15.9)
≥ 1.1
14 (20.3)
Unknown
26 (37.7)
Type of M protein
IgG
18 (72.0)
Light chain only
2 (8.0)
Unknown
5 (20.0)
Bence Jones proteinuria
Absent
32 (46.4)
Present
6 (8.7)
Unknown
31 (44.9)
Serum free light chain ratio
Normal
11 (15.9)
Abnormal
11 (15.9)
Unknown
47 (68.1)
Treatment
RT only
32 (46.4)
Surgery only
17 (24.6)
Surgery + RT
12 (17.4)
Chemotherapy alone
3 (4.3)
RT + chemotherapy
2 (2.9)
Surgery + chemotherapy
1 (1.4)
Surgery + RT + chemotherapy a)
2 (2.9)
Radiation therapy b)
No radiation therapy
22 (31.9)
< 45 GyEQD2
31 (44.9)
≥ 45 GyEQD2
15 (21.7)
Surgical resection
No surgical resection
36 (52.2)
Partial resection
3 (7.7)
Complete resection
30 (43.5)
a)

b)

Table 2 .
2Risk factors associated with treatment outcome of solitary plasmacytoma (univariate analysis) LC, local control; MMFS, multiple myeloma-free survival; FFS, failure-free survival; OS, overall survival; RT, radiation therapy.Number of
patients
5-yr LC (%) p-value 5-yr MMFS (%) p-value 5-yr FFS (%) p-value 5-yr OS (%) p-value

Age (yr)
< 60
32
68.7
0.150
40.8
0.680
37.3
0.550
89.5
0.320
≥ 60
37
94.3
47.4
45.9
81.5
Sex
Male
41
72.5
0.130
37.4
0.220
34.0
0.098
85.8
0.190
Female
28
94.4
53.4
53.4
84.2
Tumor location
Extramedullary
18
80.2
0.820
80.2
0.007
80.2
0.004
94.4
0.120
Bone
51
82.9
32.1
29.4
81.2
Tumor size (cm)
< 5
33
83.8
0.710
70.2
< 0.001
63.7
0.001
93.2
0.076
≥ 5
30
87.1
22.6
22.8
74.9
Anaplastic histology
No
67
85.4
< 0.001
45.5
0.001
43.1
< 0.001
84.6
0.260
Yes
2
0.0
0.0
0.0
100.0
Serum M protein present
No
18
90
0.470
55.7
0.220
55.7
0.130
87.1
0.650
Yes
25
85.2
27.3
29.2
79.8
Unknown
26
77.0
47.8
43.1
88.1
Bence Jones proteinuria
Absent
32
88.0
0.160
37.9
0.820
39.2
0.790
85.2
0.890
Present
6
100.0
50.0
50.0
100.0
Unknown
31
74.1
49.1
43.2
82.9
Serum free light chain ratio
Normal
11
100.0
0.320
67.5
0.046
67.5
0.019
90
0.910
Abnormal
11
78.8
21.8
18.2
75.0
Unknown
47
79.6
45.2
43.0
86.3
Treatment
RT alone
32
79.8
0.340
31.3
0.560
31.4
0.530
83.6
0.260
Surgery alone
17
72.9
55.8
49.0
94.1
Surgery + RT
12
100
50.9
50.9
75.0

Table 3 .
3Risk factors associated with treatment outcome of solitary plasmacytoma (multivariate analysis)LC
MMFS
FFS
HR (95% CI)
p-value
HR (95% CI)
p-value
HR (95% CI)
p-value
Tumor location
Extramedullary
-
Ref.
0.036
Ref.
0.023
Bone
-
8.63 (1.15-64.9)
10.28 (1.37-76.9)
Size of lesion (cm)
< 5
-
Ref.
0.012
Ref.
0.043
≥ 5
-
2.84 (1.26-6.42)
2.18 (1.03-4.65)
Anaplastic histology
No
Ref.
< 0.001
Ref.
0.002
Ref.
0.001
Yes
50.6 (7.01-366)
50.9 (4.14-626)
66.6 (5.39-822)

LC, local control; MMFS, multiple myeloma-free survival; FFS, failure-free survival; HR, hazard ratio; CI, confidence interval.

Table 5 .
5Correlation of various increases of serum M proteins with treatment failureNumber of patients (%) Sensitivity (%) Specificity (%) AUC Median time to treatment failure (mo)
Increase ≥ 0.1 g/dL from current nadir
16 (40.0)
61.9
84.2
0.731
16.9
Two increases
13 (37.1)
55.6
82.4
0.690
20.8
Two consecutive increases
12 (34.3)
50.0
82.4
0.662
17.4
Increase double above nadir
9 (22.5)
33.3
89.5
0.614
4.0

AUC, area under the curve.

Table 3 )
3. The rates of
disappearance of serum M protein were 25.0% and 66.7% with RT
dose < 45 GyEQD2 and ≥ 45 GyEQD2, respectively.

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Noninvasive, Transient and Selective Blood-Brain Barrier Opening in Non-Human Primates In Vivo
2011

F Marquet
Y-S Tung
T Teichert
V P Ferrera
E E Konofagou
Noninvasive, Transient and Selective Blood-Brain Barrier Opening in Non-Human Primates In Vivo

PLoS ONE
67225982011Received March 4, 2011; Accepted June 30, 2011;Editor: Martin W. Brechbiel, National Institute of Health, United States of America Funding: This study was supported in part by the National Institutes of Health (NIH) R01EB009041 (E.E.K.) and National Science Foundation (NSF) CAREER 0644713 (E.E.K.), NIH R01MH059244 (V.P.F.) and the Kavli Foundation (E.E.K. and V.P.F.). Additional support came from DFG 819/1-1 to T.T. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. *
The blood-brain barrier (BBB) is a specialized vascular system that impedes entry of all large and the vast majority of small molecules including the most potent central nervous system (CNS) disease therapeutic agents from entering from the lumen into the brain parenchyma. Microbubble-enhanced, focused ultrasound (ME-FUS) has been previously shown to disrupt noninvasively, selectively, and transiently the BBB in small animals in vivo. For the first time, the feasibility of transcranial ME-FUS BBB opening in non-human primates is demonstrated with subsequent BBB recovery. Sonications were combined with two different types of microbubbles (customized 4-5 mm and DefinityH). 3T MRI was used to confirm the BBB disruption and to assess brain damage.

Introduction

The main limiting factor towards the development of novel treatments of neurological and neurodegenerative diseases is the blood-brain barrier (BBB): more than 98% of small-molecule drugs and nearly all large-molecule drugs do not cross this anatomic barrier [1,2]. Several techniques exist to circumvent the BBB, such as intracranial injections, mixing or attaching agents to BBB-modifying chemicals, and the chemical alteration of agents to be delivered through endogenous transport systems [2,3]. However, these techniques are either invasive, drug-specific or are plagued by very poor spatial specificity. Even the latest advances in brain gene therapy [4] provide cell specific drug delivery but are not region specific. Global breaching of the BBB can be a risky process, as it increases influx of all molecules and therapeutic agents in untargeted areas of the brain [5] even if this approach has been proven to be successful for some applications such as metastatic lung cancer [6]. An ideal method would ensure drug-independent, reversible, localized and noninvasive delivery through the BBB to minimize potential hazards. Previously, our group has shown, along with others, that microbubble-enhanced, focused ultrasound (ME-FUS) is capable of disrupting the BBB noninvasively, transiently and selectively in small animals [7,8,9] typically at frequencies above 1 MHz. Although the exact mechanism of BBB opening remains to be determined, the interaction of the ultrasound beam with microbubbles results in mechanical perturbation of the microvasculature and thereby changes in the integrity of the BBB components. Previous studies suggest that both paracellular and transcellular barriers are affected during and after ME-FUS exposure [10].

Until now, however, the method has mainly been confined to research laboratories without specific prospects for clinical translation [1] because feasibility in large mammals has not been shown. Demonstration in large animals can open new avenues in targeted delivery of BBB-impermeable therapeutic agents (e.g., nerve growth factor, gene therapy), which have been shown effective in the treatment of neurodegenerative diseases such as Alzheimer's and Parkinson's [11,12,13] and have been shown feasible in small animals [14], especially since early detection has been shown lately to be feasible using PET imaging [15], warranting focal and noninvasive treatment methodologies. This technique could also prove useful for basic neuroscience research, replacing invasive techniques such as intracranial microinjections.

The BBB is a complex regulatory system within the neurovascular unit, which controls the flow of nutrients and chemicals into and out of the brain parenchyma maintaining the brain homeostasis necessary for proper neuronal firing [16]. The BBB hinders the effective systemic delivery of neurological agents and biomarkers to the brain through a combination of passive, transport and metabolic barriers. Determining factors for the passage of molecules across the BBB are lipid solubility, charge and molecular size (threshold range spans between 50 Da and 400 Da) [17]. Therefore, potential therapeutic agents, such as inhibitors to enzymes (100-1,000 Da) and proteins (30-3,000 kDa), do not efficiently cross the BBB when administered systemically. Such delivery and efficacy are critical in inducing therapeutic effects and triggering biological pathways.

Applying ME-FUS BBB opening in large animals is very challenging as focusing inside the brain is impeded by the presence of the skull along the beam path. The big difference between the high speed of sound through the skull and the low speed through the underlying brain tissue, combined with a severe attenuation of ultrasound waves through the skull bone, strongly distorts the beam shape especially at higher frequencies [18]. High intensity focused ultrasound (HIFU), a promising technique used in noninvasive tumor ablation, has led the way of transcranial focusing since the early 1950s [19]. The interest of HIFU on CNS diseases treatment was initiated in the 1950s [20], but for both of these applications total craniotomy was performed to circumvent the skull attenuation and aberration effects. The introduction in the 1990s of piezoceramic and piezocomposite transducers capable of being driven at high voltages and the improvement of multichannel electronics [19] have allowed to overcome these effects and provide accurate focusing through the skull in the late 1990s [21,22,23]. Transcranial HIFU research has recently paved the way for the development of complex, high cost and efficient methods requiring prior knowledge of the skull topology to perform accurate focusing [24,25] with transcranial feasibility shown for ablation shown in humans [26,27,28]. While HIFU therapy use continuous wave (CW) and relies on thermal effects in order to induce a thermal necrosis, ME-FUS BBB opening uses short pulsed-wave (PW) and relies mostly on mechanical effects such as cavitation (be it stable or inertial) [29,30].

An alternative to correcting for the aberrations induced by the skull is to operate at lower frequencies, but the focus can become very wide due to diffraction effects, thus decreasing the spatial resolution. Sonothrombolysis studies use transcranial CW ultrasound to dissolve clots in the brain, at typically lower frequencies (around 200 kHz), which are less prone to phase aberrations and absorption but may enhance cavitational effects [31,32,33]. The beam is generally loosely focused to cover a large volume of the brain in each application. However, one of these studies showed large, secondary hemorrhage [34], which has been hypothesized to be linked to unexpected enhanced cavitation effects caused by standing waves generated within the skull [35,36]. Standing waves are known to be capable of trapping microbubbles in the antinodes and decreasing their inertial cavitation threshold [36,37]. ME-FUS BBB opening also relies on mechanical effects to transiently and locally increase the trans-BBB permeability but uses PW sequences with very short duty cycles (from 0.1% to 2%). Therefore, the safety should be easier to ensure despite the use of low frequencies (around 200 kHz).

Our group has thus selected a middle solution to the aforementioned tradeoff, i.e., operate at intermediate frequencies (500 kHz) that allows transcranial propagation and sufficiently high spatial resolution with a single-element transducer, warranting a sufficiently wide safety window. Until now, feasibility with this system has been shown in simulations and in vitro [38,39].

Results and Discussion

In this study, transcranial ME-FUS is shown for the first time to induce BBB disruption in non-human primates (NHP). A total of four locations were disrupted in two animals (see Methods). Two neighboring regions in the visual cortex (V3), the caudate and the hippocampus were targeted. Pressures ranging from 0.3 MPa to 0.6 MPa were investigated. Previous studies have shown that a pressure increase results in a larger BBB opening extent and higher BBB permeability [41] while a safety window exists within the pressure range of 0.30 MPa (threshold of opening) and 0.60 MPa [42]. For all experiments, T1-weighted MRI at 3.0T was used to confirm the BBB disruption, tracking the diffusion of intravenous (IV) injected gadodiamide in the brain. Figure 1 depicts the results obtained for the visual cortex targets. The spatial selectivity of ME-FUS was hereby investigated by inducing BBB disruption in two neighboring, distinct, small sites in the visual cortex region at two different ultrasonic pressures (0.3 MPa and 0.45 MPa). The contrast agent cannot penetrate the BBB, therefore the deposition of the gadodiamide in the parenchyma confirmed local BBB disruption by ME-FUS (  Fig. 1D). The peak MR intensity enhancement at the BBB-opened region relative to the average value in the parenchyma was increased by 119% and 48% at 0.3 MPa and 0.45 MPa, respectively. The volume of the BBB disruption was equal to 24.6 mm 3 and 30.5 mm 3 , respectively. The two distinct opened sites were separated by 4.74 mm. A higher density of microbubbles at the ME-FUS focus for the 0.3 MPa site may have been caused by the proximity to a larger vessel, explaining the higher MRI contrast enhancement. The location of the induced BBB disruption areas were shifted from the expected location of respectively 0.8 mm and 0.7 mm laterally and 8.1 mm and 7.9 mm axially towards the transducer. The same MRI sequence and IV contrast agent injection were repeated six days after BBB opening (Fig. 1B). No intensity enhancement was observed indicating that the BBB was closed or reinstated. Two other MRI sequences (T2-weighted and susceptibility-weighted) were used to assess potential brain damage after ME-FUS and both of them indicated absence of detectable damage such as edema or hemorrhage (Fig. 2).

The same protocol was repeated for the two following sessions applying 0.6 MPa and two different kinds of microbubbles. The results are shown in Fig. 3. T1-weighted MR sequences were used to track the diffusion of gadodiamide. Using both of these microbubbles we obtained larger BBB disruption areas (Fig. 3A,B,D,E). This is mainly because by increasing the peak pressure, a larger portion of the brain reaches the disruption threshold. The peak MR intensity enhancement at the BBBopened region relative to the average value in the parenchyma was increased by 68% and 41% using customized and DefinityH microbubbles, respectively. The volume of the BBB disruption was  equal to 285.5 mm 3 and 116.3 mm 3 , respectively. The BBB opening regions at the caudate and the hippocampus were shifted from the targeted location by respectively 0.6 mm and 0.9 mm laterally and 6.5 mm and 7.2 mm axially. T2-weighted MR sequences were also used to assess potential damages in the brain (Fig. 3C,F). An edematous region was detected using custom made microbubbles while no damage was detected using DefinityH. All the animals have been survived and therefore histological findings are not available at this time. Even though no in-depth cognitive tests have been performed thus far, qualitative assessment of the animal basic behavior has been monitored. Normal cognitive behavior has been noted following ME-FUS procedures at moderate pressures and using DefinityH. In the case of 0.6 MPa and customized microbubbles, the animal with the edema exhibited a weakness in the contra-lateral arm over four days after treatment, most likely due to the induced edema, but then fully recovered after that four-day period.

Passive cavitation detector (PCD) recordings were performed during all experiments and are depicted in Fig. 4. Spectrograms depicted the frequency content of the bubble response during ME-FUS application and helped classify the cavitation behavior. Using moderate pressures (Fig. 4A,B), the PCD recordings showed the nonlinear modes due to the bubble oscillations induced by the acoustic excitation (stable cavitation). Very little broadband response was detected. This noise is induced by bubble collapse and jet, more generally described as inertial cavitation. In those two cases, the cavitation behavior was mainly dominated by stable cavitation. While increasing the pressure to 0.6 MPa, a large broadband signal was recorded for both customized (Fig. 4C) and DefinityH (Fig. 4D) microbubbles. Previous work from our group has shown that inertial cavitation is one of the main causes of induced damage during treatment [41]. We believe that the discrepancy observed in the last two experiments is mainly due to the bubble size dependence. A recent study [43] showed that contrary to current belief, liquid jets are directed away from the nearest vessel wall. Therefore, using smaller microbubbles, those jets might not actually be able to puncture and damage the vessels while using larger microbubbles trapped between vessel walls, jets are more likely to induce damage. Customized and DefinityH microbubbles do not only differ in size. The gas is also different (perfluorobutane for customized and perfluoropropane for Defi-nityH) but since the solubility and diffusivity of those two gases are similar, it is not expected to significantly affect the bubble oscillation during ME-FUS exposure. The different carbon chains (DSPC and DPPC, respectively), however, may change the shell property. The effect of shell property is part of ongoing work in our lab in order to assess the role of microbubbles in BBB opening.

Achieving drug-independent, localized, reversible and noninvasive BBB disruption in non-human primates can pave the way towards novel brain drug delivery and gene therapy techniques. The volume of the BBB opening was shown to be small enough using moderate pressures (24.6 mm 3 and 30.5 mm 3 ) to ensure potential therapeutic agents in untargeted regions. This technique was also proven to be highly selective with two distinct BBB opening sites in the visual cortex separated by a distance on the order of a few millimeters. Increasing the pressure resulted in larger opening regions (285.5 mm 3 and 116.3 mm 3 ). Those preliminary results have also enlightened the brain site and microbubbles type dependences. The targeting quality was also assessed. These results were found to be in good agreement with previous in silico and in vitro findings [38,39]. These previous studies showed that as long as we choose a good targeting vector (with an incidence angle close to normal), the distortion and lateral shift are very low, the attenuation and axial shift are also reproducible in vitro (with less than 1 dB and 1 mm of standard deviation respectively) at the same angle of incidence. The initial in vivo results suggest that the shift induced by the skull interface may also be reproducible in vivo under the same angle of incidence condition. Other targeting vectors might require a more complex way of determining the position of the focus and the global attenuation (e.g., simulations based on prior 3D CT skull scans). Ongoing work will be achieved in order to assess those dependences in a statistically significant way. At this point, no quantification of the contrast agent diffusion was performed. In future work, a permeability quantification technique reported in mice by our group [44] will be applied as part of the primate study.

In conclusion, initial feasibility of noninvasive, highly selective, drug-independent and reversible BBB opening was demonstrated in non-human primates in vivo for the first time. High spatial selectivity of this technique was also shown. This study is a major step toward clinical translation of this emerging technology that can be combined with any type of pharmacological treatment to the brain. Ongoing investigations entail optimization of the procedure including safety and efficacy of the trans-BBB drug delivery.

Materials and Methods

Initial feasibility studies were performed on two male rhesus macaques over the course of three sessions. In the first two sessions, the monodispersed 4-5 mm microbubbles were manufactured in-house and size-isolated using differential centrifugation [40] while in the last session DefinityH microbubbles were used (Lantheus Medical Imaging, MA, polydispersed, mean diameter 1.1-3.3 mm, 98% below 10 mm, maximum diameter 20 mm). In the first session, a region of the visual cortex (ventral V3), i.e., the medio-ventral wall of the occipital cortex, was targeted. In the second animal (second session), the caudate was targeted and the pressure was increased to 0.6 MPa to investigate potential damage induced using customized microbubbles. The third and final session was performed in the first animal targeted the hippocampus using 0.6 MPa and DefinityH microbubbles.

A 500-kHz center frequency focused ultrasound transducer was used for this experiment (Riverside Research Institute, NY, USA). The acoustic parameters used for the three protocols were the following: focal maximum estimated pressure of 0.3 MPa and 0.45 MPa, pulse length of 10 ms, pulse repetition frequency of 2 Hz, total sonication duration of 2 min. The single-element transducer was mounted on a standard monkey stereotactic frame for accurate positioning (Fig. 5). In vitro pressure measurements were realized in another study [39]. This study determined the global attenuation (absorption, reflexion and scattering) due to the presence of the skull (around -5.7 dB at 500 kHz). The attenuation in the skin [45] was assumed to be around 20.9 dB.cm 21 and its thickness was estimated to be equal to 0.5 cm. The attenuation in the monkey brain tissue [45] was assumed to be around 20.5 dB.cm 21 and the thickness of this layer was estimated to be equal to 2 cm. Therefore, the emission amplitude has to be raised by 7.15 dB (approximately a factor 2.28) compared to the calibration measurements in water to compensate for the energy loss along the path.

The first animal had previously participated in several electrophysiological and fMRI experiments. It had a surgically implanted head post to restrain head movements. The head post was embedded within a dental-acrylic implant which was held in place by ceramic screws. The screws penetrated the skull plate but did not protrude more than a millimeter into the skull-cavity. A previously implanted scleral search coil had been removed prior to the experiments reported here. During the electrophysiological experiments, single unit activity was recorded from the frontal eye fields of both hemispheres. No recording from the animal's brain targeted regions had been previously performed. The dentalacrylic implant covered a large portion of the skull. However, the occipital pole of the skull was not covered and enabled a sufficient acoustic window to the visual cortex without interference from the implant.

For the application of the FUS, all animals were anesthetized with 2% isoflurane (carrier gas: oxygen). The heart rate was held at approximately 120 beats per minute and the respiratory rate at around 60 breaths per minute. Prior to sonication, the scalp hair was removed with a depilatory cream to ensure maximal acoustic transmission. The animal's head was then placed in a stereotactic frame to enable careful targeting of the ultrasound. The sonication was performed immediately after intravenous (IV) injection of 500 mL microbubbles for all experiments (5610 9 numbers/mL for customized microbubbles and 1.2610 10 numbers/mL for Defini-tyH). Targeting was ensured using a manipulator and a positioning rod indicating the position of the focus relatively to the stereotactic coordinates (Fig. 6). Targeted regions of visual cortex (V3) were determined using a monkey brain atlas.

MRI was used to confirm BBB opening using gadiodiamide contrast agent. 3D Spoiled Gradient-Echo (SPGR) T1-weighted sequences (TR/TE = 20/1.4 ms; flip angle: 30u; NEX = 2; spatial resolution: 5006500 mm 2 ; slice thickness: 1 mm with no interslice gap) were applied after intravenous (IV) injection of gadodiamide (OmniscanH, molecular weight 573.66 Da, GE Healthcare, Princeton, NJ, USA) 1 h after sonication. The dose applied was 0.2 mL/kg and the IV injection was performed 2 minutes before the SPGR T1-weigthed scan (scan duration: 18 minutes). Gadodiamide presence in the brain parenchyma was induced by BBB opening. 3D T2-weighted sequence (TR/TE = 3000/80; flip angle: 90u; NEX = 3; spatial resolution: 4006400 mm 2 ; slice thickness: 2 mm with no interslice gap) and 3D Susceptibility-Weighted Image (SWI) sequence were applied (TR/TE = 19/ 27 ms; flip angle: 15u; NEX = 1; spatial resolution: 4006400 mm 2 ; slice thickness: 1 mm with no interslice gap) and were used to assess brain damage.

A single-element PCD (center frequency: 7.5 MHz, focal length: 60 mm, Olympus NDT, Waltham, MA, USA) was positioned through the center hole of the FUS transducer. The two transducers were aligned so that their focal regions fully overlapped within the confocal volume. The PCD transducer, which was connected to a digitizer (Gage Applied Technologies, Inc., Lachine, QC, Canada) through a 20-dB amplification (5800, Olympus NDT, Waltham, MA, USA), was used to passively acquire acoustic emissions from microbubbles. A time-frequency map of the acoustic emission was generated using a customized spectrogram function (8-cycles, i.e., 16 ms, Chebyshev window; 98% overlap; 4096-point FFT) in MATLABH (2010a, Mathworks, Natick, MA). The spectrogram can then clearly indicate how the frequency content of the signal changes over time. Therefore, the presence of broadband response can help classify cavitation behavior.

Ethical statement

All animal studies were approved by the Institutional Animal Care and Use Committee at Columbia University and the New York State Psychatric Institute (protocol ID: CU1066, NYSPI279). All animals, housed and handled in strict accordance with good animal practice under supervision of veterinarians, received environmental enrichment and were monitored for evidence of disease and changes in attitude, appetite, or behavior suggestive of illness. Every effort was made to alleviate animal discomfort and pain by appropriate and routine use of anesthetic and/or analgesic agents.

Fig. 1A,C,D). The MR images indicated that the BBB was opened at both 0.3 MPa(Fig. 1A bottom site and Fig. 1C) and 0.45 MPa(Fig. 1A top site and

Figure 1 .
1BBB opening in V3. A 3D Spoiled Gradient-Echo (SPGR) T1-weighted sequence was applied after intravenous (IV) injection of gadodiamide 1 h after sonication. Gadodiamide presence in the brain parenchyma is induced by BBB opening. (A) Coronal MR images confirming the local disruption of the BBB, two opening sites are circled confirmed by the diffusion of the contrast agent (purple circle 0.3 MPa and orange circle 0.45 MPa). (B) Coronal MR images 6 days, after sonication, confirming closing of the BBB and proving reversibility of the procedure. (C,D) Sagittal MR images of the two sonication sites at two different pressures 0.3 MPa (C) and 0.45 MPa (D). (E,F) Magnification of the corresponding color boxes. doi:10.1371/journal.pone.0022598.g001

Figure 2 .
2Damage assessment. (A,C,D) 3D T2-weighted sequence. Edemas should appear brighter in these images. (B,E,F) 3D Susceptibility-Weighted Image (SWI) sequence was applied. Hemorrhages, as well as large vessels should appear in black in these images. (A,B) Same reconstructed coronal slice as shown in figure 1. The two opening sites are circled with the corresponding colors. There is no difference between the two hemispheres. (C,D,E,F) Corresponding reconstructed sagittal slices for the two opening sites. No oedemas or hemorrhages are visible. doi:10.1371/journal.pone.0022598.g002

Figure 3 .
3BBB opening in caudate and hippocampus & damage assessment. (A,B,C) BBB opening experiment targeting caudate using customized microbubbles and applying 0.6 MPa (purple dashed line shows region of interest). (D,E,F) BBB opening experiment targeting hippocampus using DefinityH microbubbles and applying 0.6 MPa (orange dashed line shows region of interest). (A,B,D,E) 3D Spoiled Gradient-Echo (SPGR) T1-weighted sequence was applied after intravenous (IV) injection of gadodiamide 1 h after sonication. (A,D) Sagittal slices at the region of interest. (B,E) Corresponding coronal slices. (C,F) 3D T2-weighted sequence, an edema was visible using customized microbubbles while no damage was detected using DefinityH microbubbles. doi:10.1371/journal.pone.0022598.g003

Figure 4 .
4PCD recordings during ME-FUS exposure. The corresponding spectrograms of the first pulse with microbubbles administration shows that very few broadband acoustic emissions are detected at 0.3 MPa (A) and 0.45 MPa (B) using customized microbubbles. This suggests that in those cases, the mechanism is mainly dominated by stable cavitation. Increasing the pressure to 0.6 MPa, using both customized (C) and DefinityH (D) microbubbles, a large amplitude broadband signal(4)(5)(6)(7)(8) is detected which is the signature of inertial cavitation. doi:10.1371/journal.pone.0022598.g004

Figure 5 .
5Experimental setup for in vivo FUS-induced BBB opening in the operating room. (A) A single-element, circular focused ultrasound transducer with a hole in the center was driven by a function generator (Agilent Technologies, Palo Alto, CA, USA) through a 50-dB power amplifier (ENI Inc., Rochester, NY, USA). The center frequency, focal depth, outer radius and inner radius of FUS were 500 kHz, 90 mm, 30 mm and 11.2 mm, respectively. (B) Closer view of the transducer mounted on the stereotactic frame with a manipulator allowing precise positioning of the transducer in the stereotactic referential. (C) Monkey placed in the stereotactic frame. The monkey head is shaved and a degassed echographic gel container is placed on the top of its head to insure maximal acoustic transmission. doi:10.1371/journal.pone.0022598.g005

Figure 6 .
6Targeting procedure for in vivo FUS-induced BBB opening. (A) A positioning rod (black arrow), indicating the position of the focus (5 cm away from the edge of the transducer), was used to target. (B) This positioning rod was mounted on the manipulator in order to locate the origin of the stereotactic coordinates. (C) The origin of the stereotactic coordinates indicated by the engraved cross on the metal piece between the ear-bars is targeted with the tip of the positioning rod. doi:10.1371/journal.pone.0022598.g006
PLoS ONE | www.plosone.org
July 2011 | Volume 6 | Issue 7 | e22598
AcknowledgmentsThe authors especially wish to thank the Riverside Research Institute for providing the transducer that was used in this study; Mark Borden, Ph.D., and Jameel A. Feshitan, for the microbubble manufacture; and Fotios Vlachos, Ph.D., and Stephen M. Dashnaw, Department of Biomedical Engineering, Columbia University, for their help in MRI scanning. The authors appreciate Girma Asfaw, Department of Neuroscience, Columbia University, for his surgical assistance.Author Contributions
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