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Article 11.2.9 What could transform an underdog into a winner
Many feedbacks to my last post reflected radiologists’ perception of ultrasound as a low-tech, unreliable imaging device.
Ultrasounds most manifested limitation by radiologists is that its performance is too-much user-dependent. This opinion finds support in numerous clinical studies concluding that ultrasound-based assessment of a cancer patient varies with the operator.
How come that an imaging technology that is not only low-cost, simple to operate and risk-free to the patient, but has also gained a leading position in certain domain, like obstetrics, is perceived as the underdog when it comes to cancer assessment? Could it be because of its positioning as a “multi-purpose” system, which requires only very basic training?
If indeed this is the case, it doesn’t require “rocket-science” to turn it around. It only needs designing dedicated ultrasound machines who offer a comprehensive solution to one specific clinical need. Using such machines will require highly skilled operators who will enjoy a superior workflow, reporting tools and proven clinical guidelines.
The unsatisfactory reality of mammography-based breast cancer screening, as evident by epidemiology data and expert-panels’ reports, opens the opportunity to transform ultrasound into a winner in the niche-market of breast cancer screening and diagnosis. It’s a significant market that justifies the investment in ultrasound systems dedicated to detection and characterisation of breast cancer lesions.
No doubt, that the ability to provide accurate and standardized interpretation of such ultrasound systems’ scans is a pre-requisite. Ultrasound-based tissue characterisation is a must for any application aiming at standardized image interpretation. A sample out-of present ultrasound-based technologies aiming at providing some level of tissue-characterisation are listed below. Recent clinical studies show promising results using these technologies. It is worth watching carefully to see if any of those could be part of a future ultrasound-based solution to breast cancer screening.
Results: Of the 136 biopsied lesions, 54 were carcinomas and 82 were benign. The average total hemoglobin concentration in the malignant group was 223.3 μmol/L ± 55.8 (standard deviation), and the average hemoglobin concentration in the benign group was 122.5 μmol/L ± 80.6 (P = .005). When the maximum hemoglobin concentration of 137.8 μmol/L was used as the threshold value, the sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of DOT with US localization were 96.3%, 65.9%, 65.0%, 96.4%, and 76.5%, respectively. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of conventional US were 96.3%, 92.6%, 89.7%, 97.4%, and 93.4%, respectively. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of conventional US combined with DOT were 100%, 93.9%, 91.5%, 100%, and 96.3%, respectively.
Conclusion: US-guided DOT combined with conventional US improves accuracy compared with DOT alone.
Results: All breast lesions were detected at Supersonic Shear Imaging. Malignant lesions exhibited a mean elasticity value of 146.6 kPa ± 40.05 (standard deviation), whereas benign ones had an elasticity value of 45.3 kPa ± 41.1 (P < .001). Complicated cysts were differentiated from solid lesions because they had elasticity values of 0 kPa (no signal was retrieved from liquid areas).
Conclusion: Supersonic Shear Imaging provides quantitative elasticity measurements, thus adding complementary information that potentially could help in breast lesion characterization with B-mode US.
Results: The Az of B-mode US, US elastography, and Doppler US (average, 0.844; range, 0.797–0.876) was greater than that of B-mode US alone (average, 0.771; range, 0.738–0.798) for all readers (P = .001 for readers 1, 2, and 3; P < .001 for reader 4; P = .002 for reader 5). When both elastography and Doppler scores were negative, leading to strict downgrading, the specificity increased for all readers from an average of 25.3% (75.4 of 298; range, 6.4%–40.9%) to 34.0% (101.2 of 298; range, 26.5%–48.7%) (P < .001 for readers 1, 2, 4, and 5; P = .016 for reader 3) without a significant change in sensitivity.
Conclusion: Combined use of US elastography and color Doppler US increases both the accuracy in distinguishing benign from malignant masses and the specificity in decision-making for biopsy recommendation at B-mode US.
A 57-year-old woman with a no-palpable lesion in the outer upper quadrant of left breast. (a) Gray scale image show an indistinct, hypo-echoic lesion. (b) Contrast enhanced image obtained 35 s after contrast agent injection showing a homogeneously and hyper-enhanced lesion. (c) Micro flow image obtained 38 s after contrast agent injection showing the enhanced mass with several radial vessels (arrow). (d) The time-intensity curve analysis show the peak intensity is 145.69 (intensity/1000), time to peak is 15.08 s, ascending slope is 8.98, descending slope is 1.03, the area under the curve is 7783.34. Pathologic analyses show this is an invasive ductal carcinoma.
Results: Histopathologic analysis of the 91 lesions revealed 44 benign and 47 malignant. For qualitative analysis, benign and malignant lesions differ significantly in enhancement patterns (p < 0.05). Malignant lesions more often showed heterogeneous and centripetal enhancement, whereas benign lesions mainly showed homogeneous and centrifugal enhancement. The detectable rate of peripheral radial or penetrating vessels was significantly higher in malignant lesions than in benign ones (p < 0.001). For quantitative analysis, malignant lesions showed significantly higher (p = 0.031) and faster enhancement (p = 0.025) than benign ones, and its time to peak was significantly shorter (p = 0.002). The areas under the ROC curve for qualitative, quantitative and combined analysis were 0.910 (Az1), 0.768 (Az2) and 0.926(Az3) respectively. The values of Az1 and Az3 were significantly higher than that for Az2 (p = 0.024 and p = 0.008, respectively). But there was no significant difference between the values of Az1 and Az3 (p = 0.625).
Conclusions: The diagnostic performance of qualitative and combined analysis was significantly higher than that for quantitative analysis. Although quantitative analysis has the potential to differentiate benign from malignant lesions, it has not yet improved the final diagnostic accuracy.
Results: In 17 normal testing volumes, 3% of isolated voxels were classified as abnormal. In 15 abnormal testing volumes, the subclassifiers differentiated between malignant and benign tissue. BHS in benign tissue showed <1% abnormal voxels in cyst, hamartoma, papilloma and benign fibrosis. The fibroadenomas differed showing <5% and <24% abnormal voxels. Abnormal voxels in cancers increased with the volume of cancer at pathology.
Conclusions: HistoScanning reliably discriminated normal from abnormal tissue and could distinguish between benign and malignant lesions.
Automated Breast Ultrasound System (‘ABUS’) for full breast scanning: The beginning of structuring a solution for an acute need!
Writer: Dror Nir, PhD
Word Cloud By Danielle Smolyar
GE Healthcare announced this week the acquisition of U-Systems, Inc. U-systems has developed the first and only Automated Breast Ultrasound System (ABUS) on the market – somo•v®, to receive FDA approval as an adjunct to mammography screening for breast cancer of; “asymptomatic women, with greater than 50 percent dense breast tissue and no prior breast interventions.”
somo•v® screen shot, showing mass in upper-outer quadrant of the left breast. Image courtesy of U-Systems.
I became aware of somo•v® already in 2004, when Prof. André Grivegnée, head of the breast screening unit at Jules Bordet – European oncology center in Brussels, Belgium, invited me to participate in a technology assessment of U-Systems’ somo•v® product. On that occasion, I also shared with U-System’s developers the idea of incorporating tissue characterisation into their product, an idea which they did not take on board. There is nothing more vivid to fully understand the meaning of this acquisition for breast cancer screening then the following quote from AuntMinnie’s report “GE taps interest in ABUS with U-Systems acquisition”: “You know you’re onto something when the big boys come calling. GE Healthcare today announced its acquisition of automated breast ultrasound (ABUS) developer U-Systems, a move that highlights the rapid evolution of ABUS from a niche technology into a promising adjunct to screening mammography. “ First savvy: The reality of medical device startups is that it doesn’t matter how real and large is the need for your technology. Until one of the big boys will adopt it, it is prone to be considered as niche technology. I discussed the potential role of ABUS in future breast screening in my recent posts: Closing the Mammography gap; Introducing smart-imaging into radiologists’ daily practice. As noted, in recent years, several ABUS systems were developed. An intriguing question is; why did GE choose to buy the somo•v® and not one of the other systems? Why now and not 2 or 3 years ago? The answer must have to do with the fact that in September 2012, somo•v® became the first ABUS system to receive premarket approval (PMA) for its application to use the system in a breast cancer screening environment. Until then, somo·v was indicated for use as an adjunct to mammography for B-mode ultrasonic imaging of a patient’s breast when used with an automatic scanning linear array transducer or a handheld transducer. The PMA has extended somo·v’s Indication For Use (IFU) allowing a claim that it increases breast cancer detection in a certain patients population.Second savvy: Having a PMA approval for a compelling indication for use, in a significant enough patient group, will dramatically increase “big boys” interest in your product. From the information available on the FDA site, one can get an insight into U-System’s regulatory strategy. They were smart enough to be satisfied with achieving a small step; increasing the detection rate of mammography-based screening. Therefore, the same radiologist who read the mammograms also read the ultrasound image. This increases the probability that your device’s sensitivity will not be worse than that of mammography. U-Systems did not try to go all the way to become an alternative to mammography. A claim that would significantly increase the complexity of the required clinical study; e.g. will require comparison of cancer detection-rates between modalities by independent, blinded-readers. Therefore, “the device is not intended to be used as a replacement for screening mammography”. Third savvy: The most expensive component, in time and money, in a regulatory pathway are the clinical studies. A cost-effective regulatory strategy is linked to good understanding of the market segmentation. Identifying what kind of IFU differentiates your products from its competition in a large enough niche-market is key. It will also lead to the simplest clinical-study design possible. As an entrepreneur, I cannot help congratulating U-Systems’ team for pulling through continuous hurdles to reach the point all medical device startups are hoping for. They certainly picked up the right item to focus their efforts on: i.e. PMA approval for breast cancer screening. Finally, I will reiterate my vision that embedding real-time tissue characterization in an ultrasound system, capable of performing fast and standardized full breast scanning is: a. Technologically achievable; and b. in the long-term, will be an excellent alternative to mammography for breast cancer screening. Additional readings: Two studies related to somo•v® will be discussed at the 2012 RSNA meeting: “ A study led by Dr. Rachel Brem of George Washington University Medical Center: ABUS plus mammography finds cancer early in women with dense tissueBrem’s study found that ABUS enabled detection of early-stage cancers in women with dense breasts, giving healthcare providers time to start early treatment. In all, 88% of cancers found by ABUS alone in a group of 15,000 women were grade 1 or 2.” “A study presented by Maryellen Giger, PhD, of the University of Chicago: ABUS boosts mammography’s performancethis study results showthat adding ABUS to mammography for women with dense breast tissue improved sensitivity by 23.3 percentage points, from 38.8% for mammography alone to 63.1% for mammography plus ABUS.” As I mentioned already, there are other ultrasound modalities out there, some are ABUS and some are not. All are adjunct to mammography screening. Related studies will also be presented during that same meeting.
UPDATE (04-Aug-2013)
Here below is a recent publication on the use of ABUS for better detection of breast cancer in patients presented with mammographically dense breast.
Automated breast ultrasound (ABUS)was performed in 3418 asymptomatic women with mammographically dense breasts. The addition of ABUS to mammography in women with greater than 50% breast density resulted in the detection of 12.3 per 1,000 breast cancers, compared to 4.6 per 1,000 by mammography alone. The mean tumor size was 14.3 mm and overall attributable risk of breast cancer was 19.92 (95% confidence level, 16.75 – 23.61) in our screened population. These preliminary results may justify the cost-benefit of implementing the judicious us of ABUS in conjunction with mammography in the dense breast screening population.
Keywords
Breast ultrasound;
3-dimensional sonography;
Breast screening;
Dense breast;
Breast cancer;
Cancer detection
1. Introduction
Mammographic density as an independent risk factor for developing breast cancer has been documented since the 1970’s [1]. The appearance of breast tissue is variable among women. The appearance of density on mammography is the result of the relative proportion of breast stroma, which is less radiolucent compared to fat, accounting for increased breast density. Wolfe classified breast density as an independent risk factor for breast cancer in women [2] and [3]. Approximately 70 to 80% of breast cancers occur in women with no major predictors [4], [5] and [6]. Population-based screening for early detection of breast cancer is therefore the primary strategy for reducing breast cancer mortality. Mammography has been used as the standard imaging method for breast cancer screening, with reduction in breast cancer mortality [7]. Breast density significantly reduces the ability to visualize cancers on mammography. The number of missed cancers is substantially increased in mammographically dense breasts, where the sensitivity is reported as low as 30 to 48%. [8]; and the odds of developing breast cancer 17.8 times higher [9]. Hand held ultrasound (HHUS) has been used to optimize the detection of cancers in mammographically dense breasts, but is limited due to technical factors, such as breast size, considerable user variability and reproducibility, technical skill, and time constraints, precluding HHUS as an effective screening modality for breast cancer [10], [11] and [12]. Kelly described the use of 3D-automated breast ultrasound (ABUS) as an adjunct to mammography in the evaluation of non-palpable breast cancers in asymptomatic women. ABUS with mammography resulted in an increase in diagnostic yield from 3.6 per 1,000 with mammography alone, to 7.2 per 1,000 by adding ABUS, resulting in a mammography miss rate of 3.6 per 1,000 [13]. However, one of the limitations of the study was that it did not isolate dense breasts as an independent risk factor for developing breast cancer, where the detection rate should be expected to be higher. ABUS is FDA-approved in the United States for screening of women with dense breast parenchyma [14]. The purpose of this study was to demonstrate that ABUS increases the detection of non-palpable breast cancers in mammographically dense breasts when used as an adjunct diagnostic modality in asymptomatic women. This resulted in the subsequent detection of cancers missed by mammography of smaller size and stage, justifying the basis for the judicious use of implementing ABUS in conjunction with mammography in the dense breast screening population. The tabulated data was extrapolated based on known mammography screening utilization to show a cost-benefit of additional ABUS as a population based screening method.
2. Methods
2.1. Selection of participants
This study and the use of patient electronic health records were approved by an ethics committee appointed by the institute Board of Directors. The study design included two study groups, the control and test groups, in successive years. Each group was followed prospectively for 1 year. The control group consisted of women screened by digital mammography alone and stratified for breast density based on a Wolf classification of 50% or greater breast density (defined as the ‘mammographically dense breast’ for the purpose of this study). The second group consisted of women initially screening by digital mammography as having mammographically dense breasts, followed by automated breast ultrasound (ABUS). Each group was carefully selected on the basis of breast density and having no major pre-existing predictors of breast cancer, such personal or family history of breast cancer, or BRCA gene positive. In addition, the test group patients were not included in the screening group so as to eliminate impact on the results of the test group patients. The control group consisting of 4076 asymptomatic women designated as Wolf classification of 50% or greater breast density underwent stand-alone screening digital mammography between January 2009 and December 2009 using digital mammography (Selenia, Hologic Inc., Bedford, MA USA). The sensitivity, specificity, positive predictive value, and negative predictive value for biopsy recommendation were determined, in addition to data collection regarding the size and stage of cancers missed by mammography. The test group, consisting of 3418 asymptomatic women designated as Wolf classification of 50% or greater breast density, underwent stand-alone screening digital mammography between January 2010 and May 2011 using digital mammography (Selenia, Hologic Inc., Bedford, MA USA). This was followed by automated whole breast ultrasound (Somo-V. U-Systems, Sunnyvale, CA USA). The mammography-alone results were not used as control results in order to eliminate potential bias introduced by ABUS results on the mammography interpretations. In addition, mammography results were interpreted independently from ABUS results so as not to introduce bias. The sensitivity, specificity, positive predictive value, and negative predictive value for biopsy recommendation were determined, in addition to derived statistical data regarding the relative risk, and odds ratio for developing breast cancer.
2.2. Assessment of mammographic density
Mammographic density was assessed independently by radiologists on a dedicated mammography viewing workstation equipped with 5-Megapixel resolution. The radiologists were FDA-qualified in mammography, with at least 10 years experience in breast ultrasound, 24 months of which included ABUS. Two radiologists interpreted both the mammography and ABUS examinations under identical viewing conditions of 5-Megapixel resolution. The mammograms and ABUS studies were double read by two radiologists, with final consensus determination for each case. Mammograms were evaluated according to one of five categories of density (0%, 1 to 24%, 25 to 49%, 50 to 74%, and 75 to 100%) and only mammograms with breast density of 50% or greater were included in the control and test study groups.
2.3. 3D-Automated breast ultrasound evaluation
3D-Automated Breast Ultrasound (ABUS) is a computer-based system for evaluating the whole breast. The whole breast ultrasound system (Somo-V, U-Systems, Sunnyvale, CA USA) was used in combination with a 6 to 14 MHz broadband mechanical transducer attached to a rigid compression plate and arm, producing over 300 images per image acquisition obtained as coronal sweeps from the skin to the chest wall. The mechanical arm controls transducer speed and position, while a trained ultrasound technologist maintains appropriate contact pressure and vertical orientation to the skin. Interpretation and reporting time for an experienced radiologist is approximately 10 minutes per examination. The radiologist has cine functionality to simultaneously view breast images in the coronal, sagittal, and axial imaging planes.
2.4. Data collection
ABUS scan data was collected for location and size of breast masses and recorded in a radial or clock orientation consistent with American College of Radiology reporting lexicon. Studies were reported according to the American College of Radiology Breast Imaging Reporting and Data System (BI-RADS) six-point scale (0=incomplete, needs additional assessment; 1=normal; 2=benign; 3=probably benign; 4=suspicious; 5=highly suggestive of malignancy) [15] and [16]. For BI-RADS scores of 1, 2, and 3 on ABUS, patients were followed prospectively for 1 year to exclude cancers missed on both mammography and ABUS. For BI-RADS scores of 4 and 5, stereotactic hand held ultrasound (HHUS) biopsy was performed using 14 gauge or larger percutaneous biopsy. HHUS was employed because ABUS is presently not equipped with biopsy capability. If a benign non-high risk lesion was diagnosed, such as simple breast cysts, no further tissue sampling was performed. All non-cystic lesions were biopsied. Cystic lesions were identified as anechoic, thin walled lesions with posterior acoustic enhancement. All pathology proven breast malignancies were further staged using contrast volumetric/whole breast MR imaging (1.5T HDe Version 15.0/M4 with VIBRANT software, GE Medical Systems, Waukesha, WI USA.) with computer assisted detection (CADStream software, Merge Healthcare, Belleview WA USA). A final pathological stage was assigned by the pathologists in the usual manner in accordance with the American Joint Committee on Cancer (AJCC) TNM system guidelines. The pathologists were blinded to patient participation in the study and the method of cancer detection.
2.5. Statistical analysis
Calculations were made of the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), relative risk, odds risk, and attributable risk of breast cancer using MedCal version 12.2.1 software. Exact 95% confidence intervals (CI) were calculated for diagnostic yield. Statistical methods involved the Chi-square test statistic, which was used to compare the number of cancers detected by ABUS, based on the size of cancer. P-values of less than .05 were considered to indicate statistical significance. Attributable risk (AR) was calculated according to the following formula: AR=(RR − 1)Pc ÷ RR, where RR denotes relative risk of greater than 50%, and Pc prevalence of density of greater than 50% in case patients[17], [18] and [19].
3. Results
Comparable interobserver diagnostic reliability (Kappa value of 0.98) was observed with mammography and ABUS examinations. In the control group (N=4076), the median age of participants with breast cancer (N=19) at the time of biopsy was 54 years, distributed as follows: 26% (5 out of 19) cancers occurred in women younger than age 50; 63% (12 out of 19) in women 50 to 69 years; and 11% (2 out of 19) over the age of 70 years. All cancers (N=19) were biopsy proven invasive ductal carcinoma. The sensitivity and specificity of stand-alone digital mammography were 76.00% (95% CI: 54.87% – 90.58%) and 98.2% (95% CI: 97.76% – 98.59%). The positive predictive value was 20.43% (95% CI: 12.78% – 30.05%) with a breast cancer prevalence rate of 0.60% (95% CI: 12.78% – 30.05%). The cancer detection rate was 4.6 per 1,000, with mean tumor size detected by mammography (N=19) of 21.3 mm. The average size of missed breast cancer (N=6) was 22.3 mm. The node positivity rate was 5% (1 of 19 cases). In the ABUS study group (N=3418), the median age of participants with breast cancer (N=42) at the time of biopsy was 57 years, distributed as follows: 17% (7 out of 42) cancers occurred in women younger than age 50; 64% (27 out of 42) in women 50 to 69 years; and 19% (8 out of 42) over the age of 70 years. The sensitivity and specificity of ABUS were 97.67% (95% CI: 87.67% – 99.61%) and 99.70%, (95% CI=99.46% – 99.86%), respectively, in mammographically dense breasts. The positive predictive value of ABUS was 80.77% (95% CI=67.46% – 90.36%), with a breast cancer prevalence rate of 1.25% (95% CI: 0.91% – 1.69%). The odds ratio of breast cancer in mammographically dense breasts determined by ABUS was 2.65 (95% CI: 1.54 – 4.57; P=0.0004). The cancer detection rate was 12.3 per 1,000. A 2.6-fold increase in cancer detection rate was observed between ABUS added to digital screening mammography compared to stand-alone digital screening mammography. Invasive breast cancer accounted for 81% (42 out of 52) solid breast masses detected by ABUS, of which 93% (39 out of 42) were invasive ductal carcinomas, and 7% (3 out of 42) were invasive lobular carcinomas. The mean tumor size detected by ABUS in patients with breast cancer (N=42) was 14.3 mm, distributed as follows: Stage 1A disease accounted for 83% (35 out of 42) of cases; 12% were Stage 2A (5 out of 42), and 5% were Stage 3A (2 out of 42). Stage 3A disease was associated with multifocal disease in both cases, one of which also was Level 1 axillary lymph node positive. The node positivity rate was 2% (1 in 42) of cases. The false positive rate of ABUS was 19.3%, with a negative predictive value of 99.97% (95% CI 99.83% – 100.00%). The pathologies associated with false positive results (N=10) were fibroadenomas and atypical epithelial neoplasms. We also used our data to extrapolate the theoretical cost-benefit of ABUS screening applied to a large screening population in the United States. Our analysis relied on the following assumptions: (1) Global Centers for Medicare and Medicaid reimbursement rate of breast ultrasound of $71 [20]; and (2) Estimated mean doubling time of a missed cancer of 250 days at the 95th percentile [21] and [22]. According to previously cited cancer kinetics models, a missed breast cancer should be clinically evident within 9 months[23]. When we considered the mean breast cancer size in our positive test subject group, 14.3 mm (N=42), we extrapolated a theoretical missed cancer size of 29.2 mm at 9 months in mammographically dense breasts, representative of Stage 2 or greater disease. In control subjects, a mean breast cancer size of 22.3 mm was consistent with stage 2 breast cancer. Incremental treatment cost assumptions, based on the global Centers for Medicare and Medicaid reimbursement rate between Stage 1 and Stage 2 breast cancer, were $24,002 and $34,469, respectively, for a cost differential of $10,467 [24]. Accordingly, the aggregate costs of screening 3418 ABUS patients in this study were $239,260, compared to the estimated aggregate costs of additional treatment in 26 potentially missed cancers (based on previously noted theoretical assumptions) of $275,557 based on a cancer miss rate of 0.77% (or 7.7 per 1,000).
4. Discussion
Table 1 shows the clinical indications for ordering an ABUS examination. Table 2 shows the distribution of breast cancer size according to age in the control and test study groups. The test group showed no statistical difference between size of the cancer and patient age at presentation. A significant increase in tumor size in the over 70 patients in control subjects was attributed to the more advanced tumor stage at presentation.Table 3 shows that stand-alone digital mammography was less sensitive than ABUS in breast cancer detection, with a 4-fold increase in positive predictive value of ABUS compared to stand-alone mammography in dense breasts. Our results showed that mammographic density of 50% or more was associated with an increased risk of breast cancer and resulted in a significant miss rate in asymptomatic women. Table 4 shows a statistically significant age-related attributable risk of developing breast cancer for mammographic density of 50% or greater. These observations are consistent with other studies which have shown an increased risk of breast cancer in dense breasts following negative mammography screening [2],[3], [8] and [9]. We observed that breast cancer risk was highest in patients over age 70, where increased breast density was associated with an attributable risk of 29.6 (95% CI, 21.5 – 40.8). Fig. 1 shows box plots comparing case patients and control subjects according to age, with tumor sizes shown as a function of the odds ratio, relative risk, and attributable risk for each age category.
Table 1. Clinical criteria for ABUS screening
• As a supplement to mammography, screening for occult cancers in certain populations of women (such as those with dense fibroglandular breasts and/or with elevated risk of breast cancer);
• Imaging evaluation of non-palpable masses in women under 30 years of age who are not at high risk for development of breast cancer, and in lactating and pregnant women; and
• BI-RADS (American College of Radiology Breast Imaging Reporting and Data System) scoring classification class III, heterogeneously dense, with 50% to 74% or 75% to 100% breast density on mammography, without palpable mass.
Table 2. Breast cancer size according to method detection
Table 3. Detection of breast cancer according to method
Table 4. Risk of breast cancer according to method detection
Fig. 1. Breast Cancer Staging and Risk Assessment by Screening Method Detection. Box plots comparing case patients and control subjects according to age (boxes A through D). Tumor sizes are shown as a function of the odds ratio, relative risk, and attributable risk for each age category. Bars represent the highest and lowest observed values with respect to individual variables (individually labeled with arrows).
Our study also showed that 3D-Automated Breast Ultrasound (ABUS) was an effective screening modality in mammographically dense breasts. Our extrapolated data suggest a breast cancer miss rate of 7.7 per 1,000 in mammographically dense breasts in asymptomatic women, which is higher compared to the cancer miss rate of 3.6 per 1,000 reported by Kelly using ABUS [13]. We attribute the increased breast cancer miss rate due to breast density, which was isolated as the principal risk factor in our study. Other studies have shown that the attributable risk of breast cancer for a mammographic density of 50% or greater was 40% for all cancers detected less than 12 months after a negative screening mammogram, and as high as 50% in women less than the age of 50. This marked increase in the risk of breast cancer associated with mammographic density of 50% or greater up to 12 months following screening directly reflects cancers that were present at the time of screening but went undetected due to masking by dense breast parenchyma [25],[26], [27], [28] and [29]. In the final analysis, there is the issue of the theoretical cost-benefit of adding ABUS screening to mammography in an otherwise healthy population. The importance of screening mammographically dense breasts with ABUS has particular relevance based on the small size and early stage of breast cancers. Our study showed a mean tumor size of 14.3 mm, representing stage 1 disease, which was present in 81% of patients. From our data, we derived theoretical population-based costs as a basis for the cost-benefit of ABUS in the United States population. Our study compared the incremental costs of screening versus the costs of added treatment related to a change in the staging of missed cancers from Stage 1 to Stage 2. The costs of additional treatment outweighed the costs of screening by $32,808, which calculated to $9.60 added healthcare cost per patient in the 3418 participants in the study. In the United States, 48 million mammograms were performed annually, with a reported estimated miss rate of 10% [30]. When comparing control versus test patients, our study suggests a theoretical miss rate of 7.7 cancers per 1,000 mammograms, or 0.77%, which is considerably lower than the reported missed rate of 10%. Based on these theoretical assumptions, annual added ABUS screening of the entire U.S. population would cost $3.40-billion. However, in actual practice, ABUS would be used only in the mammographically dense breast, which would potentially reduce the screening costs by at least a factor of 0.8, bringing the cost closer to $2.72-billion. By contrast, the incremental costs of added treatment associated with stage 2 compared to stage 1 breast cancer in the U.S. population would be $3.82-billion, assuming a conservative cost basis of $10,467 per patient.. The cost-benefit of early detection of stage 1 disease results in a theoretical per capital annual cost savings of $22.75 per screened patient in the U.S. population, according to our model. However, we have no actual or derived data to support improved breast cancer mortality with the addition of ABUS as a universal screening modality. This is one of the major limitations of our study because actuarial analyses used to justify screening modalities are typically based on mortality statistics. With respect to five year survival statistics between stage 1 and stage 2 breast cancers, of 98% and 80%, respectively, one could construe the potential for a theoretical quality-of-life benefit based on judicious ABUS screening. Another limitation of our study is the relatively small screening population used in our study, emphasizing the need for continued research in order to validate ABUS as a viable and cost-effective population-based screening modality, which should be stratified for other risk factors for breast cancer, such as: personal or family history of breast cancer, BRCA genetic results, environmental factors (late parity, previous exposure to ionizing radiation, exogenous estrogen, smoking, and alcohol use), early menarche/late menopause, and ethnic/racial differences. At most imaging centers, mammography is the only screening method for breast cancer detection. Our study corroborates with the data derived from other studies that the principal mechanism for breast cancer in dense breast parenchyma is not rapid growth, but rather, the masking of coincident cancers that are missed on screening mammograms [9]. These findings further suggest that the addition of mammographic screening in patients with dense breast parenchyma is likely not to increase diagnostic yield in the detection of breast cancers. Therefore, emphasis should be placed on alternative imaging techniques for such women. To conclude, our study of a small representative dense breast screening population showed that the addition of ABUS was more effective than digital mammography alone. This study provides a platform for using ABUS as cost-effective approach to breast cancer detection in the judicious screening of asymptomatic women with excessive mammographic density, in whom the greatest risk is between screening mammography examinations.
Corresponding author. Breast Cancer Research Institute, Nova Southeastern University College of Medicine, 5732 Canton Cove, Winter Springs, FL 32708, USA. Tel.: 1 407 699 7787.
New biomedical imaging technology could enhance pathologists’ ability to examine tissue samples via fluorescence microscopy
Scientists at Harvard University’s Wyss Institute for Biologically Inspired Engineeringhave developed a new DNA, barcoding technique. The fluorescence microscopy approach has significant implications for the imaging community.
Beyond imaging, however, pathologists will be able to use this same technology when evaluating tissue specimens.
The new method could enable simultaneous imaging of many different types of molecules in a single cell, according to Peng Yin, Ph.D., Associate Professor of Systems Biology at Harvard Medical School and Core Faculty Member at Wyss Institute. The developers expect the method to provide researchers with a richer, more accurate view of cell behavior than is possible using current techniques.
Pathologists Could Adopt DNA Barcoding for In Vitro Diagnostics
“We hope this new method will provide much-needed molecular tools for usingfluorescence microscopy to study complex biological problems,” stated Yin, the study’s co-author, in a recent press release.
1903 Siedentopf Fluorescence Microscope (Photo credit: Carl Zeiss Microscopy)
Using DNA Origami to Create Fluorescent Linear DNA Barcodes
The newly engineered DNA barcode harnesses the natural ability of DNA to self-assemble. The basis of the new technology is a process called DNA origami. This enables scientists to arrange colored dots, or fluorophores, into geometric patterns, or fluorescent linear DNA barcodes.
These imaging probes translate a cell’s invisible biological information, such asproteins or RNA molecules, into detectable signals, noted a summary of Yin’s research on the Wyss website. These signals help researchers better understand the role of cell behavior in the onset and progression of disease.
New Barcode Could Offer a Virtually Unlimited Number of Styles
Scientists currently use fluorescence microscopy to pair fluorescent elements—the barcodes—with molecules they know will attach to the part of the cells they want to investigate. When they illuminate the sample, it triggers each kind of barcode to fluoresce at a particular wavelength of light, which indicates the location of the molecules of interest.
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Researchers at Harvard’s Wyss Institute recently engineered a new DNA barcode. Labeled DNA samples appear as multi-colored barcodes under fluorescent light at certain wavelengths. Pathologists and clinical laboratory professionals will recognize the potential of this technology in the examination of tissue specimens. (Photo credit: Rick Groleau, Harvard University.)
However, the multiplexing ability of fluorescence microscopy is limited by the number of spectrally distinguishable fluorophores, a story in Nature Chemistry explained. The barcodes that scientists currently use have only three or four colors available, such as red, blue, or green. And sometimes those colors blur. This limits the number of objects scientists have been able to study in a cell sample at one time.
Multiplex Capability with 216 Readable Color Combinations
Using the new method, Yin was able to demonstrate 216 color combinations resulting from attaching just three colors to a DNA nanotube, the press release stated. With the new barcode, the combinations are almost limitless. This will significantly advance the ability to fluoresce more cellular structure than previously possible.
DNA origami works by programming a long strand of DNA to self-assemble by folding in on itself, the release stated. Shorter strands, called staples, help it to create predetermined forms. Researchers then attach fluorescent molecules to the desired spots on the now more structurally complex DNA nanostructures. In this way, they use origami technology to generate a large pool of barcodes out of only a few fluorescent molecules.
“We can essentially use DNA joints to assemble the nanostructures into long rods, and we can modify the rods with fluorescence at different locations,” declared Yin. “We then use these tiny rods to arrange the fluorescent spots into colorful barcodes. Basically now using three or four colors, we can have hundreds of different barcodes,” he observed in a story in The Harvard Crimson.
A New Tool in the Cellular Imaging—and In Situ Examination—Toolbox
“[The technique] holds great promise for using the method to study cells in their native environments,” Yin observed. Additionally, the technique is low-cost, easy to do, and more robust compared to current methods, according to Yin.
Pathologists and clinical laboratory managers will recognize the range of potential of this new technology, from developing targeted drug-delivery mechanisms to improving the scope of cellular and molecular activities scientists are able to observe at a disease site.
The researchers found that a protein called PDGFRB is important to the development of a common form of ALCL. PDGFRB, a growth factorreceptor protein, is a target of imatinib. Imatinib had anticancer effects in both a mouse model of ALCL and a patient with the disease, Dr. Lukas Kenner of the Medical University of Vienna in Austria and his colleagues reported October 14 in Nature Medicine.
VIEW VIDEO byLeading CLL researcher Dr. Thomas Kipps discusses the latest in the course of the disease including: symptoms, causes, and risk factors. Dr. Kipps also highlights discoveries made in his lab that may lead to developing new therapies to treat this disease. Series: “Stein Institute for Research on Aging” [9/2010] [Health and Medicine] [Show ID: 19345]
Understanding the Molecular Basis of Imatinib Mesylate Therapy in Chronic Myelogenous Leukemia and the Related Mechanisms of Resistance1
Commentary re: A. N. Mohamed et al., The Effect of Imatinib Mesylate on Patients with Philadelphia Chromosome-positive Chronic Myeloid Leukemia with Secondary Chromosomal Aberrations. Clin. Cancer Res., 9: 1333–1337, 2003.
Division of Hematology and Oncology, Department of Internal Medicine [G. M., M. A. C.], Division of Human Cancer Genetics, Department of Molecular Virology, Immunology and Medical Genetics [G. M., D. P., M. A. C.], The James Cancer Hospital and The Comprehensive Cancer Center at The Ohio State University, Columbus, Ohio 43210
Introduction
CML3 is a myeloproliferative disorder with an incidence of approximately 1–1.5 cases/100,000 population/year, a slight male predominance, and a peak between 50 and 60 years of age (1) . This condition arises from a transformed pluripotent hematopoietic precursor associated with t(9;22)(q34;q11.2) that gives rise to the Ph′ chromosome. At the molecular level, this cytogenetic aberration results in the fusion of the ABL gene at chromosome band 9q34 with the BCR gene at chromosome band 22q11.2. The resultantBCR/ABL fusion gene encodes a chimeric protein necessary and sufficient to confer the leukemic phenotype. In a minority of the cases, despite absence of the Ph′ chromosome, molecular methodologies (i.e., fluorescence in situ hybridization, Southern, or RT-PCR) can still detect BCR/ABL as the product of complex cytogenetic aberrations involving other chromosomes in addition to 9q34 and 22q11.2, or cryptic genomic rearrangements of the BCR and ABL genes. Taken all together, >95% of the CML cases are associated with BCR/ABL expression that, therefore, represents the hallmark of this condition.
CML is a bi- or triphasic disease (2) . Patients usually present in a chronic proliferative phase characterized by splenomegaly and accumulation of neutrophils in their various stages of maturation. Basophils and blasts are normally <20% and 10%, respectively, in CP CML. Evolution to the BP (or blast crisis) is defined by an increase (≥20%) in myeloid or lymphoid blasts in blood, BM, or extramedullary locations. In many cases, the transformation is characterized by a passage through an AP, in which the failure of a patient to respond to therapy is accompanied by an increase in percentage of blasts (10–19%); a ≥20% increase in basophils, uncontrolled thrombocytosis, or thrombocytopenia; and acquisition of new cytogenetic abnormalities such as trisomy 8, isochromosome 17q, or a second Ph chromosome and/or genetic inactivation of the p53 gene.
The ultimate goal of treatment for CML is prevention of blast crisis, because, once this occurs, the prognosis is dismal. Although a rapid reduction of the white cell count can be achieved with chemotherapy (i.e., busulfan or hydroxyurea) in CP CML, these drugs usually fail to prevent disease progression. IFN-α was the first agent proven capable of modifying the biological history of CML by prolonging survival in patients who achieved CHR and MCR (<35% of Ph′+cells; Ref. 3 ). A second breakthrough in the treatment of CML occurred when 60–80% of patients undergoing alloBMT in CP were reported to be disease free at 5 years (4, 5, 6) . However, both IFN-α and alloBMT have considerable treatment-induced toxicity that attenuated the initial enthusiasm for these results, and novel, less toxic therapeutic strategies are being explored. In 2001, Druker et al.(7)reported the first Phase I study with imatinib mesylate (STI571, Gleevec), a specific inhibitor of BCR/ABL oncogenic activity. In this study, the authors demonstrated that high rates of CHR and MCR were achieved with relatively few side effects in patients with CML refractory or intolerant to IFN-α, opening new avenues for molecularly targeted therapies in this disease. Subsequently, encouraging results were also obtained for CML patients in BP (8) .
Is BCR/ABLthe Driving Force for Leukemogenesis in CML?
The transforming activity of BCR/ABL has long been demonstrated using in vitro and in vivo models. In initial studies, transfection of the BCR/ABL gene fusion resulted in malignant transformation of normal fibroblasts, and induced independent survival and proliferation in growth factor-dependent cell lines. Expression of BCR/ABL was also shown to be necessary and sufficient to induce leukemogenesis in animal models (reviewed in Ref. 9 ). Expression of BCR/ABL in mice was achieved by either introduction (“knock-in”) of the fusion gene in the mouse genome or by infecting murine stem cells with BCR/ABL-containing retroviral vectors. In knock-in transgenic mice with conditionalBCR/ABL expression, a low penetrance phenotype of acute B- or T-cell leukemia was reported. In contrast, in mice sublethally radiated and transplanted with syngeneic retrovirally transfected BCR/ABL+ stem cells, a condition mimicking human myeloproliferative disorders with neutrophil increase, BM expansion, hepatosplenomegaly, extramedullary hematopoiesis, and pulmonary leukostasis was observed.
How Does BCR/ABL Transform Cells?
The fusion partner ABL is a member of the nonreceptor tyrosine kinase family (10) . This gene encodes a protein containing a tyrosine kinase activity domain (SH1) in addition to two other regulatory domains (SH2 and SH3) that mediate protein-protein interaction and modulate activation of signal transduction. A nuclear localization domain is also present, supporting a shuttling activity of the ABL protein between cytoplasm and nucleus. Genetic disruption of ABL in mice results in lymphopenia, runting, and perinatal mortality. The other fusion partner, BCR, is a protein with multiple functional domains involved in oligomerization, SH-2 binding, serine/threonine kinase activity, and activation of members of the Rho small GTP-ase family of proteins (10) . Structural analysis of this gene suggests a role as a mediator of signaling transduction. With targeted disruption of BCR, mice have increased susceptibility to septic shock, but normal hematopoiesis.
In CML, each of the two partner genes are disrupted at specific breakpoints and fuse to create the chimeric BCR/ABL gene (11) . The most common rearrangements give rise to fusion transcripts identified as b2a2 or b3a2, which, in turn, are translated into a 210 kDa protein. Alternative BCR breakpoints can be located in minor cluster (m-BCR) or micro (μ) cluster (μ-BCR) regions and result in fusion transcripts that encode smaller (190 kDa) or larger (230 kDa) products, respectively. Although usually associated with Ph′+ acute lymphoblastic PiQO leukemia, the protein can also be detected in ≥90% of the CML patients from alternative splicing of p210. p230 is instead usually associated with CML patients presenting with an unusual predominance of neutrophils, resembling chronic neutrophilic leukemia. In each of these BCR/ABL variants, the ABL tyrosine kinase domain autophosphorylates and becomes constitutionally activated. Such BCR/ABLderegulated tyrosine kinase activity is, in fact, responsible for transformation of the hematopoietic stem cell and maintenance of the leukemic phenotype by recruiting and activating transducing signal pathways (i.e., RAS, RAF, extracellular signal-regulated kinase, c-Jun NH2-terminal kinase, phosphatidylinositol 3′-kinase, cCbl, CRKL, Janus-activated kinase-signal transducers and activators of transcription, PKC and PLCγ) involved in: (a) enhanced gene transcription (i.e., c-myc, c-Jun, reviewed in Ref. 12 ); (b) altered mRNA processing, nuclear export, and translation (i.e., bcl-xL, CAAT/enhancer binding protein α, and p53; reviewed in Ref. 13, 14 ); and (c) increased or decreased protein stability (i.e., Abi proteins; DNA-PKcs; FUS, and hnRNP A1; reviewed in Ref. 13). This, in turn, leads to enhanced proliferative potential and survival, altered motility and trafficking, and suppression of granulocytic differentiation (1, 13, 15, 16) .
Mechanisms of Action of Imatinib Mesylate
BCR/ABL is an ideal target for molecular targeted therapy, because this fusion protein is present in all of the CML cells, is absent from nonmalignant cells, and is necessary and sufficient to induce leukemia. Imatinib mesylate is a 2-phenylaminopyrimidine tyrosine kinase inhibitor with specific activity for ABL, platelet derived growth factor receptor, c-kit, and Albeson-related gene(17) . The pharmacological basis of this interaction has been elucidated by crystallographic studies. Imatinib mesylate binds to the amino acids of the BCR/ABL tyrosine kinase ATP binding site and stabilizes the inactive, non-ATP-binding form of BCR/ABL, thereby preventing tyrosine autophosphorylation and, in turn, phosphorylation of its substrates. This process ultimately results in “switching-off” the downstream signaling pathways that promote leukemogenesis. Preclinical in vitro and in vivo data indicated an impressive selective activity of imatinib mesylate on cells expressing BCR/ABL, and supported a rapid transition of this compound from the bench to the clinic.
To date, imatinib mesylate has been evaluated in several Phase I and II clinical trials of patients with IFN-α-resistant chronic, accelerated, or BP CML (7, 8, 18, 19, 20) . From the collective analysis of these studies, imatinib mesylate appears to effectively induce high CHR and cytogenetic response rates with relatively few side effects. In patients with CP CML who have failed IFN-α the CHR was 95%, MCR 60%, and complete cytogenetic remission 46%. Notably, in these patients achievement of MCR at the 3-month time point correlated with improved progression-free survival. In AP and in blast crisis, the CHRs were 34% and 8%, MCRs were 24% and 16%, and complete cytogenetic remissions were 17%, and 7%, respectively. Disease progression was 11% at 18 months for CP, 40% at 12 months for AP, and 80% at 18 months for BP. Finally, preliminary data from an interim analysis of a phase III study of untreated CML patients randomized between imatinib mesylate versus IFN-α and ARA-C indicate a significantly better CHR, complete cytogenetic remission, and progression-free survival for the imatinib mesylate group after a median follow-up of 14 months (21) . However, a longer follow-up will be necessary to assess whether this compound can also impact on the natural history of the disease and prevent or delay transformation to blast crisis.
Mechanisms of Resistance to Imatinib Mesylate
During disease progression, CML progenitor cells acquire a number of genetic alterations, most probably because of increased genomic instability, that may explain the aggressive phenotype, chemotherapeutic drug resistance, and poor prognosis of CML in BP. Despite the exciting results obtained with imatinib mesylate noted above, CML patients eventually show resistance at a rate of 80% in BP, 40–50% in AP, and 10% in CP post-IFNα failure, at 2 years (19) . Identification of the molecular basis of resistance is important, because it could provide insight into disease progression and into the design of novel therapeutic strategies to prevent and overcome treatment resistance.
CML patients with imatinib mesylate resistance can be stratified into those with primary refractory disease, most frequently in accelerated or BPs, and those who relapse after initial response, who are most frequently in CP. On the basis of the presence or absence of BCR/ABL tyrosine kinase activity in leukemia cells, it is also possible to discriminate between cases with BCR/ABL-dependent and -independent mechanisms of imatinib mesylate resistance. Notably, because the BCR/ABL enzymatic activity cannot be easily measured in blood or BM patient samples, levels of phosphorylation of the BCR/ABL substrate CRKL have been used as a surrogate end point for the tyrosine kinase activity(22) .
In patients with higher levels of CRKL phosphorylation despite treatment with imatinib mesylate, resistance has been found to result from at least three different BCR/ABL-dependent mechanisms: BCR/ABL gene amplification, BCR/ABL mutations, and high plasma levels of AGP (reviewed in Ref. 23 ).
The association of BCR/ABL gene amplification with resistance to imatinib mesylate is consistent with the reliance of CML blast crisis cells on BCR/ABL expression/activity for their proliferation and survival, and with the reported enhanced expression of BCR/ABL in these cells (24) . Indeed, high levels of BCR/ABL expression, which are detected frequently in CML-blast crisis but not CP cells, appear to be required for suppression of myeloid differentiation and increased resistance to chemotherapy-induced apoptosis ofBCR/ABL-expressing cells. Specifically, high levels of BCR/ABL kinase activity are required for hnRNP E2-dependent inhibition of CAAT/enhancer binding protein α, the major regulator of granulocytic differentiation (25) , and for the La-dependent enhancement of MDM2 expression, which, in turn, results in functional inactivation of p53(14) , also required for myeloid blastic transformation (26, 27) Although the mechanisms underlying such an increase of BCR/ABL expression are unclear, a double Ph′ chromosome is likely to be responsible for the enhanced BCR-ABL levels in some cases.
Other mechanisms of imatinib mesylate resistance involve mutations in the BCR/ABLgene itself. Several different mutations have been detected in at least 13 amino acids of the ATP-binding site or other regions of the tyrosine kinase domain, and the list is growing (23, 28, 29) . These mutations usually prevent imatinib mesylate from binding to BCR/ABL, thereby resulting in lack of inhibition of the tyrosine kinase activity. Among these mutations, substitution of a threonine to isoleucine at position 315 of ABL that prevents imatinib mesylate from binding to the ATP-binding domain, is the first described and one of the most frequent (22) .
A third mechanism of resistance relies on plasma levels of AGP. It has been shown that AGP binds imatinib mesylate at physiological concentrations in vitro and in vivo, and blocks the ability of imatinib mesylate to inhibit BCR/ABL kinase activity in a dose-dependent manner (reviewed in Ref. 23 ). Finally, in patients with primary refractoriness to imatinib mesylate, resistance more often occurs in absence of significant CRKL phosphorylation, suggesting activation of BCR/ABL-independent leukemogenic pathways.
In this issue, Mohamed et al.(30) hypothesized that clonal evolution, defined as acquisition of additional cytogenetic abnormalities other than t(9;22)(q34;q11), is a marker for genomic instability, and thereby, in this setting, additional “hits” can occur to activate BCR/ABL-independent leukemogenic mechanisms. Given this premise, CML patients with a more complex karyotype were expected to be more resistant to imatinib mesylate. To test their hypothesis, these authors analyzed 58 BCR/ABL-positive patients with IFN-α-resistant CP (n = 13), AP (n = 24), or BP (n = 21) CML with additional cytogenetic abnormalities who were each treated with imatinib mesylate. Of the 58 patients, 15 (CP = 46%; AP = 25%; BP = 14%) achieved a cytogenetic response, and 12 had a complete cytogenetic remission. With a follow-up of 17–30 months, 7 (12%) remained in complete remission on imatinib mesylate, supporting the notion that a subset of CML patients with t(9;22)(q34;q11) and additional cytogenetic abnormalities can achieve a sustained response. Because the molecular mechanisms of resistance in these patients were not fully evaluated, the contribution of additional cytogenetic abnormalities as an independent predictor of resistance to imatinib mesylate could not be directly addressed. Similar results were also reported in the imatinib mesylate initial studies (18, 20) , and more recently by Schoch et al.(31) . These authors reported on 31 patients with additional chromosomal abnormalities present before the start of imatinib mesylate therapy. Additional cytogenetic abnormalities were less frequent in chronic than other phases of the disease (35.5% versus 68.8%; P = 0.0008), and when corrected for the difference in disease stage, they did not influence response to imatinib mesylate. These results have been confirmed recently by a larger study reported by Cortes et al.(32) where 498 CML patients in CP or AP were treated with imatinib mesylate. Of the 498, 121 had additional cytogenetic abnormalities. In a multivariate analysis at the 3-month time point, lack of cytogenetic response, but not presence of additional cytogenetic abnormalities, was found to be a negative prognostic factor for survival
Although a longer follow-up is necessary to draw definitive conclusions, these findings suggest that additional cytogenetic aberrations do not appear to impact on disease response, and, therefore, karyotype analysis should not be used to stratify patients for therapeutic alternatives to imatinib mesylate. Furthermore, despite clonal evolution, the oncogenic potential of BCR/ABL appears to remain the driving force for leukemogenesis, and, therefore, may continue to serve as a therapeutic target. Finally, in patients with additional cytogenetic abnormalities who are resistant to imatinib mesylate, evaluation for levels of CRKL-phosphorylation should be done to sort out the nature of the resistance to this treatment and to understand the interplay between BCR/ABL-dependent and -independent mechanisms in disease progression.
Concluding Remarks
The results obtained with imatinib mesylate to date are truly impressive, but longer follow-up will be necessary to establish whether prevention or delay of blast crisis can be achieved and whether an improved overall survival for the majority of patients with CML can be obtained. It is clear that complete cytogenetic remission is achievable in most patients with CP CML, and that those who do not achieve this important end point between 3 and 6 months are likely to have a poor outcome. However, in patients with complete cytogenetic remission, other challenges remain. In these patients, for instance, molecular remission defined as the absence of BCR/ABL fusion transcript by RT-PCR is usually not achieved. The reasons for persistent low levels of residual disease after imatinib mesylate and the prognostic significance of these findings are unknown. It is possible that small numbers of mutated and resistant BCR/ABL-positive subclones remain essentially unaffected by this treatment, and if a proliferation advantage is subsequently acquired in these cells, they may drive disease recurrence. Therefore, in patients with complete cytogenetic remission, monitoring of the BCR/ABL fusion transcript levels by quantitative RT-PCR has been suggested to predict impending relapse, and if rising levels of BCR/ABL expression are detected, consideration could be given to alternative strategies including alloBMT.
However, emerging data support the notion that hematologic or cytogenenetic remission can be achieved in imatinib-relapsed CP CML patients with higher doses of this agent. Kantarjian et al. reported on 54 patients with CML in CP who were initially treated with 400 mg of imatinib mesylate and, subsequently, with a higher dose (i.e., 800 mg) when hematologic or cytogenetic resistance or relapse developed (33) . Among 20 patients treated for hematologic resistance or relapse, 13 (65%) achieved CHR without complete cytogenetic remission, and among 34 patients treated for cytogenetic resistance or relapse, 19 (56%) achieved a complete cytogenetic remission or MCR. However, the mechanisms of resistance to standard dose were not evaluated in these patients and, therefore, correlation of these mechanisms with clinical response to the higher doses was not possible. Nevertheless, these data are intriguing and pose the question of whether a higher dose of imatinib mesylate could be used at the time of the initial diagnosis or during molecular relapse to prevent overt leukemia recurrence or blast transformation. Future studies will no doubt address these important issues. Regardless, it is undeniable that imatinib mesylate has changed our approach to CML and paved the way for additional molecular targeted strategies in leukemia.
Footnotes
↵1 Supported in part by P30-CA16058, and K08-CA90469 Grants from the National Cancer Institute, Bethesda, MD, The Elsa U. Pardee Cancer Research Foundation, and The Coleman Leukemia Research Foundation.
↵2 To whom requests for reprints should be addressed, at The Ohio State University, 458A Starling-Loving Hall, 320 West 10th Avenue, Columbus, OH 43210. Phone: (614) 293-7597; Fax: (614) 293-7527; E-mail: marcucci-1@medctr.osu.edu.
↵3 The abbreviations used are: CML, chronic myelogenous leukemia; Ph′, Philadelphia; ABL, Abelson; RT-PCR, reverse transcription-PCR; BM, bone marrow; CHR, complete hematologic remission; MCR, major cytogenetic response; alloBMT, allogeneic bone marrow transplantation; AGP, α1 glycoprotein; CP, chronic phase; AP, accelerated phase; BP, blastic phase.
Based on the results of a new study, researchers are developing a clinical trial to test imatinib (Gleevec) in patients with anaplastic large cell lymphoma (ALCL), an aggressive type of non-Hodgkin lymphoma that primarily affects children and young adults.
The researchers found that a protein called PDGFRB is important to the development of a common form of ALCL. PDGFRB, a growth factor receptor protein, is a target of imatinib. Imatinib had anticancer effects in both a mouse model of ALCL and a patient with the disease, Dr. Lukas Kenner of the Medical University of Vienna in Austria and his colleagues reported October 14 in Nature Medicine.
The authors decided to investigate the effect of imatinib after finding a link between PDGFRB and a genetic abnormality that is found in many patients with ALCL. Previous work had shown that this genetic change—a translocation that leads to the production of an abnormal fusion protein called NPM-ALK—stimulates the production of two proteins, transcription factors called JUN and JUNB.
In the new study, experiments in mice revealed that these proteins promote lymphoma development by increasing the levels of PDGFRB.
Because imatinib inhibits PDGFRB, the authors tested the effect of the drug in mice with the NPM-ALK change and found that it improved their survival. They also found that imatinib given together with the ALK inhibitor crizotinib (Xalkori) greatly reduced the growth of NPM-ALK-positive lymphoma cells in mice.
To test the treatment strategy in people, they identified a terminally ill patient with NPM-ALK-positive ALCL who had no other treatment options and agreed to try imatinib. The patient began to improve within 10 days of starting the therapy and has been free of the disease for 22 months, the authors reported.
The observation that inhibiting both ALK and PDGFRB “reduces lymphoma growth and alleviates relapse rates” led the authors to suggest that the findings might be relevant to lymphomas with PDGFRB but without the NPM-ALK protein. “Our findings suggest that imatinib is a potential therapeutic option for patients with crizotinib-resistant lymphomas.”
A planned clinical trial will be based on the expression of PDGFRB in tumors.
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Cortes J. E., Talpaz M., Giles F., O’Brien S., Rios M. B., Shan J., Garcia-Manero G., Faderl S., Thomas D. A., Wierda, W., Ferrajoli A., Jeha S., Kantarjian H. M. Prognostic significance of cytogenetic clonal evolution in patients with chronic myelogenous leukemia on imatinib mesylate therapy. Blood, : 2003.
Kantarjian H. M., Talpaz M., O’Brien S., Giles F., Garcia-Manero G., Faderl S., Thomas D., Shan J., Rios M. B., Cortes J. Dose escalation of imatinib mesylate can overcome resistance to standard-dose therapy in patients with chronic myelogenous leukemia. Blood, 101: 473-475, 2003.
RNA Sequencing led to Targeting FLT3 Gene on Chromosome 13 for Receptor Blockage causing REMISSION in Adult Acute Lymphoblastic Leukemia (ALL), Aviva Lev-Ari, PhD, RN
There are 40 million women seeking mammography breast-screening every year in the USA, out of which 15 million are women with heterogeneously dense or extremely dense breasts. USA epidemiology statistics show that 6 out of 7 missed cancers at mammography occur in women with dense breasts. It is also known that the majority of women presenting with mammography-dense breasts are below 45 years old.
The Oct. 22 issue of the American Journal of Roentgenology ( AJR) publishes results of a study showing that ultrasound is superior to mammography in evaluating symptomatic women 30-39 years of age [1].
This study was conducted by researchers at the Seattle Cancer Alliance and University of Washington. Patients were recruited between January 2002 and August 2006. 954 women ranging from 30 to 39 years old who presented for diagnostic breast imaging evaluation were examined, and it was found that sensitivity (probability for cancer detection) of ultrasound was 95.7 percent compared to 60.9 percent for mammography. A very important result of this study is the calculated Negative Predictive Value (the probability to have negative pathology if the imaging-test is negative) which was similar for both modalities: 99.9% for ultrasound and 99.2% for mammography.
Show case in images (All images courtesy of the American Roentgen Ray Society.):
35-year-old woman who presented with a palpable left breast lump. Whole-breast craniocaudal (above left) and mediolateral oblique (above right) and spot-magnification craniocaudal (below left) and mediolateral (below right) mammographic images show no abnormality at area of clinical concern, marked by BB.
Zoom-in on the region of interest
Targeted ultrasound image above reveals solid mass with irregular shape and indistinct and angular margins. BI-RADS 5 assessment was made. Histopathology from ultrasound-guided core needle biopsy showed invasive ductal carcinoma.
In regards to which imaging modality should be used when screening such a population, the conclusion of the investigators is very clear: “Ultrasound has high sensitivity (95.7%) and high NPV (99.9%) in this setting and should be the primary imaging modality of choice. The added value of adjunct mammography is low.”
When reading this article I noted a gap to overcome if we want to successfully replace mammography with ultrasound. The Positive Predictive Value (the probability of detecting a cancer) calculated for ultrasound in these study settings was lower than that calculated for mammography: 13.2% for ultrasound and 18.4% for mammography. This is because mammography detected one additional malignancy in an asymptomatic area in a 32-year-old woman who was subsequently found to have a BRCA2 gene mutation. Mammography could do that because it scans the whole breast, whereas the investigators in this study used ultrasound just for scanning the suspicious lumps. A solution is offered in using the recently introduced ultrasound modalities, which are able to perform automatic full breast ultrasound scans [2], preferably enhanced by real-time tissue characterisation capability – a technology I’m working to develop.
1 Department of Radiology, University of Washington School of Medicine, Seattle WA.2 Seattle Cancer Care Alliance, G2-600, 825 Eastlake Ave E, Seattle, WA 98109.
2. Using Automated Breast Sonography as Part of a Multimodality Approach to Dense Breast Screening, Vincenzo Giuliano, MD, RDMS, RVT1, Concetta Giuliano, DO1, Journal of Diagnostic Medical SonographyJuly/August 2012 28: 159-165,
1Novasoutheastern University, Winter Springs, FL, USA Written by: Dror Nir, PhD.
Otto Heinrich Warburg (October 8, 1883 – August 1, 1970), son of physicist Emil Warburg, was a German physiologist, medical doctor and Nobel laureate.
Otto Heinrich Warburg was born on October 8, 1883, in Freiburg, Baden. His father, the physicist Emil Warburg, was President of the Physikalische Reichsanstalt, Wirklicher Geheimer Oberregierungsrat. He was a member of the Warburg family, a prominent family and financial dynasty of German Jewish descent, noted for their varied accomplishments in physics, classical music, art history, pharmacology, physiology, finance, private equity and philanthropy. They are believed to be descended from the Venetian Jewish del Banco family, in the early 1500s one of the wealthiest Venetian families. The Warburgs fled from Italy to Warburg in Germany in the 16th century before moving to Altona, near Hamburg in the 17th century taking their surname from the city of Warburg. The brothers Moses Marcus Warburg(1763 – 1830) and Gerson Warburg (1765 – 1826) founded the M. M. Warburg & Co. banking company in 1798 that is still in existence.
Otto studied chemistry under the great Emil Fischer, and gained the degree, Doctor of Chemistry (Berlin), in 1906. He then studied under von Krehl and obtained the degree, Doctor of Medicine (Heidelberg), in 1911.
He served as an officer in the elite Uhlan (cavalry regiment) during the First World War, and won the Iron Cross (1st Class) for bravery. Warburg was one of the 20th century’s leading biochemists. [1] He won the Nobel Prize of 1931. In total, he was nominated an unprecedented three times for the Nobel prize for three separate achievements.
While working at the Marine Biological Station, Warburg performed research on oxygen consumption in sea urchin eggs after fertilization, and proved that upon fertilization, the rate of respiration increases by as much as sixfold. His experiments also proved iron is essential for the development of the larval stage.
In 1918, Warburg was appointed professor at the Kaiser Wilhelm Institute for Biology in Berlin-Dahlem (part of the Kaiser-Wilhelm-Gesellschaft). By 1931 he was named director of the Kaiser Wilhelm Institute for Cell Physiology, which was founded the previous year by a donation of the Rockefeller Foundation to the Kaiser Wilhelm Gesellschaft (since renamed the Max Planck Society).
Warburg’s early researches with Fischer were in the polypeptide field.
At Heidelberg he worked on the process of oxidation. His special interest in the investigation of vital processes by physical and chemical methods led to attempts to relate these processes to phenomena of the inorganic world. His methods involved detailed studies on the assimilation of carbon dioxide in plants, the metabolism of tumors, and the chemical constituent of the oxygen transferring respiratory ferment. Warburg was never a teacher, and he has always been grateful for his opportunities to devote his whole time to scientific research. His later researches at the Kaiser Wilhelm Institute have led to the discovery that the flavins and the nicotinamide were the active groups of the hydrogen-transferring enzymes.
This, together with the iron-oxygenase discovered earlier, gives a complete account of the oxidations and reductions in the living world. Warburg investigated the metabolism of tumors and the respiration of cells, particularly cancer cells, and in 1931 was awarded the Nobel Prize in Physiology for his “discovery of the nature and mode of action of the respiratory enzyme.”[2]
The award came after receiving 46 nominations over a period of nine years beginning in 1923, 13 of which were submitted in 1931, the year he won the prize. This discovery opened up new ways in the fields of cellular metabolism and cellular respiration. He hypothesized, among other things, that cancerous cells can live and develop, even in the absence of oxygen. Warburg also wrote about oxygen’s relationship to the pH of cancer cells’ internal environments, since fermentation was a major metabolic pathway of cancer cells.
Three scientists who worked in Warburg’s lab, including Sir Hans Adolf Krebs, went on to win the Nobel Prize. Among other discoveries, Krebs is credited with the identification of the citric acid cycle (or Szent györgyi-Krebs cycle).
In 1944, Warburg was nominated for a second Nobel Prize in Physiology by Albert Szent-Györgyi, for his work on nicotinamide, the mechanism and enzymes involved in fermentation, and the discovery of flavine (in yellow enzymes). Although he was considered a worthwhile candidate, he was not selected for the prize.
References
Krebs, HA (1972), “Warburg Heinrich Warburg. 1883-1970”, Biographical Memoirs of Fellows of the Royal Society (The Royal Society) 18: 628–699,doi:10.1098/rsbm.1972.0023
Som P; Atkins HL; Bandoypadhyay D et al. (1 July 1980), “A fluorinated glucose analog, 2-fluoro-2-deoxy-D-glucose (F-18): nontoxic tracer for rapid tumor detection”, J. Nucl. Med.21 (7): 670–5, PMID7391842
Chernow, Ron (1993), The Warburgs: The Twentieth-Century Odyssey of a Remarkable Jewish Family, New York, NY: Random House, ISBN0-679-41823-7
Advances in medicine, especially imaging technology have made the identification of small Acoustic Neuromas (AN) possible. After routine auditory tests reveal loss of hearing and speech discrimination (i.e. “I can hear sound in that ear, but can’t understand what’s being said”) a special test for hearing which records responses from the brain-stem called the auditory brainstem response test (ABR, BAER, BSER) maybe done. The results of this test detect the cause of a poorly functioning 8th nerve. If an abnormality in the ABR test suggests an AN, imaging is done to confirm the diagnosis. I do not perform the ABR test in all patients to diagnose an acoustic neuroma because imaging techniques (MRI/CT scans) are the gold standard for diagnosis. CT scan has proven to be a powerful tool in locating AN’s. The only drawback is that small tumors confined to the internal auditory canal (IAC) may not show on plain CT scan. Such cases require air or contrast materials to be introduced into the body in order to enhance the tumor. Therefore, the MRI, a more recently developed diagnostic test, has become the gold standard for diagnosis of AN. Gadolinium is the contrast material used to define & enhance the tumor.
Small tumorsA small tumor is also called intracanalicular because it is confined within the bony internal auditory canal (figure). A patient with such a tumor may have hearing loss, ringing in the ear or ear noise, and vertigo or dizziness.
Medium tumorsA medium sized acoustic neuroma is one that has extended from the bony canal into the brain cavity, but has not yet produced pressure on the brain itself (figure). Patients with such tumors have worsening of their hearing, difficulty in balance, in addition to dizziness, and occasionally, the onset of headaches due to irritation of the lining of the brain called dura. Some patients may experience numbness of the mid-face or diminished sensation in the eye during the later stages.
Large tumorsA large tumor is one that is extended out of the internal auditory canal in to the brain cavity and is sufficiently large to produce pressure on the brain and disturb vital centers in the brain (figure). During this stage, all previous symptoms worsen; facial twitch and weakness may occur, and finally patient may develop hydrocephalus due to the blockage of the cavity which contains CSF-the resultant symptoms are headache, visual loss and double vision.
Microscopy
The AN usually arises within the nerve trunk of the vestibular part of the 8th nerve. It gradually grows out of the nerve as it increases in size and assumes a peripheral position. The AN’s usually arise halfway along the length of the vestibular nerve, which corresponds to the transition zone of the nerve structure. The typical microscopic appearance of AN’s has two distinct features of arrangement of the cells-either tightly packed (Antoni A) or loosely packed (Antoni B) fibers. The distinction of these two cell types is of no clinical importance. Indeed, regions of Antoni A and B may coexist in the same tumor. As the tumor grows, it follows the direction of least resistance, usually towards the brain (cerebellopontine angle) and may reach considerable size. Thus, most tumors consist of 2 parts: a stalk or stem within the internal auditory canal (IAC) and another portion near the brain region. Microscopic investigations into the effect of AN’s on adjacent facial or 7th nerve have shown tumor involvement in some cases. This involvement may not be recognized by the surgeon during removal of the AN.
This picture shows the microscopic appearance of a normal vestibular (8th) nerve passing through the internal auditory canal (IAC) to supply the organ of balance. The facial (7th) nerve runs along with the 8th nerve in the IAC. The organ of hearing (cochlea) is also seen in this picture.
This picture shows an AN tumor arising from the 8th nerve, within the IAC.
This is a higher magnification of the above picture showing the junction of the tumor and the VII nerve. The arrows indicate the sheath (covering layer) of the tumor.
This picture is a high magnification of the same tumor showing the arrangement of the fibers within the AN. The arrow indicates a whorled appearance of the fibers while the upper part of the tumor shows loosely packed (Antoni B) fibers.
Origin and Cause
ACOUSTIC NEUROMA-THE BASIC FACTS
Origin and Cause
What is an acoustic neuroma?
An acoustic neuroma (sometimes also termed a neurinoma or vestibular schwannoma) is a benign or non-cancerous growth that arises from the 8th or vestibulo-cochlear nerve. The 8th nerve is actually 2 separate nerves, the vestibular nerve and the cochlear nerve. The vestibular nerve is responsible for balance while the cochlear nerve is responsible for hearing. The vestibular nerve has 2 parts-the superior vestibular nerve (SVN) and the inferior vestibular nerve (IVN).These nerves lie adjacent to each other as they pass through a bony canal, from the inner ear to the brainstem. This bony canal is called the internal auditory canal (IAC) and it varies in length from 0.4 to 1.2 cm. We have two figures of a temporal bone (that part of the skull which has the ear in it) dissection to the right.The first figure is a view from the top showing the middle ear and the internal auditory canal (IAC) with the nerves passing through it. The organ of hearing (cochlea) and the dura lining the IAC can be seen clearly.The second figure is a magnification of the IAC region showing the different nerves passing through it. This figure also demonstrates clearly, the cochlear nerve supplying the cochlea. Acoustic neuromas usually arise from the cells of the VIII nerve within the internal auditory canal (third figure).
The third figure is a schematic drawing showing an acoustic neuroma arising from the vestibular nerve within the IAC. The facial or 7th nerve that is responsible for facial movement, along with important blood vessels, also passes with the 8th nerve in the canal (figures).
The cause of acoustic neuroma is unknown. A small percentage of individuals have a hereditary condition called neurofibromatosis type 2 (NF-2). These patients may have an acoustic neuroma on both sides with an aggressive growth pattern and often involve adjacent nerves.
What is the growth pattern?
Acoustic neuromas usually grow very slowly over a period of many years. Once the tumor fully occupies the internal auditory canal, it often begins to erode the walls of the canal and enlarges it. This bony erosion however, does not always occur. They typically remain within their capsule or lining and displace the surrounding nerves and brain tissue very slowly. This is why the body has ample time to accommodate the abnormal growth. The tumor first distorts the 8th nerve, and then presses on the adjacent 7th nerve. The 7th nerve is gradually stretched into a ribbon like structure over the enlarging tumor (figure; cross section of the 7th nerve is shown in the right half of the figure). As the tumor slowly enlarges towards the brain, it protrudes from the internal auditory canal into an area of the skull called cerebello-pontine angle. The tumor is now pear or mushroom shaped with the smaller end within the canal and the larger part towards the brain (figure). It is at this stage that the tumor presses adjacent nerves like the trigeminal or 5th nerve responsible for facial sensation. Ultimately, with increasing tumor size, it can press on the brainstem which can be life threatening.
How often do acoustic neuromas occur?
Acoustic neuromas have been known to occur in all areas of the world without any predilection for individuals of any ethnic background. Small AN’s without any symptoms, have been found on autopsy in 2.5% of the general population. Estimates of symptomatic AN range from 1 in every 3,500 to 5 in every million people. It appears that women are more affected than men and most AN’s are diagnosed between the ages of 30 & 60 years.
Symptoms
Early symptoms of AN can occur in other conditions of the ear that can be easily overlooked. Early diagnosis of AN is quite challenging because there is no typical pattern. However, there are symptoms that act as indicators to the possibility if an AN. Patients with “inner ear” problems should be completely evaluated to rule out AN as a cause of these symptoms. It is possible that Meniere’s disease or hardening of the bone of the middle ear (otosclerosis) could be causing these symptoms. Patients with AN may present the following symptoms:
Hearing loss
Ringing in the ears (tinnitus)
Dizziness (vertigo)
Difficulty in balance (imbalance or dysequilibrium)
Fullness or pressure in the ears
Facial numbness or paralysis (for very large tumors)
HEARING LOSS
In over 90 percent of the patients with AN, the first symptom is a reduction in hearing in one ear due to involvement of the VIII nerve. This is usually accompanied by ringing in the ears or ear noise-also called “tinnitus”. The hearing loss is usually subtle and worsens very slowly over a period of time. In some cases, the hearing loss may be sudden. Some patients may experience a sense of fullness in the affected ear. Unfortunately, since hearing loss is often mild and there is no pain, patients tend to ignore the change in hearing and merely shift the phone to the opposite ear or make other compromises for the one-sided hearing loss rather than seek medical attention.
VERTIGO & IMBALANCE The tumor usually arises from the vestibular or balance nerve. As a result, unsteadiness or balance problems may be one of the earlier symptoms in the growth of the tumor. Since the remainder of the balance system compensates for this loss, balance problems may be forgotten after some time.
If the tumor grows larger in size it may start to press on other nerves, mainly the trigeminal nerve, causing facial sensation to become affected. Patients may then experience constant or intermittent numbness and facial tingling. Patients may also have facial tics or spasms. If the tumor grows larger and presses on the brainstem raised intracranial pressure may cause headaches, facial weakness, vertigo and an unsteady gait to ensue.
Treatment There are 3 treatment options available for AN
1) Observation
2) Microsurgical removal (partial or total)
3) Stereotactic radiation therapy (radiosurgery)
Observation AN are occasionally discovered incidentally while evaluating another problem or when the tumor is very small with subtle symptoms. Since AN are benign tumors and produce symptoms due to pressure on surrounding structures, careful observation over a period of time may be appropriate for some patients. For instance, a small tumor diagnosed in an elderly patient may only require observation to study the growth rate of the tumor if acute symptoms are not present. If it appears that the tumor will not need to be treated during the patient’s normal life expectancy, treatment and its potential risks and complications maybe avoided. In these patients, MRI is performed periodically to monitor growth of the tumor. If there is no growth, observation is continued. On the other hand, if the tumor shows increase in size, treatment may become necessary. Another group of patients for whom observation is preferred is in patients who have a tumor in their only or better hearing ear, particularly if it is a size where hearing preservation is unlikely. In such cases, periodic MRI is done to monitor growth and surgery is considered only if the hearing is lost or the tumor size becomes life threatening.
Microsurgical removal At the present time, the only treatment that can cure the patient is removal of the tumor by surgery. Within the last 2 decades, microsurgical techniques have been pioneered and refined. Use of the operating microscope, finely scaled surgical instruments, alternate cutting & tumor reducing tools, and better anesthesia, have reduced the death rate extremely. In addition, results have improved as surgeons have gained experience in the delicate removal process of the tumor.
Three main surgical approaches are used depending upon the location, tumor size and hearing level of the patient. They are- middle fossa (MF), sub-occipital (SO), and the trans-labyrinthine (TL) approach. Surgery for AN’s is done under general anesthesia using an operating microscope. Postoperatively, one to several days may be spent in the intensive care with careful monitoring. Problems that may develop in the immediate postoperative period including headache, dizziness, imbalance, vomiting and decreased mental alertness due to the development of a blood clot causing obstruction to the flow of cerebrospinal fluid (CSF).
Other early complications may include cerebrospinal fluid leak and meningitis, an infection controlled with antibiotics that will require a longer hospitalization. Some patients and their surgeons prefer incomplete removal of an AN in order to reduce the risk of complications, realizing that further surgery maybe needed in the future. Occasionally in cases with large tumors, disturbances in the vital brain centers during surgery require ending the surgery prior to complete tumor removal. In these cases, the tumor which was left behind is followed with MRI scans and if tumor growth is demonstrated, further surgery maybe necessary to remove the growing tumor. On the other hand, if the tumor shows no growth, observation is continued. Partial tumor removal maybe also be required in a patient with an only hearing ear such as a Neurofibromatosis-2 (NF 2) patient. Unfortunately, partial removal may result in substantial hearing loss in these patients and this risk must be considered.
Small tumor
If the hearing is still preserved in such tumors, a middle fossa approach, incision for which is in front of the ear (figure) may be considered. A small square piece of bone from the side of the skull is then removed (blue shaded area in the figure). The tumor is removed completely in most cases. On rare occasions, partial removal is possible. This approach attempts to preserve the hearing in all cases while removing the tumor. In about half of the patients, the tumor involves the hearing nerve or the artery supplying the inner ear and in such cases, total loss of hearing occurs in the operated ear. In addition, the risk to the facial nerve is far greater in this approach/
Medium tumor
The operation for medium sized tumors is performed by the sub-occipital and/or the trans-labyrinthine approach. The incision for these approaches is behind the ear, overlying the mastoid, the bony projection felt behind the ear (figures). The mastoid and the inner ear structures are removed to expose the tumor, and remove it completely. The opening created in the mastoid bone is closed with fat taken from the abdomen. The translabyrinthine approach sacrifices the hearing and balance mechanism since the inner ear is entered. Consequently, the ear is made permanently deaf. In such cases, the balance mechanism of the opposite ear compensates for the non-functioning operated ear and provides stabilization for the patient within few weeks to months.
Large tumor
Surgery for large tumors requires extensive removal of bone to properly expose the tumor and control the large blood vessels that make access to the tumor difficult. For this reason, special studies of the arteries (arteriograms) may be required in addition to the other investigations, in order to diagnose and establish the size of the acoustic tumor. The operation for large tumors is performed by the TL-SO approach as described for medium tumors. The figure to the right shows the area of the skull approached via the TL and the SO approaches. In these patients, total removal is attempted unless changes in vital signs occur. If there are changes in blood pressure, pulse rate, or respiratory rate, the surgery must be terminated even if the tumor has not been totally removed. The opening in the mastoid is closed with abdominal fat. For large tumors, it is often necessary to monitor the patient’s general status by inserting a small tube (arterial line) into an artery in the arm or leg. In these cases, occasionally a blood clot may form in the artery following surgery. In case this complication occurs, further surgery maybe required to remove the blood clot. A very rare complication of this arterial line monitoring is the loss of a finger, toe, or even a hand or a foot.
Stereotactic Radiation Therapy (Radiosurgery): This is a technique based on the principle that a single relatively high dose of radiation delivered precisely to a small area will arrest or kill the tumor while minimizing injury to the surrounding nerves & brain tissue. The source of radiation is from either radioactive cobalt (called gamma ray) or a linear accelerator (LINAC). The treatment team consists of a neurosurgeon, radiophysicist and a radiation oncologist working together to develop a treatment plan based on the size & shape of the tumor. Radiation, even at relatively high doses such as those used in radiosurgery, does not kill or injure cells immediately. Some tumor cells die in weeks while others die more gradually over 6-18 months after radiation. This treatment usually arrests growth of the tumor and some tumors shrink, but they rarely disappear.
Follow-up of these patients is important because approximately 20% of tumors continue to grow after radiosurgery or at some time in the future. A tumor that has been irradiated and grows may be more difficult to remove than an un-radiated tumor. Symptoms such as dizziness & disturbances in balance typically improve earlier after microsurgical tumor removal than after radiosurgery. This is because effects of radiosurgery may require up to 18 months. Residual dizziness & imbalance may be less after microsurgical treatment. The side effects of radiosurgery may be headache, dizziness, nausea, facial numbness, or rarely, cranial nerve paralysis. In the long term requires follow-up MRI’s over the years and there is a potential for additional treatment in cases of continued growth or later re-growth.
Microsurgery requires follow-up MRI’s suggested at perhaps 1 and 5 years if the tumor has been completely removed. Radiosurgery may be considered in selected patients in whom the risk of surgery is excessive because of advanced age or pre-existing health problems, patients having small to moderate sized tumors or patients with tumors on both sides, or in the only hearing ear.
Postoperative
Microsurgery of an AN is a complex and delicate procedure. The smaller the tumor at the time of surgery, the fewer the chances are for complications. As the tumor size increases, the chances of complications become greater. Thus, there may be problems with the cranial nerves affected by the tumor (like facial paralysis or hearing loss) following surgery that may or may not have been present before tumor removal. Here is a list of some of the more common post-operative issues and problems encountered.
Residual problems
This period is the days or perhaps weeks following surgery. There is a possibility of fatigue or tiredness and increased drowsiness, although some patients may experience “survival euphoria” and a renewed sense of energy and vigor. A period of emotional lows is common as the patient adjusts to physical changes. One symptom that may occur after discharge is a nasal drip of clear colorless fluid, which is particularly noticeable when bending over. This may indicate a cerebrospinal fluid leak and should be reported to the surgeon right away due to the risk of infection.
Follow-up period :After being discharged from the hospital, patients operated for an AN are followed up regularly (every 2-3 months for the first year, every 6 months for the 2nd year, and every year thereafter). These follow up visits are important to monitor the hearing (in patients operated by the MF or SO approach), facial nerve paralysis if any and for recurrence of tumor.
HEARING LOSS With small tumors, it may be possible to save hearing. In larger tumors, especially those that have extended into the brain cavity, the hearing has usually been partially or totally lost and cannot be restored. This loss means the patient will continue having problems locating sound, hearing on the deaf side and understanding speech over high background noise. Consultation with an audiologist is required for these patients for amplification options like traditional hearing aids or a CROS hearing aid (a device which crosses sound over from the operated ear to the opposite ear) or a BAHA.
TINNITUS Ear noises usually remain the same as before surgery, though in a few cases noises may increase or begin after surgery. A masking device may help some people affected by tinnitus.
FACIAL WEAKNESS OR PARALYSIS Since the facial nerve which controls muscles of facial expression is in close proximity with the AN, it is usually necessary to manipulate and at times remove the portion of the nerve. In some cases however, even though the nerve is intact after surgery, nerve damage or swelling may cause temporary or in some cases permanent facial paralysis. Regrowth of the nerve is a slow process that may take up to a year for recovery to be noticeable. If recovery is not observed by 1 year, a second operation may be required to connect the healthy portion of the facial nerve to a nerve in the neck usually the one supplying one side of the tongue. This procedure is called the hypoglossal-facial nerve anastamosis and can restore some but not all facial movement. Spontaneous movements like laughing are asymmetric. There may be loss of tongue function. There are some other procedures that adapt available muscles and nerves to help in toning or reanimating the sagging face. If it becomes necessary to remove a portion of the facial nerve during surgery, the facial nerve may be reconnected directly or by inserting a nerve graft. Usually, the result is asymmetric but will provide some spontaneous movement.
EYE PROBLEMS
Studies have shown that at least half of those who have had an acoustic neuroma removed develop long term eye discomfort and other eye problems, particularly if the tumor was medium or large. Loss of eyelid function and/or altered tear production can cause irritation and scratchiness in the eye because it is dry & unprotected. To deal with this problem, there are various surgical procedures that can be done to protect the cornea. They include canthoplasty (bringing together tendons in either or both corners of the eye), a spring implantation in the upper lid, an elastic prosthesis secured around the upper and lower lids, a gold weight implant in the upper lid; and a tarsorapphy (sewing the lids together). Artificial tears or eye lubricants maybe needed for a short time or permanently. Taping part of the lids together, using protective glasses and moisture chamber, using bandage contact lenses and avoiding eye irritants may be helpful. In a few patients, double vision may be present due to pressure on the 6th cranial nerve that controls the muscles that move the eyes.
TASTE DISTURBANCE AND MOUTH DRYNESS OR EXCESSIVE SALIVATION There maybe some changes in taste and amount of saliva secretion for a short time following surgery. In some cases this may be prolonged. In the others, increased salivation occurs while chewing or there maybe increased tearing while eating. The appetite maybe affected for some time.
SWALLOWING, THROAT AND VOICE PROBLEMS In a small number of patients, AN surgery affects the nerves which control the throat, swallowing and voice production leading to hoarseness & difficulty in swallowing. These symptoms usually improve slowly over time.
BALANCE PROBLEMS The vestibular portion of the VIII nerve is almost always removed during surgery. Usually this part of the nerve is non-functional and has already been destroyed because of the AN. Dizziness is common following surgery and maybe severe for a time. After a while, the balance apparatus of the opposite or normal ear compensates for this loss, and balance improves. This compensation may not be perfect, particularly in darkness, when the patient is fatigued, when there is a sudden change in body position, or while walking on uneven surfaces. Maintaining a good general physical health through proper diet and moderate exercise, can improve balance & general vitality to a great extent.
FATIGUE Fatigue sometimes remains a prolonged problem for some patients after some of the other symptoms have subsided. It is important in such patients to adjust their pace of life in harmony with their energy level.
HEADACHE
Headaches can be a problem for some patients while still in the hospital. This maybe related to tension from holding the head rigidly, changes in intracranial pressure, muscle spasm, or anxiety. Headaches are almost never related to tumor recurrence. Treatment is with analgesics & muscle relaxation. If severe headaches persist after hospital discharge, medical help should be sought.
DENTAL CARE If the patient has facial paralysis, food tends to get lost in the mouth on the affected side and can lead to dental problems. Washing and rinsing the mouth is therefore necessary, as well as brushing & flossing the teeth several times a day is important.
PROTECTING THE OTHER EAR It is important to provide sensible protection to the opposite or good ear that has the remaining hearing apparatus. This is done by avoiding extreme or sudden exposure to loud noises like firearms or some cordless phones near the good ear. Some physicians suggest follow-up MRI scans and/or audiograms for some time following AN removal.
PSYCHOLOGICAL COPING For some patients, adjustment to a new self after AN removal can be a challenging task. This is because in addition to changes in hearing, the appearance may now be altered along with the presence of other impairments. Return to normal activity may be slow. Concentrating on strengths rather than on weaknesses will help such patients to return to all former activities and also expand their abilities in new areas.
Department of Radiation Oncology, Massachusetts General Hospital, Boston, Massachusetts, USA. damien.weber@psi.ch
Abstract
OBJECTIVE:
We sought to determine the tumor control rate and cranial nerve function outcomes in patients with vestibular schwannomas who were treated with proton beam stereotactic radiosurgery.
METHODS:
Between November 1992 and August 2000, 88 patients with vestibular schwannomas were treated at the Harvard Cyclotron Laboratory with proton beam stereotactic radiosurgery in which two to four convergent fixed beams of 160-MeV protons were applied. The median transverse diameter was 16 mm (range, 2.5-35 mm), and the median tumor volume was 1.4 cm(3) (range, 0.1-15.9 cm(3)). Surgical resection had been performed previously in 15 patients (17%). Facial nerve function (House-Brackmann Grade 1) and trigeminal nerve function were normal in 79 patients (89.8%). Eight patients (9%) had good or excellent hearing (Gardner-Robertson [GR] Grade 1), and 13 patients (15%) had serviceable hearing (GR Grade 2). A median dose of 12 cobalt Gray equivalents (range, 10-18 cobalt Gray equivalents) was prescribed to the 70 to 108% isodose lines (median, 70%). The median follow-up period was 38.7 months (range, 12-102.6 mo).
RESULTS:
The actuarial 2- and 5-year tumor control rates were 95.3% (95% confidence interval [CI], 90.9-99.9%) and 93.6% (95% CI, 88.3-99.3%). Salvage radiosurgery was performed in one patient 32.5 months after treatment, and a craniotomy was required 19.1 months after treatment in another patient with hemorrhage in the vicinity of a stable tumor. Three patients (3.4%) underwent shunting for hydrocephalus, and a subsequent partial resection was performed in one of these patients. The actuarial 5-year cumulative radiological reduction rate was 94.7% (95% CI, 81.2-98.3%). Of the 21 patients (24%) with functional hearing (GR Grade 1 or 2), 7 (33.3%) retained serviceable hearing ability (GR Grade 2). Actuarial 5-year normal facial and trigeminal nerve function preservation rates were 91.1% (95% CI, 85-97.6%) and 89.4% (95% CI, 82-96.7%). Univariate analysis revealed that prescribed dose (P = 0.005), maximum dose (P = 0.006), and the inhomogeneity coefficient (P = 0.03) were associated with a significant risk of long-term facial neuropathy. No other cranial nerve deficits or cancer relapses were observed.
CONCLUSION:
Proton beam stereotactic radiosurgery has been shown to be an effective means of tumor control. A high radiological response rate was observed. Excellent facial and trigeminal nerve function preservation rates were achieved. A reduced prescribed dose is associated with a significant decrease in facial neuropathy.
The Proton Beam Unit was founded in 1962 and has the largest experience with stereotactic radiosurgery of any center in the United States. Information regarding non-invasive proton beam radiosurgery and fractionated radiosurgery for brain and spinal tumors and arteriovenous malformations.The Purpose of this Center is to provide a complete range of services for the diagnosis, and treatment with non-invasive proton beam radiosurgery and fractionated radiosurgery for brain and spinal tumors and arteriovenous malformations. Patients may be referred for consultation only, care in partnership with referring physician, or complete management.
Bragg Peak Proton Beam Radiosurgery Unit – The Proton Beam Unit was founded in 1962 and has the largest experience with stereotactic radiosurgery of any center in the United States. Proton beam offers certain theoretical advantages over other modalities of stereotactic radiosurgery (i.e. gamma knife and linear accelerators) because it makes use of the quantum wave properties of protons to reduces doses to surrounding tissue beyond the target to a theoretical minimum of zero. In practice, the proton facility offers advantages for the treatment of unusually shaped brain tumors and arteriovenous malformations. The homogeneous doses delivered also makes fractionated therapy possible. Proton beam radiosurgery also has the ability to treat tumors outside of the cranial cavity. These properties make it the ideal post-resection therapy for many chordomas and certain chondrosarcomas of the spine and skull base as well as an excellent mode of therapy for many other types of tumors.
NPTC: Information, proton radiosurgery treatments and support services have been transfered to the new The Northeast Proton Therapy Center (NPTC). Located on the main hospital campus of the Massachusetts General Hospital (MGH), the NPTC represents the forefront of technological advancement in radiation therapy. The construction of the facility was jointly funded by the hospital and the National Cancer Institute to meet the increasing medical demand for high precision radiation therapy provided by proton therapy. The program builds on more than forty years of pioneering work and experience gained by the physicians, physicists, and clinical support personnel at Harvard University’s Cyclotron Laboratory where more than nine thousand patients were treated with proton therapy from 1961 to it’s closing in 2002.
Rabinov JD, Brisman JL, Cole AJ, Lee PL, Bussiere MR, Chapman PH, Loeffler JS, Cosgrove GR, Chaves T, Gonzalez RG.: MRI changes in the rat hippocampus following proton radiosurgery. Stereotact Funct Neurosurg. 2004;82(4):156-64.
Brisman JL, Cole AJ, Cosgrove GR, Thornton AF, Rabinov J, Bussiere M, Bradley-Moore M, Hedley-Whyte T, Chapman PH.: Radiosurgery of the rat hippocampus: magnetic resonance imaging, neurophysiological, histological, and behavioral studies. Neurosurgery. 2003 Oct;53(4):951-61; discussion 961-2.
Weber DC, Chan AW, Bussiere MR, Harsh GR 4th, Ancukiewicz M, Barker FG 2nd, Thornton AT, Martuza RL, Nadol JB Jr, Chapman PH, Loeffler JS.: Proton beam radiosurgery for vestibular schwannoma: tumor control and cranial nerve toxicity. Neurosurgery. 2003 Sep;53(3):577-86; discussion 586-8.
Barker FG 2nd, Butler WE, Lyons S, Cascio E, Ogilvy CS, Loeffler JS, Chapman PH.: Dose-volume prediction of radiation-related complications after proton beam radiosurgery for cerebral arteriovenous malformations. J Neurosurg. 2003 Aug;99(2):254-63.
Harsh GR, Thornton AF, Chapman PH, Bussiere MR, Rabinov JD, Loeffler JS.: Proton beam stereotactic radiosurgery of vestibular schwannomas. Int J Radiat Oncol Biol Phys. 2002 Sep 1;54(1):35-44.
Barker FG 2nd, Amin-Hanjani S, Butler WE, Lyons S, Ojemann RG, Chapman PH, Ogilvy CS.: Temporal clustering of hemorrhages from untreated cavernous malformations of the central nervous system. Neurosurgery. 2001 Jul;49(1):15-24; discussion 24-5.
Chapman PH, Tarbell: Proton beam therapy. In: Pediatric Neurosurgery. Surgery of the Developing Nervous System, 4th ed. Ed: McLone DG: WB Saunders: Philadelphia, pp. 1255-1262, 2001.
Loeffler JS, Singer RJ, Chapman PH, Ogilvy CS: Proton-beam radiation therapy. In: LINAC and Gamma Knife Radiosurgery. Ed: Germano IM. The American Association of Neurological Surgeons: Park Ridge, IL, pp. 71-74, 2000.
Harsh G, Loeffler JS, Thornton A, Smith A, Bussiere M, Chapman PH: Stereotactic Proton Radiosurgery. Neurosurg Clin N Am 1999; 10:243-256.
Tatter SB, Butler WE, Chapman PH. Technical and clinical aspects of proton-beam stereotactic radiosurgery. In: Textbook of Stereotactic and functional Neurosurgery. Eds: Gildenberg PL, Tasker RR. McGraw-Hill, New York pp. 705-710, 1998.
Serago CF, Thornton AF, Urie MM, Chapman P, Verhey L, Rosenthal SJ, Gall KP, Niemierko A: Comparison of proton and x-ray conformal dose distributions for radiosurgery applications. Med Phys 22:2111-16, 1995.
Butler WE, Ogilvy CS, Chapman PH, Verhy L , Zervas NT. “Stereotactic alignment for Bragg peak radiosurgery.” In Radiosurgery: Baseline and Trends, ed. L. Steiner. 85-91. New York: Raven Press, 1992.
Chapman PH, Ogilvy CS , Butler WE. “A new stereotactic alignment system for charged-particle radiosurgery at the Harvard Cyclotron Laboratory, Boston.” In Stereotactic Radiosurgery, ed. Eben Alexander III, Jay S. Loeffler, and L. Dade Lunsford. 105-108. New York: McGraw-Hill, 1993.
De Salles AA, Asfora WT, Abe M, Kjellberg RN: Transposition of target information from the magnetic resonance and computed tomography scan images to conventional X-ray stereotactic space. Applied Neurophysiology 50: 23-32, 1987.
Gall KP, Verhey LJ, Wagner M: Computer-assisted positioning of radiotherapy patients using implanted radiopaque fiducials. Medical Physics 20: 1153-9, 1993.
Kjellberg RN, Hanamura T, Davis KR, Lyons SL , Adams RD: Bragg-peak proton-beam therapy for arteriovenous malformations of the brain. New England Journal of Medicine 309: 269-74, 1983.
Kjellberg RN, Shintani A, Frantz AG, Kliman B: Proton-beam therapy in acromegaly. New England Journal of Medicine 278: 689-95, 1968.
Urie MM, Fullerton B, Tatsuzaki H, Birnbaum S, Suit HD, Convery K, Skates , Goitein M: A dose response analysis of injury to cranial nerves and/or nuclei following proton beam radiation therapy. International Journal of Radiation Oncology, Biology, Physics 23: 27-39, 1992.
Department of Radiation Oncology, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH 44195, USA. murphye3@ccf.org
Abstract
Vestibular schwannomas are slow-growing tumors of the myelin-forming cells that cover cranial nerve VIII. The treatment options for patients with vestibular schwannoma include active observation, surgical management, and radiotherapy. However, the optimal treatment choice remains controversial. We have reviewed the available data and summarized the radiotherapeutic options, including single-session stereotactic radiosurgery, fractionated conventional radiotherapy, fractionated stereotactic radiotherapy, and proton beam therapy. The comparisons of the various radiotherapy modalities have been based on single-institution experiences, which have shown excellent tumor control rates of 91-100%. Both stereotactic radiosurgery and fractionated stereotactic radiotherapy have successfully improved cranial nerve V and VII preservation to >95%. The mixed data regarding the ideal hearing preservation therapy, inherent biases in patient selection, and differences in outcome analysis have made the comparison across radiotherapeutic modalities difficult. Early experience using proton therapy for vestibular schwannoma treatment demonstrated local control rates of 84-100% but disappointing hearing preservation rates of 33-42%. Efforts to improve radiotherapy delivery will focus on refined dosimetry with the goal of reducing the dose to the critical structures. As future randomized trials are unlikely, we suggest regimented pre- and post-treatment assessments, including validated evaluations of cranial nerves V, VII, and VIII, and quality of life assessments with long-term prospective follow-up. The results from such trials will enhance the understanding of therapy outcomes and improve our ability to inform patients.
Radiosurgery is focused delivery of radiation to an image-defined target performed in 1 to 5 sessions.
When used as an alternative to or in conjunction with open neurosurgical techniques, radiosurgery is an effective, less invasive option for treating many benign intracranial tumors, including meningiomas, vestibular schwannomas, and pituitary adenomas.
The challenge
Benign intracranial tumors occur about as often as primary malignant brain tumors. Most benign tumors are noninvasive, well defined and well visualized on MRI, and have a slow rate of progression. Each of these features makes them good candidates for radiosurgery.
Radiosurgery can deliver a destructive dose of radiation to the target with little or no radiation effects on adjacent structures. Proper patient selection for this procedure is critical.
Defining selection criteria
With 2 decades of experience performing radiosurgery, Mayo Clinic neurosurgeons have accumulated a depth of expertise and a vast database that includes patient characteristics, radiosurgical dosimetry, and outcomes.
After reviewing more than 1,400 cases of meningiomas, vestibular schwannomas, and pituitary adenomas, Mayo clinicians observe that radiosurgery is an excellent choice when these types of benign tumors are small, occur in critical locations, or have recurred following previous surgery.
Radiosurgery is also well tolerated and of particular utility in elderly patients with medical conditions that put them at risk for an open procedure. Additionally, radiosurgery does not preclude an open procedure, should that be necessary at a later time.
Radiosurgery for meningiomas
The rate of recurrence for a surgically removed meningioma is about 18% to 25% at 10 years. For this reason, Mayo neurosurgeons recommend maintaining extended surveillance of meningiomas. In contrast, radiosurgery has been found to reduce the risk of recurrence or progression.
Tumor progression outside the field of radiation and tumor histology can affect both long- and short-term outcomes. Tumors that can be clearly imaged and those that are benign and without atypical histology have a far greater rate of 5-year progression-free survival.
Radiosurgery is also an effective therapy for cavernous sinus meningiomas, except when there is symptomatic mass effect, an unusual clinical presentation, or nontypical features on imaging.
Radiosurgery is typically not recommended for convexity and parasagittal meningiomas.
Radiosurgery for vestibular schwannomas
Several studies report that radiosurgery for small to moderate-sized vestibular schwannomas is associated with higher rates of hearing preservation and improved facial nerve outcomes when compared to surgical removal. This conclusion was supported by a Mayo Clinic study comparing surgical resection and radiosurgery for vestibular schwannomas with an average diameter of less than 3 cm. These Mayo investigators also found that the radiosurgical patients experienced less postprocedure dizziness.
MRI of patient’s brain with parathyroid carcinoma before radiosurgery
Radiosurgery is considered safe and effective for hormone-secreting pituitary adenomas. When compared with radiotherapy, radiosurgery appears to shorten by more than half the time required to achieve biochemical remission and normal hormone levels.
Controversy remains over whether pituitary-suppressive medications at the time of surgery have a negative impact on tumor control. Several studies, however, including a series of 46 acromegaly cases at Mayo Clinic, found that patients were more than 4 times as likely to reach normal hormone levels if they were taken off such medications before surgery.
At Mayo Clinic, patients with oversecretion of growth hormone or adrenocorticotropic hormone and patients who experience new or progressing visual field deficits are referred for surgical resection. Patients with tumors that extend into the cavernous sinuses and patients with recurrent tumors after prior surgery, however, are generally treated with radiosurgery if the tumor does not directly involve the optic nerves and chiasm.
Across Mayo Clinic’s 3 sites in Arizona, Florida, and Minnesota, patients are seen by neurosurgeons with expertise in both open procedures and radiosurgery. When used as an alternative to or in conjunction with traditional neurosurgery, radiosurgery is an effective, noninvasive option for treating benign intracranial tumors.
Department of Radiation Oncology, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH 44195, USA. murphye3@ccf.org
Abstract
Vestibular schwannomas are slow-growing tumors of the myelin-forming cells that cover cranial nerve VIII. The treatment options for patients with vestibular schwannoma include active observation, surgical management, and radiotherapy. However, the optimal treatment choice remains controversial. We have reviewed the available data and summarized the radiotherapeutic options, including single-session stereotactic radiosurgery, fractionated conventional radiotherapy, fractionated stereotactic radiotherapy, and proton beam therapy. The comparisons of the various radiotherapy modalities have been based on single-institution experiences, which have shown excellent tumor control rates of 91-100%. Both stereotactic radiosurgery and fractionated stereotactic radiotherapy have successfully improved cranial nerve V and VII preservation to >95%. The mixed data regarding the ideal hearing preservation therapy, inherent biases in patient selection, and differences in outcome analysis have made the comparison across radiotherapeutic modalities difficult. Early experience using proton therapy for vestibular schwannoma treatment demonstrated local control rates of 84-100% but disappointing hearing preservation rates of 33-42%. Efforts to improve radiotherapy delivery will focus on refined dosimetry with the goal of reducing the dose to the critical structures. As future randomized trials are unlikely, we suggest regimented pre- and post-treatment assessments, including validated evaluations of cranial nerves V, VII, and VIII, and quality of life assessments with long-term prospective follow-up. The results from such trials will enhance the understanding of therapy outcomes and improve our ability to inform patients.
Department of Otolaryngology–Head and Neck Surgery, Maggiore Hospital, Bologna, Italy.
Abstract
A comprehensive literature search identified only 14 well-documented cases of intracanalicular meningioma. A case is presented of meningioma confined to the internal auditory canal which was excised using a sub-occipital retrosigmoid approach. Preoperative MRI and CT scans were suggestive of intracanalicular vestibular schwannoma. Only the intraoperative findings, which were confirmed by the histological data, revealed that the tumor was a meningioma. We review the literature and discuss the diagnostic and therapeuticissues relating to these tumors.
Department of Otorhinolaryngology, Sunnybrook and Women’s College Health Science Centre, University of Toronto, Canada. malcolmhilton@hotmail.com
Abstract
Pathological lesions confined to the internal auditory canal (IAC) commonly present with cochleovestibular symptoms; sensorineural hearing loss, tinnitus and balance disturbance. The commonest lesion of the IAC is vestibular schwannoma. Other lesions include meningioma, facial neuroma, cavernous haemangioma, lipoma and arachnoid cyst. Presentation with facial palsy and an intracanalicular lesion is suggestive of pathology other than acoustic neuroma. Magnetic resonance imaging (MRI) cannot reliably distinguish intracanalicular vestibular schwannomas from meningiomas. Particular care is required for surgery of these lesions: the facial nerve typically does not lie in a protected anterior position within the IAC.
Department of Neurosurgery, Nordstadt Hospital, Teaching Hospital Hannover Medical School, Hannover, Germany. mnakamura@web.de
Abstract
OBJECTIVE:
Meningiomas arising primarily within the internal auditory canal (IAC) are notably rare. By far the most common tumors that are encountered in this region are neuromas. We report a series of eight patients with meningiomas of the IAC, analyzing the clinical presentations, surgical management strategies, and clinical outcomes.
METHODS:
The charts of the patients, including histories and audiograms, imaging studies, surgical records, discharge letters, histological records, and follow-up records, were reviewed.
RESULTS:
One thousand eight hundred meningiomas were operated on between 1978 and 2002 at the Neurosurgical Department of Nordstadt Hospital. Among them, there were 421 cerebellopontine angle meningiomas; 7 of these (1.7% of cerebellopontine angle meningiomas) were limited to the IAC. One additional patient underwent surgery at the Neurosurgical Department of the International Neuroscience Institute, where a total of 21 cerebellopontine angle meningiomas were treated surgically from 2001 to 2003. As a comparison, the incidence of intrameatal vestibular schwannomas during the same period, 1978 to 2002, was 168 of 2400 (7%). There were five women and three men, and the mean age was 49.3 years (range, 27-59 yr). Most patients had signs and symptoms of vestibulocochlear nerve disturbance at presentation. One patient had sought treatment previously for total hearing loss before surgery. No patient had a facial paresis at presentation. The neuroradiological workup revealed a homogeneously contrast-enhancing tumor on magnetic resonance imaging in all patients with hypointense or isointense signal intensity on T1- and T2-weighted images. Some intrameatal meningiomas showed broad attachment, and some showed a dural tail at the porus. In all patients, the tumor was removed through the lateral suboccipital retrosigmoid approach with drilling of the posterior wall of the IAC. Total removal was achieved in all cases. Severe infiltration of the facial and vestibulocochlear nerve was encountered in two patients. There was no operative mortality. Hearing was preserved in five of seven patients; one patient was deaf before surgery. Postoperative facial weakness was encountered temporarily in one patient.
CONCLUSION:
Although intrameatal meningiomas are quite rare, they must be considered in the differential diagnosis of intrameatal mass lesions. The clinical symptoms are very similar to those of vestibular schwannomas. A radiological differentiation from vestibular schwannomas is not always possible. Surgical removal of intrameatal meningiomas should aim at wide excision, including involved dura and bone, to prevent recurrences. The variation in the anatomy of the faciocochlear nerve bundle in relation to the tumor has to be kept in mind, and preservation of these structures should be the goal in every case.
Surgical management of jugular foramen schwannomas with hearing and facial nerve function preservation: a series of 23 cases and review of the literature.
Gruppo Otologico, Piacenza-Rome, Rome, Italy. mario.sanna@gruppotologico.it
Abstract
OBJECTIVE:
Schwannomas of the jugular foramen are rare lesions and controversy regarding their management still exists. The objective of this retrospective study was to analyze the management and outcome in a series of 23 cases collected at a single center.
SETTING:
This study was conducted at a quaternary private otology and skull base center.
METHODS:
Charts belonging to patients with a diagnosis of jugular foramen schwannoma attending our center between May 1988 and April 2006 were examined retrospectively.
RESULTS:
The study group consisted of 23 patients. One patient (a 73-year-old woman) with normal lower cranial nerves function was managed with watchful expectancy and regular clinical and radiologic follow ups. The infratemporal fossa approach-type A (IFTA-A) was performed in 3 cases. One patient underwent a transcochlear-transjugular approach. Of the 22 patients surgically treated, 12 patients were operated on by the petrooccipital transsigmoid approach (POTS). In one patient with a preoperative dead ear, a combined POTS-translabyrinthine approach was adopted. Two patients were operated on through the POTS approach combined with the transotic approach. In another case (a 67-year-old woman), a subtotal tumor removal through a transcervical approach was planned to resect a 10-cm mass in the neck. One patient underwent a first-stage combined transcervical-subtotal petrosectomy approach to remove a huge tumor in the neck; the second-stage intradural removal of the tumor was accomplished through a translabyrinthine-transsigmoid-transjugular approach. The last patient underwent a first-stage combined transcervical-subtotal petrosectomy approach to remove the neck tumor component; this patient is now waiting for the second-stage intradural removal of the tumor. Complete tumor removal was accomplished in 21 cases and in one case, a residual schwannoma was left in place in the area of the jugular foramen. The 3 patients who were operated on by IFTA-A underwent permanent anterior transposition of the facial nerve. At 1-year follow up, 2 of these patients had House-Brackmann grade I and 1 reached grade IV. The patient who underwent a transcochlear-transjugular approach had a permanent posterior transposition of the facial nerve. At 1-year follow up, he had grade III facial nerve function. Postoperative facial nerve function was normal (House-Brackmann grade I) in all patients operated on by the POTS approach. Twelve patients had hearing-preserving surgery using the POTS approach. Good hearing was preserved in 10 cases (83.3%), the majority of whom (58.3%) maintained their preoperative hearing level. There was no perioperative mortality. One patient (4.5%) experienced a postoperative cerebrospinal fluid leak. After surgery, all patients did not recover the function of the preoperatively paralyzed lower cranial nerves. A new deficit of one or more of the lower cranial nerves was recorded in 50% of cases. So far, no patient has experienced recurrence during the follow-up period as ascertained by computed tomography or magnetic resonance imaging.
CONCLUSIONS:
Surgical resection is the treatment of choice for jugular foramen schwannomas. The POTS approach allowed single-stage, total tumor removal with preservation of the facial nerve and of the middle and inner ear functions in the majority of cases. Despite the advances in skull base surgery, new postoperative lower cranial nerve deficits still represent a challenge.
Unidad de Investigación, Hospital Universitario La Paz, 28046 Madrid, Spain. vmartinezg.hulp@salud.madrid.org
Abstract
Microarray gene expression profiling is a high-throughput system used to identify differentially expressed genes and regulation patterns, and to discover new tumor markers. As the molecular pathogenesis of meningiomas and schwannomas, characterized by NF2 gene alterations, remains unclear and suitable molecular targets need to be identified, we used low density cDNA microarrays to establish expression patterns of 96 cancer-related genes on 23 schwannomas, 42 meningiomas and 3 normal cerebral meninges. We also performed a mutational analysis of the NF2 gene (PCR, dHPLC, Sequencing and MLPA), a search for 22q LOH and an analysis of gene silencing by promoter hypermethylation (MS-MLPA). Results showed a high frequency of NF2 gene mutations (40%), increased 22q LOH as aggressiveness increased, frequent losses and gains by MLPA in benign meningiomas, and gene expression silencing by hypermethylation. Array analysis showed decreased expression of 7 genes in meningiomas. Unsupervised analyses identified 2 molecular subgroups for both meningiomas and schwannomas showing 38 and 20 differentially expressed genes, respectively, and 19 genes differentially expressed between the two tumor types. These findings provide a molecular subgroup classification for meningiomas and schwannomas with possible implications for clinical practice.
Meningiomas account for up to 30% of all primary intracranial tumours. They are histologically classified according to the World Health Organization (WHO) classification of tumours of the nervous system. Most meningiomas are benign lesions of WHO grade I, whereas some meningioma variants correspond with WHO grades II and III and are associated with a higher risk of recurrence and shorter survival times. Mutations in the NF2 gene and loss of chromosome 22q are the most common genetic alterations associated with the initiation of meningiomas. With increase in tumour grade, additional progression-associated molecular aberrations can be found; however, most of the relevant genes are yet to be identified. High-throughput techniques of global genome and transcriptome analyses and new meningioma models provide increasing insight into meningioma biology and will help to identify common pathogenic pathways that may be targeted by new therapeutic approaches.
Centre for Research in Neuroscience, McGill University, Montreal, Canada.
Abstract
Schwannomas are tumors arising from schwann cells surrounding peripheral nerves. Although most schwannomas are sporadic, they are seen in approximately 90% of individuals with neurofibromatosis type 2 (NF2), an autosomal dominantly inherited disease with an incidence of 1:40000 live births. The NF2 gene has recently been isolated on chromosome 22 and encodes a putative membrane organizing protein named schwannomin. It is believed to act as a tumor suppressor gene based on the high frequency of loss of heterozygosity (LOH) on this autosome in both sporadic and NF2 associated schwannomas and meningiomas and the identification of inactivating mutation in NF2 patients. In this study we examined 61 schwannomas including 48 sporadic schwannomas (46 of which are vestibular schwannomas) and 12 schwannomas obtained from NF2 patients, for mutations in 10 of the 16 coding exons of the NF2 gene. Twelve inactivating mutations were identified, 8 in sporadic tumours and 4 in tumors from people with NF2. These results support the hypothesis that loss of function of schwannomin is a frequent and fundamental event in the genesis of schwannomas.
Offering imaging-guided therapy to cancer patients is a natural evolutionary step in imaging-based management of cancer patients. Moreover, as imaging-based detection of cancer becomes common, the number of patients presenting with small and localized cancer lesions increases. This serves as an incentive to treat the disease with a more targeted approach or, in today’s jargon, “Focal (or Focused) Treatment”.
This means pushing the role of imaging during treatment beyond the classical support it provides to the practitioner in controlling what he does towards:
Limiting treatment only to the life-threatening cancerous tissue.
Controlling and minimizing undesired damage to surrounding tissues.
Providing evidence that treatment has reached its goal. This is a clear technological challenge in non-chirurgical interventions, although one might argue that predicting clear surgical margins is not any less challenging.
Since this is a post J and not an essay, for each of the above requirements I will only discuss one technology component that I perceive as the most difficult to develop.
In order to treat only the cancerous lesion, or, even more ambitiously in the case of some cancers (e.g. prostate, breast), to treat just the life-threatening (aggressive) part of the lesion, we need a device that will reliably characterize the lesion, map the aggressive parts inside the lesion and, based on 3D imaging, enable accurate volume segmentation of the tissue we want to treat. For example, I can think of solutions in the form of a device that incorporates imaging-based tissue characterisation, or a device that relies on accurate registration between an image and bio-markers’ detector. Efforts to develop such solutions are ongoing [1-5].
Minimizing damage to surrounding tissues requires real-time feedback to the practitioner during the treatment, regarding the location he is treating. This becomes even more crucial when the intervention is not chirurgical but some sort of non-invasive or minimally invasive treatment such as external beam radiation, HIFU, photothermal ablation etc. These devices still suffer from limited control over treatment location (e.g. due to limitation of accuracy between imaging modalities and treating modalities, patient’s movements etc..) and the spatial deposition of energy [6-10]. It would be my preference to combine the source of energy and the imaging-guidance under one device, e.g. when using HIFU as an ablation method, conduct the treatment, the treatment navigation and the treatment efficacy control using ultrasound-based tissue characterisation and avoid the complexity such as the one offered in [7]:
Being able to receive feedback on treatment efficacy in a timely and noninvasive manner will enable not only the acceptance of focal treatments but will also be a game-changer in the way cancer is being treated by drugs [8, 9]. The potential technological route in this case will require development of reproducible imaging work-flow that will enable reliable identification and comparable measures of the same lesion over and over again. It will definitely rely on imaging-based real-time tissue characterisation which will enable measuring temporal changes in a certain tissue characteristic, e.g. tracking changes in tissue temperature through using ultrasound for tracking changes in its density or using MRI for tracking change in response to magnetic fields [7].
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Advances in Separations Technology for the “OMICs” and Clarification of Therapeutic Targets
Curator, Reporter, EAW: Larry H Bernstein, MD, FCAP
Article 1.5 Advances in Separations Technology for the “OMICs” and Clarification of Therapeutic Targets
This discussion is a continuation of an earlier piece on the technologic framework for , proteomics, nutrigenomics, and translational medicine. The last decade has seen the emergence of a genomic science that is changing the trajectory of biological sciences and medicine. It has not resolved all of our problems by any means, but it has begun to redraw the map, which began with the elucidation of major metabolic pathways in the first half of the 20th century, was then captured by the transformation of genetics with the discovery of the “Watson-Crick Model”, and then later was recharged with the discovery of the Toll-like receptor and the drawing of “signaling pathways”. What we have seen in an unraveling of protein-genome interactions, small peptide regulators, and dynamic changes in pathway dominance, bloackage, and reentry, depending on genetic, dietary, and environmental conditions, mostly expressed in what we refer to as “oxidative stress”.
Unraveling the multitude of nutrigenomic, proteomic, and metabolomic patterns that arise from the ingestion of foods or their bioactive food components will not be simple but is likely to provide insights into a tailored approach to diet and health. The use of new and innovative technologies, such as microarrays, RNA interference, and nanotechnologies, will provide needed insights into molecular targets for specific bioactive food components and how they harmonize to influence individual phenotypes. A challenging aspect of omic technologies is the refined analysis of quantitative dynamics in biological systems.
In recent years, nutrition research has moved from classical epidemiology and physiology to molecular biology and genetics. The new era of nutrition research translates empirical knowledge to evidence-based molecular science. Following this trend, Nutrigenomics has emerged as a novel and multidisciplinary research field in nutritional science that aims to elucidate how diet can influence human health. It is already well known that bioactive food compounds can interact with genes affecting transcription factors, protein expression and metabolite production. The study of these complex interactions requires the development of advanced analytical approaches combined with bioinformatics. The Institute of Medicine recently convened a workshop to review the state of the various domains of nutritional genomics research and policy and to provide guidance for further development and translation of this knowledge into nutrition practice and policy. Nutritional genomics holds the promise to revolutionize both clinical and public health nutrition practice and facilitate the establishment of
genome-informed nutrient and food-based dietary guidelines for disease prevention and healthful aging,
individualized medical nutrition therapy for disease management, and
better targeted public health nutrition interventions (including micronutrient fortification and supplementation) that maximize benefit and minimize adverse outcomes within genetically diverse human populations.
For metabolomics, gas and liquid chromatography coupled to mass spectrometry are well suited for coping with high sample numbers in reliable measurement times with respect to both technical accuracy and the identification and quantitation of small-molecular-weight metabolites. This potential is a prerequisite for the analysis of dynamic systems. Thus, metabolomics is a key technology for systems biology. The bioavailability of bioactive food constituents as well as dose-effect correlations are key information to understand the impact of food on defined health outcomes. Both strongly depend on appropriate analytical tools to identify and quantify minute amounts of individual compounds in highly complex matrices–food or biological fluids–and to monitor molecular changes in the body in a highly specific and sensitive manner. Based on these requirements, mass spectrometry has become the analytical method of choice with broad applications throughout all areas of nutrition research.
Dynamic Construct of the –Omics
Metabolomics is a term that encompasses several types of analyses, including
metabolic fingerprinting, which measures a subset of the whole profile with little differentiation or quantitation of metabolites;
metabolic profiling, the quantitative study of a group of metabolites, known or unknown, within or associated with a particular metabolic pathway; and
target isotope-based analysis, which focuses on a particular segment of the metabolome by analyzing only a few selected metabolites that comprise a specific biochemical pathway.
Any unifying concept of the metabolome was incomplete or debatable in the first 30 years of the 20th century. It was only known that insulin is anabolic and that insulin deficiency (or resistance) would have consequences in the point of entry into the citric acid cycle, which generates 28-32 ATPs. In fat catabolism, triglycerides are hydrolyzed to break them into fatty acids and glycerol. In the liver the glycerol can be converted into glucose via dihydroxyacetone phosphate and glyceraldehyde-3-phosphate by way of gluconeogenesis. In the case of this cycle there is a tie in with both catabolism and anabolism.
For bypass of the Pyruvate Kinase reaction of Glycolysis, cleavage of 2 ~P bonds is required. The free energy change associated with cleavage of one ~P bond of ATP is insufficient to drive synthesis of phosphoenolpyruvate (PEP), since PEP has a higher negative DG of phosphate hydrolysis than ATP. The two enzymes that catalyze the reactions for bypass of the Pyruvate Kinase reaction are the following:
Pyruvate Carboxylase (Gluconeogenesis) catalyzes pyruvate + HCO3- + ATP — oxaloacetate + ADP + Pi
PEP Carboxykinase (Gluconeogenesis) catalyzes: oxaloacetate + GTP —- phosphoenolpyruvate + GDP + CO2
Many high throughput methods have been employed to get some insight into the whole process and several examples of successful research. Proteomics and metabolomics need to encompass large numbers of experiments and linked data. Due to the nature of the proteins, as well as due to the properties of various metabolites, experimental approaches require the use of comprehensive high throughput methods and a sufficiency of analysed tissue or body fluids.
An important and revolutionary aspect of ‘The 2010 Project’ is that it implicitly endorses the allocation of resources to attempts to assign function to genes that have no known function. This represents a significant departure from the common practice of defining and justifying a scientific goal based on the biological phenomena. The rationale for endorsing this radical change is that for the first time it is feasible to envision a whole-systems approach to gene and protein function. I shall not discuss the emerging field of bioinformatics that makes this possible. In this review, the end-of-the line “detector will be considered having been covered. The entire focus proceeds to a discussion of separation methods. Separation methods have always been tricky, time consuming, and a multiple step process that depended on using anionic and cationic resins as intermediate steps in bulk separation, and then molecular size separation. Therapeutic Targets will be identified as they are seen.
Affinity Chromatography The rapid development of biotechnology and biomedicine requires more reliable and efficient separation technologies for the isolation and purification of biopolymers such as therapeutic proteins, antibodies, enzymes and nucleic acids. In particular, monoclonal antibodies are centrally important as therapeutics for the treatment of cancer and other diseases, leading to recombinant monoclonal antibodies that dominate today’s biopharmaceutical pipeline. The large-scale production of therapeutic biopolymers requires
a manufacturing process that delivers reliability and in high-yield, as well as
an effective purification process affording extremely pure products.
Because of its high selectivity, affinity chromatography has been used extensively to isolate a variety of biopolymers. The retention of solutes is based on specific, reversible interactions found in biological systems, such as the binding of an enzyme with an inhibitor or an antibody with an antigen. These interactions are exploited in affinity chromatography by immobilizing an affinity ligand onto a support, and using this as a stationary phase. Non-porous particles having an average diameter of 2.1 mm were prepared by co-polymerization of styrene, methyl methacrylate and glycidyl methacrylate, which was abbreviated as P(S–MMA–GMA). The particles were mechanically stable due to the presence of benzene rings in the backbone of polymer chains, and could withstand high pressures when a column packed with these particles was operated in the HPLC mode.
The polymer particles were advantaged by immobilization of ligands via the epoxy groups on the particle surface that were introduced by one of the monomers, glycidyl methacrylate. As a model system, Cibacron Blue 3G-A was covalently immobilized onto the non-porous copolymer beads. The dye-immobilized P(S–MMA–GMA) particles were slurry packed into a 1.0 cm30.46 cm I.D. column. This affinity column was effective for the separation of turkey egg white lysozyme from a protein mixture. The bound lysozyme could be eluted to yield a sharp peak by using a phosphate buffer containing 1 M NaCl. For a sample containing up to 8 mg of lysozyme, the retained portion of proteins could be completely eluted without any slit peak. Due to the use of a shorter column, the analysis time was shorter in comparison with other affinity systems reported in the literature. The retention time could be reduced significantly by increasing the flow-rate, while the capacity factor remained at the same level. CH Chen, WC Lee. Affinity chromatography of proteins on non-porous copolymerized particles of styrene, methyl methacrylate and glycidyl methacrylate. Journal of Chromatography A 2001; 921: 31–37.
Affinity separation membranes, consisting of electrospun nanofibers, have been developed recently. Affinity ligands are attached to the surface of the constituent fibers, offering a potential solution to some of the problems of traditional, column-based, affinity chromatography. Electrospun fibers are good candidates for use in affinity separation because of their
unique characteristics of high surface area to volume ratio, resulting in
high ligand loading, and
their large porosity, resulting in
high throughput operation.
A number of polymers have been used for electrospun fiber mesh-based affinity membrane separations including poly (ether-urethane-urea), cellulose, poly(ethylene terephthalate, polysulphone, and polyacrlonitrile. Typically, very thin electrospun fiber meshes are produced by electrostatically collecting negatively charged fibers on a collector electrode. These very thin 2D electrospun fiber mesh mats provide excellent solution permeability as compared to 3D column packed with affinity beads. M Miyauchi, J Miao, TJ Simmons, JS Dordick and RJ Linhardt. Flexible Electrospun Cellulose Fibers as an Affinity Packing Material for the Separation of Bovine Serum Albumin. J Chromatograph Separat Techniq 2011; 2:2 http://dx.doi.org/10.4172/2157-7064.1000110
Dye Affinity Chromatography Biomimetic Dyes Affinity adsorbents based on immobilized triazine dyes offer important advantages circumventing many of the problems associated with biological ligands. The main drawback of dyes is their moderate selectivity for proteins. Rational attempts to tackle this problem are realized through the biomimetic dye concept according to which new dyes, the biomimetic dyes, are designed to mimic natural ligands. Biomimetic dyes are expected to exhibit increased affinity and purifying ability for the targeted proteins.
Biocomputing offers a powerful approach to biomimetic ligand design. The successful exploitation of contemporary computational techniques in molecular design requires the knowledge of the three-dimensional structure of the target protein, or at least, the amino acid sequence of the target protein and the three-dimensional structure of a highly homologous protein. From such information one can then design, on a graphics workstation,
the model of the protein and also
a number of suitable synthetic ligands which mimic natural biological ligands of the protein.
There are several examples of enzyme purifications
Interactions between Cibacron Blue F3GA (CB F3GA), as a model of triazine dye, and 2-hydroxypropyl-b-cyclodextrin (HP-b-CD), as a model of cyclodextrin, were investigated by monitoring the spectral shift that accompanies the binding phenomena. Matrix analysis of the difference spectral titration of CB F3GA with HP-b-CD revealed only two absorbing species, indicating a host–guest ratio of 1:1. The dissociation constant for this HP-b-CD–CB F3GA complex, K , was found d to be 0.43 mM. The data for HP-b-CD forming inclusion complexes with CB F3GA were used to develop the concept of competitive elution by inclusion complexes in dye-affinity chromatography. When this concept was applied to the elution of L-lactate dehydrogenase from a CB F3GA affinity matrix, it was shown to be an effective elution strategy. It provided a 15-fold purification factor with 89% recovery and sharp elution profile (0.8 column volumes for 80% recovery), which is as good as that obtained by specific elution with NADH (16-fold, 78% recovery and 1.8 column volumes). In addition, the new elution strategy showed a better purification factor and sharper elution profile than traditional non-specific. JA Lopez-Mas, SA Streitenberger, F Garcıa-Carmona, AA Sanchez-Ferrer. Cyclodextrin biospecific-like displacement in dye-affinity chromatography. Journal of Chromatography A 2001; 911: 47–53.
Affinity chromatography uses biospecific binding usually between an antibody and an antigen, an enzyme and a substrate or other pairs of key-lock type of matching molecules. Due to its high selectivity, it is able to purify proteins and other macromolecules from very dilute solutions. In this work, a general rate model for affinity chromatography was used for scale-up studies. Parameters for the model were estimated from existing correlations, or from experimental results obtained on a small column with the same packing material. As anexample, Affi-Gel with 4.5mol cm−3 Cibacron Blue F-3GA as immobilized ligands covalently attached to cross-linked 6% agarose was used for column packing. Cibacron Blue F-3GA was also used as a soluble ligand in the elution stage. Satisfactory scale-up predictions were obtained for a 98.2 ml column and a 501 ml column based on a few experimental data obtained on a 7.85 ml small column. T. Gu, K.-H. Hsu and M.-J. Syu, “Scale-Up of Affinity Chromatography for Purification of Enzymes and Other Proteins.” Enzyme and Microbial Technology 2003; 33:433-437.
Affinity Column with AAAA as a Model Sense Ligand The degeneracy of antisense peptides was studied by high-performance affinity chromatography. A model sense peptide (AAAA) and its antisense peptides (CGGG, GGGG, RGGG, SGGG) were designed and synthesized according to the degeneracy of genetic codes. An affinity column with AAAA as the ligand was prepared. The affinity chromatographic behaviors of antisense peptides on the column were evaluated. The results indicated that model antisense peptides have clear retention on the immobilized AAAA affinity column. RGGG showed the strongest affinity interaction. R Zhao, X Yu, H Liu, L Zhai, S Xiong, et al. Study on the degeneracy of antisense peptides using affinity chromatography. Journal of Chromatography A 2001; 913: 421–428.
Frontal AC for Biomolecular Interactions Frontal affinity chromatography is a method for quantitative analysis of biomolecular interactions. We reinforced it by incorporating various merits of a contemporary liquid chromatography system. As a model study, the interaction between an immobilized Caenorhabditis elegans galectin (LEC-6) and fluorescently labeled oligosaccharides (pyridylaminated sugars) was analyzed. LEC-6 was coupled to N-hydroxysuccinimide-activated Sepharose 4 Fast Flow (100 mm diameter), and packed into a miniature column (e.g., 1034.0 mm, 0.126 ml). The volume of the elution front (V) determined graphically for each sample was compared with that obtained in the presence of an excess amount of hapten saccharide, lactose (V ); and the dissociation constant, K , was calculated according to the literature. This system also proved to be useful for an inverse confirmation; that is, application of galectins to an immobilized glycan column (in the present case, asialofetuin was immobilized on Sepharose 4 Fast Flow), and the elution profiles were monitored by fluorescence based on tryptophan. The newly constructed system proved to be extremely versatile. It enabled rapid (analysis time 12 min/ cycle) and sensitive (20 nM for pyridylaminated derivatives, and 1 mg/ml for protein) analyses of lectin–carbohydrate interactions. J Hirabayashi, Y Arata, K Kasai. Reinforcement of frontal affinity chromatography for effective analysis of lectin–oligosaccharide interactions. Journal of Chromatography A 2000; 890:261–271.
Immobilized Metal Ion Affinity New immobilized metal ion affinity chromatography (IMAC) matrices containing a high concentration of metal–chelate moieties and completely coated with inert flexible and hydrophilic dextrans are here proposed to improve the purification of polyhistidine (poly-His) tagged proteins. The purification of an interesting recombinant multimeric enzyme (a thermoresistant b-galactosidase from Thermus sp. strain T2) has been used to check the performance of these new chromatographic media.
IMAC supports with a high concentration (and surface density) of metal chelate groups promote a rapid adsorption of poly-His tagged proteins during IMAC. However, these supports also favor the promotion of undesirable multi-punctual adsorptions and problems may arise for the simple and effective purification of poly-His tagged proteins. For example, desorption of the pure enzyme from the support may become quite difficult (e.g., it is not fully desorbed from the support even using 200 mM of imidazole).
The coating of these IMAC supports with dextrans greatly reduces these undesired multi-point adsorptions. However, this dextran coating of chromatographic matrices seems to allow the formation of strong one-point adsorptions that involve small areas of the protein and support surface, but the dextran coating seems to have dramatic effects for the prevention of weak or strong multipoint interactions that should involve a high geometrical congruence between the enzyme and the support surface. C Mateo , G Fernandez-Lorente , BCC Pessela , A Vian, et al. Affinity chromatography of polyhistidine tagged enzymes. New dextran-coated immobilized metal ion affinity chromatography matrices for prevention of undesired multipoint adsorptions. Journal of Chromatography A 2001; 915:97–106. The underlying principle of immobilized metal ion affinity chromatography (IMAC) of proteins is the coordination between the electron donor groupings on a protein surface (histidine, tryptophan, cysteine) and chelated (iminodiacetate; IDA) transition metal ions [IDA-M(II)]. This principle of immobilized metal ion affinity (IMA) has been presented by now in some detail. The practice of IMAC in the purification of proteins has had its empirical phase. There is now a need, from the body of data, to establish somewhat more detailed ground rules that would allow for the use of IMAC in a more predictive manner. Immobilized metal ion affinity chromatography (IMAC) has been explored as a probe into the topography of histidyl residues of a protein molecule. An evaluation of the chromatographic behavior of selected model proteins-
thioredoxin
ubiquitin
calmodulin
lysozyme
cytochrome c
myoglobin
on immobilized transition metal ions
Co2+
Ni2+
Cu2+
Zn2
-allows establishment of the following facets of the histidyl side chain distribution:
either interior or surface;
when localized on the surface, accessible or unaccessible for coordination;
single or multiple;
When multiple, either distant or vicinal.
Moreover, proteins displaying single histidyl side chains on their surfaces may, in some instances, be resolved by IMAC; apparently, the microenvironments of histidyl residues are sufficiently diverse to result in different affinities for the immobilized metal ions. IMAC, previously introduced as an approach to the fractionation of proteins, has become also, upon closer examination, a facile probe into the topography of histidyl residues. This is possible because of the inherent versatility of IMAC; an appropriate metal ion (M2+) can be selected to suit the analytical purpose and a particular chromatographic protocol can be applied (isocratic pH, falling pH, and imidazole elution). We now report that IMAC may be exploited as an analytical tool in addition to its use as a protein purification technique. IMAC can be used to ascertain several facets of the status of a histidyl residue(s) in a protein molecule:
localization (interior vs. surface)
coordination potential as defined by the steric accessibility and the state of protonation
A novel, two-step preparative technique is described for the purification of authentic recombinant human prolactin (rhPRL) secreted into the periplasm of transformed Escherichia coli cells. The first step is based on immobilized metal ion affinity chromatography of periplasmic extract, using Ni(II) as a relatively specific ligand for hPRL in this system. It gives superior resolution and yield than established ion-exchange chromatography. Size-exclusion chromatography is used for further purification to .99.5% purity. The methodology is reproducible, leading to 77% recovery. Identity and purity of the rhPRL were demonstrated using sodium dodecylsulphate–polyacrylamide electrophoresis, isoelectric focusing, mass spectrometry (matrix-assisted laser desorption ionization time-of-flight), radioimmunoassay, RP-HPLC and high-performance size-exclusion chromatography. In the Nb2 bioassay, the hormone showed a bioactivity of 40.9 IU/mg.
Adenosine Affinity Ligand for Glutamine Synthase Glutamine synthetase has been purified from both procaryotic and eucaryotic sources using various types of affinity chromatography. For example, ADP-agarose has been used to purify glutamine synthetase from photosynthetic bacteria, while the related “Blue” chromatography media (e.g. Affigel Blue) have been used to purify glutamine synthetases from a variety of sources. In addition, 2’,5’-ADPSepharose 4B has been used to purify glutamine synthetase from procaryotes, plants and insects. However, this latter affinity ligand resembles NADP more than ADP, particularly with respect to the position of the phosphate moieties. This is reflected in the more general use of this affinity ligand in the purification of NADPH-dependent enzymes. In the present report, we characterize the ability of glutamine synthetase to be purified by three different adenosine-affinity ligands: 5’-ADP-agarose (an ADP analogue), 2’,5’-ADP-Sepharose 4B (an NADP analogue) and 3’,5’-ADP-agarose (a cyclic AMP analogue). We report conditions for the successful purification of insect flight muscle glutamine synthetase using each of these three different affinity ligands. The enzyme bound most strongly to the
ADP analogue (S-ADP-agarose),
followed by the NADPH analogue (2’,5’-ADP-Sepharose 4B), and least strongly to
the cyclic AMP analogue (3’J’-ADP-agarose).
In all cases, binding was strongest in the presence of Mn2+ when compared to Mg”. These results suggest that the binding of glutamine synthetase to adenosine-affinity media is related to the participation of Mn. ADP in the y-glutamyl transferase reaction that is catalyzed by glutamine synthetase. M Dowton, IR Kennedy. Purification of glutamine synthetase by adenosine-affinity chromatography. Journal of Chromatography A 1994; 664: 280-283
Aptamer Based Stationary Phase An anti-adenosine aptamer was evaluated as a stationary phase in packed capillary liquid chromatography. Using an 21 aqueous mobile phase containing 20 mM Mg , adenosine was strongly retained on the column. A gradient of increasing 21 Ni (to 18 mM), which is presumed to complex with nitrogen atoms in adenosine involved in binding to the aptamer, eluted adenosine in a narrow zone. The adenosine assay, which required no sample preparation, was used on microdialysis samples. Total analysis times were short so samples could be injected every 5 min. Q Deng, CJ Watson, RT Kennedy. Aptamer affinity chromatography for rapid assay of adenosine in microdialysis samples collected in vivo. Journal of Chromatography A 2003; 1005:123–130.
We will realize the full power of proteomics only when we can measure and compare the proteomes of many individuals to identify biomarkers of human health and disease and track the blood-based proteome of an individual over time. Because the human proteome contains an estimated 20,000 proteins – plus splicing and post-translational variants – that span a concentration range of ,12 logs, identifying and quantifying valid biomarkers is a great technical challenge. Proteomic measurements demand
extreme sensitivity
specificity
dynamic range
accurate quantification.
We describe a new class of DNA-based aptamers enabled by a versatile chemistry technology that endows nucleotides with protein-like functional groups. These modifications greatly expand the repertoire of targets accessible to aptamers. The resulting technology provides efficient, large-scale selection of exquisite protein-binding reagents selected specifically for use in highly multiplexed proteomics arrays. Aptamers are a class of nucleic acid-based molecules discovered twenty years ago, and have since been employed in diverse applications including
therapeutics
catalysis
proteomics
Aptamers are short single-stranded oligonucleotides, which fold into diverse and intricate molecular structures that bind with high affinity and specificity to
proteins
peptides
small molecules.
Aptamers are selected in vitro from enormously large libraries of randomized sequences by the process of Systematic Evolution of Ligands by EXponential enrichment (SELEX). A SELEX library with 40 random sequence positions has 440 (,1024) possible combinations and a typical selection screens 1014–1015 unique molecules. This is on the order of 105 times larger than standard peptide or protein combinatorial molecular libraries.
The interrogation of proteomes (‘‘proteomics’’) in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology and medicine. We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 mL of serum or plasma).
Our current assay measures 813 proteins with low limits of detection (1 pM median), 7 logs of overall dynamic range (,100 fM–1 mM), and 5% median coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding signature of DNA aptamer concentrations, which is quantified on a DNA microarray.
Our assay takes advantage of the dual nature of aptamers as both folded protein-binding entities with defined shapes and unique nucleotide sequences recognizable by specific hybridization probes.
This is a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine. L Gold, D Ayers, J Bertino, Christopher Bock, et al. Aptamer-Based Multiplexed Proteomic Technology for Biomarker Discovery. PlosONE 2010; 5 (12): e15004
Biomarker Discovery, Diagnostics, and Therapeutics Progression from health to disease is accompanied by complex changes in protein expression in both the circulation and affected tissues. Large-scale comparative interrogation of the human proteome can offer insights into disease biology as well as lead to
the discovery of new biomarkers for diagnostics
new targets for therapeutics
can identify patients most likely to benefit from treatment.
Although genomic studies provide an increasingly sharper understanding of basic biological and pathobiological processes, they ultimately only offer a prediction of relative disease risk, whereas proteins offer an immediate assessment of “real-time” health and disease status. We have recently developed a new proteomic technology, based on modified aptamers, for biomarker discovery that is capable of simultaneously measuring more than a thousand proteins from small volumes of biological samples such as plasma, tissues, or cells. Our technology is enabled by SOMAmers (Slow Off-rate Modified Aptamers), a new class of protein binding reagents that contain chemically modified nucleotides that greatly expand the physicochemical diversity of nucleic acid-based ligands. Such modifications introduce functional groups that are absent in natural nucleic acids but are often found in protein-protein, small molecule-protein, and antibody-antigen interactions. The use of these modifications expands the range of possible targets for SELEX (Systematic Evolution of Ligands by EXponential Enrichment), results in improved binding properties, and facilitates selection of SOMAmers with slow dissociation rates. Our assay works by transforming protein concentrations in a mixture into a corresponding DNA signature, which is then quantified on current commercial DNA microarray platforms. In essence, we take advantage of the dual nature of SOMAmers as
both folded binding entities with defined shapes and
unique nucleic acid sequences recognizable by specific hybridization probes.
Aptamers and Smart Drug delivery Targeting In this review, the strategies for using functional nucleic acids in creating smart drug delivery devices will be explained, as their has been very recent progress in controlled drug release based on molecular gating achieved with aptamers. Aptamers are functional nucleic acid sequences which can bind specific targets. An artificial combinatorial methodology can identify aptamer sequences for any target molecule, from ions to whole cells. Drug delivery systems seek to increase efficacy and reduce side-effects by concentrating the therapeutic agents at specific disease sites in the body. This is generally achieved by specific targeting of inactivated drug molecules. Aptamers which can bind to various cancer cell types selectively and with high affinity have been exploited in a variety of drug delivery systems for therapeutic purposes. Recent progress in selection of cell-specific aptamers has provided new opportunities in targeted drug delivery. Especially functionalization of nanoparticles with such aptamers has drawn major attention in the biosensor and biomedical areas.
Nucleic acids are recognized as attractive building materials in nanomachines because of their unique molecular recognition properties and structural features. An active controlled delivery of drugs once targeted to a disease site is a major research challenge. Stimuli-responsive gating is one way of achieving controlled release of nanoparticle cargoes. Recent reports incorporate the structural properties of aptamers in controlled release systems of drug delivering nanoparticles.
Nanoparticle-encapsulated drug delivery aims to deliver the active therapeutic ingredients to the disease site in stable compartments in order to reduce premature release. This ensures that the effects of drug are maximized and the side effects are reduced. An encapsulated nanoparticle system requires a specific targeting mechanism and at the same time the retention of drugs inside the container should be high. The balance between specificity of targeting and the extent of premature leakage determines the success of a given delivery system.
Nanotechnology research approaches in drug delivery include a wide variety of nanomaterials ranging from soft hydrogels to solid polymeric particles. Large surface area, high drug loading efficiency and potential combination with other organic/inorganic materials are the main properties of hollow nanostructures that are attractive for biomedical applications.
Packaging of small-molecule drugs
improves their availability
compatibility
reduces toxicity
Controlling the drug release profile is the main challenge in drug delivery development when the drug is to be successfully targeted to a specific site. Stimuli-responsive materials have been created by using biological, physical and chemical properties of materials for heat-activated, light-activated or pH-activated delivery. Nucleic acids are utilized to construct rationally designed nanostructures at molecular levels for nanotechnology applications. Integration of the properties of nucleic acids can offer many opportunities for drug delivery systems, including stimuli-responsive nanogates for nanocarriers and molecular sensors. Favorable drug release kinetics can be achieved at the target sites by aptamer-based capping systems.
Activity Based Profiling Powerful strategies for the gel-free analysis of proteomes have emerged, including isotope-coded affinity tagging (ICAT) for quantitative proteomics and multidimensional protein identification technology (MudPIT) for comprehensive proteomics, both of which utilize liquid chromatography (LC) and MS for protein separation and detection, respectively. Nonetheless, these methods, like 2DE-MS, still focus on measuring changes in protein abundance and, therefore, provide only an indirect estimate of dynamics in protein function. Indeed, several important forms of post-translational regulation, including protein–protein and protein–small-molecule interactions, may elude detection by abundance-based proteomic methods. To facilitate the analysis of protein function, several proteomic methods have been introduced to characterize the activity of proteins on a global scale. These include large-scale yeast two-hybrid screens and epitope tagging immunoprecipitation experiments, which aim to construct comprehensive maps of protein–protein interactions, and protein microarrays, which aim to provide an assay platform for the rapid assessment of protein activities. A chemical proteomic strategy referred to as activity-based protein profiling (ABPP) has emerged that utilizes active site-directed probes to profile the functional state of enzyme families directly in complex proteomes.
Recent advances in genomic and proteomic technologies have begun to address the challenge of assigning molecular and cellular functions to the numerous protein products encoded by prokaryotic and eukaryotic genomes. In particular, chemical strategies for proteome analysis have emerged that enable profiling of protein activity on a global scale. Herein, we highlight these chemical proteomic methods and their application to the discovery and characterization of disease-related enzyme activities.
Cells with fundamental metabolic alterations commonly arise during tumorigenesis, and it is these types of changes that help to establish a biochemical foundation for disease progression and malignancy. A seminal example of this was discovered in the 1920s when Otto Warburg found that cancer cells consume higher levels of glucose and secrete most of the glucose carbon as lactate rather than oxidizing it completely. Since then, studies by multiple groups have uncovered a diverse array of metabolic changes in cancer, including alterations in
glycolytic pathways
the citric acid cycle
glutaminolysis
lipogenesis
lipolysis
proteolysis
These in turn modulate the levels of cellular building blocks
lipids, nucleic acids and amino acids,
cellular energetics,
oncogenic signaling molecules
the extracellular environment to confer protumorigenic and malignant properties.
Despite these advances, our current understanding of cancer metabolism is far from complete and would probably benefit from experimental strategies that are capable of profiling enzymatic pathways on a global scale. To this end, conventional genomic and proteomic methods, which comparatively quantify the expression levels of transcripts and proteins, respectively, have yielded many useful insights. These platforms are, however, limited in their capacity to identify changes in protein activity that are caused by posttranslational mechanisms.
Annotating biochemical pathways in cancer is further complicated by the potential for enzymes to carry out distinct metabolic activities in tumor cells that might not be mirrored in normal physiology. In addition, a substantial proportion of the human proteome remains functionally uncharacterized, and it is likely that at least some of these poorly understood proteins also have roles in tumorigenesis. These challenges require new proteomic technologies that can accelerate the assignment of protein function in complex biological systems, such as cancer cells and tumors.
Metabolomics has emerged as a powerful approach for investigating enzyme function in living systems. Metabolomic experiments in the context of enzyme studies typically start with
the extraction of metabolites from control and enzyme-disrupted biological systems,
followed by metabolite detection and comparative data analysis.
For example, lipophilic metabolites can be enriched from cells or tissues by organic extraction. Mass spectrometry (MS) has become a primary analytical method for surveying metabolites in complex biological samples, with upfront separation accomplished by liquid chromatography (LC–MS) or gas chromatography (GC–MS). MS experiments can be carried out using
targeted or untargeted approaches,
depending on whether the objective is
to profile and quantitate known metabolites or
to broadly scan for metabolites across a large mass range, respectively.
As metabolomic experiments generate a large amount of data, powerful software tools are needed for identification and quantitation of ions in LC–MS data sets (see the figure; the mass to charge ratio (m/z) is indicated). One such program is XCMS95, which
aligns,
quantifies and
statistically ranks ions that are altered between two sets of metabolomic data.
This program can be used to rapidly identify metabolomic signatures of various disease states or to assess metabolic networks that are regulated by an enzyme using pharmacological or genetic tools that modulate enzyme function. Additional databases assist in metabolite structural characterization, such as HMDB96,97, METLIN98,99 and LIPID MAPS100. In this Review, we discuss one such proteomic platform, termed activity based protein profiling (ABPP), and its implementation in the discovery and functional characterization of deregulated enzymatic pathways in cancer. We discuss the evidence that, when coupled with other large scale profiling methods, such as metabolomics and proteomics, ABPP can provide a compelling, systems level understanding of biochemical networks that are important for the development and progression of cancer.
Large-scale profiling methods have uncovered numerous gene and protein expression changes that correlate with tumorigenesis. However, determining the relevance of these expression changes and which biochemical pathways they affect has been hindered by our incomplete understanding of the proteome and its myriad functions and modes of regulation. Activity-based profiling platforms enable both the discovery of cancer-relevant enzymes and selective pharmacological probes to perturb and characterize these proteins in tumour cells. When integrated with other large-scale profiling methods, activity-based proteomics can provide insight into the metabolic and signaling pathways that support cancer pathogenesis and illuminate new strategies for disease diagnosis and treatment.
Representative activity-based probes and their application to cancer research
enzyme class applications in cancer
Serine hydrolases increased KIAA1363 and MAGL
aggressive human cancer lines
uPA and tPA serine protease aggressive cancers
RBBP9 activity in pancreatic carcinoma
Metalloproteinases neprilysin activity in melanoma cell lines
Cysteine proteases cathepsin cysteine protease in pancreatic islet tumours
Kinases Inhibitor selectivity profiling of kinase inhibitors
Caspases visualization of apoptosis in colon tumour-bearing mice treated with Apomab
Deubiquitylases Identified increased carboxy-terminal hydrolase UCHL3 and UCH37 activity in HPV cervical carcinomas
Cytochrome P450s Identified the aromatase inhibitor anastrazole as an inducer of CYP1A2
Serine hydrolases KIaa1363 and MaGL regulate lipid metabolic pathways that support cancer pathogenesis. Activity-based protein profiling (ABPP) identified
KIAA1363 and
monoacylglycerol (MAG) lipase (MAGL)
as being increased in aggressive human cancer cells from multiple tumour types. Pharmacological and/or RNA interference ablation of KIAA1363 and MAGL coupled with metabolomic analysis revealed specific roles for KIAA1363 and MAGL in cancer metabolism. Disruption of KIAA1363 by the small-molecule inhibitor AS115 lowered monoalkylglycerol ether (MAGE), alkyl lysophosphatidic acid (alkyl LPA) and alkyl lysophosphatidyl choline (alkyl LPC) levels in cancer cells. Disruption of MAGL by the small-molecule inhibitor JZL184 raised MAG levels and reduced free fatty acid, lysophosphatidic acid (LPA) and prostaglandin E2 (PGE2) levels in cancer cells. Disruption of KIAA1363 and MAGL leads to impairments in cancer cell aggressiveness and tumour growth, PAF, platelet-activating factor.
At a glance
• Activity-based protein profiling (ABPP) facilitates the discovery of deregulated enzymes in cancer. • Competitive ABPP yields selective inhibitors for functional characterization of cancer enzymes. • ABPP can be integrated with metabolomics to map deregulated enzymatic pathways in cancer. • ABPP can be integrated with other proteomic methods to map proteolytic pathways in cancer. • ABPP probes can be used to image tumour development in living animals.
Activity-based protein profiling (ABPP), the use of active site-directed chemical probes to monitor enzyme function in complex biological systems, is emerging as a powerful post-genomic technology. ABPP probes have been developed for several enzyme classes and have been used to inventory enzyme activities en masse for a range of (patho)physiological processes.
ABPP uses active site–directed, small molecule–based covalent probes to report on the functional state of enzyme activities directly in native biological systems. ABPP probes are designed or selected to target a subset of the proteome based on shared principles of binding and/or reactivity and have been successfully developed for many enzyme classes, including
serine
cysteine,
aspartyl
metallo hydrolases
kinases
glycosidases
histone deacetylases and
oxidoreductases.
These probes have been shown to selectively label active enzymes but not their inactive precursor (zymogen) or inhibitor-bound forms, thus allowing researchers to capture functional information that is beyond the scope of standard proteomic methods. By presenting specific examples, we show here that ABPP provides researchers with a distinctive set of chemical tools to embark on the assignment of functions to many of the uncharacterized enzymes that populate eukaryotic and prokaryotic proteomes.
Vinyl-methylester UL from HSV-1 Deubiquitinating enzyme (DUB)
Aryl 2-deoxy-2-fluoro glycoside Cfx from C. fimi Glycosidase (β-1-4-glycanase) Fluorophosphonate SAE Serine hydrolase
Examples of enzymes assigned to specific mechanistic classes by ABPP
ABPP can also be implemented as a direct assay for inhibitor discovery, allowing researchers to develop potent and selective pharmacological probes for uncharacterized enzymes.
Examples of enzymes assigned to specific mechanistic classes by ABPP.
Probe Leu-Asp-αCA probe selectively labeled Upβ
Substrate the endogenous Upβ substrate, N-carbamoyl-β-alanine
Substrate mimicry of an ABPP probe.
Multidimensional profiling strategy for the annotation of the cancer-related enzyme KIAA1363. ABPP using fluorophosphonate probes identified KIAA1363 as a highly elevated enzyme activity in aggressive cancer cells. Competitive ABPP was then used to develop a selective KIAA1363 inhibitor (AS115). Metabolomic analysis of cancer cells treated with AS115 determined a role for this enzyme in the regulation of MAGE lipids in cancer cells. Biochemical studies confirmed that KIAA1363 acts as 2-acetyl MAGE hydrolase in a metabolic network that bridges the platelet activating factor and lysophosphatidic acid classes of signaling lipids. Assignment of enzyme mechanism by ABPP
There are multiple levels of annotation for enzymes. The most basic level is assignment to a specific mechanistic class based on the general chemical reaction catalyzed by the enzyme (for example, hydrolase, kinase, oxidoreductase and others). Additional annotation involves determining the endogenous substrates and products for the enzyme. Finally, complete annotation requires an understanding of how the specific chemical transformation(s) catalyzed by an enzyme integrate into larger metabolic and signaling pathways to influence cell physiology and behavior.
Many of the predicted enzymes uncovered by genome sequencing projects can be assigned to a mechanistic class or ascribed a putative biochemical function based on sequence homology to well-characterized enzymes. But some enzymes have insufficient sequence relatedness for class assignment or have a function different from that predicted by sequence comparisons. ABPP has facilitated class annotation for several such uncharacterized enzymes.
A principal goal of modern biomedical research is to discover, assemble, and experimentally manipulate molecular pathways in cells and organisms to reveal new disease mechanisms.
Toward this end, complete genome sequences for numerous bacteria and higher organisms, including humans, have laid the fundamental groundwork for understanding the molecular basis of life in its many forms. However, the information content of DNA sequences is limited and, on its own, cannot describe most physiological and pathological processes.
Unlike oligonucleotides, proteins are a very diverse group of biomolecules that display a wide range of chemical and biophysical features, including
membrane-binding,
hetero/homo-oligomerization, and
posttranslational modification.
The biochemical complexity intrinsic to protein science intimates that several complementary analytical strategies will be needed to achieve the ultimate goal of proteomics – a comprehensive characterization of the expression, modification state, interaction map, and activity of all proteins in cells and tissues.
A powerful LC-MS strategy for proteomics involves the use of isotope-coded affinity tags (ICAT). This approach enables the comparison of protein expression in proteomes by treating samples with isotopically distinct forms of a chemical labeling reagent. ICAT methods provide superior resolving power compared to gel-based methods and improve access to membrane-associated proteins. More recently, isotope-free MS methods for quantitative proteomics have emerged.
Reverse protein microarrays have also been described in which proteomes themselves are arrayed and the antibodies used for detection in a format analogous to Western blotting. In addition to increasing the throughput of proteomic experiments by integrating the protein separation and detection steps, microarrays consume much less material than conventional proteomic methods. Still, the general application of microarrays for proteomics is currently limited by the availability of high-quality capture reagents (e.g., antibodies, aptamers, etc).
These approaches, by measuring protein abundance provide, like genomics, only an indirect assessment of protein activity and may fail to detect important posttranslational events that regulate protein function, such as protein–protein or protein–small-molecule interactions. To address these limitations, complementary strategies for the functional analysis of proteins have been introduced. Prominent among these functional proteomic efforts is the use of chemistry for the design of active site-directed probes that measure enzyme activity in samples of high biological complexity.
Many post-translational modes of enzyme regulation share a common mechanistic foundation – they perturb the active site such that catalytic power and/or substrate recognition is impaired. Accordingly, it was hypothesized that chemical probes capable of reporting on the integrity of enzyme active sites directly in cells and tissues might serve as effective functional proteomic tools. These activity based protein profiling (ABPP) probes consist of at least two general elements:
a reactive group for binding and covalently modifying the active sites of many members of a given enzyme class or classes
a reporter tag for the detection, enrichment, and identification of probe-labeled proteins
ABPP probes have been successfully developed for more than a dozen enzyme classes, including
all major classes of proteases
kinases
phosphatases
glycosidases
GSTs
oxidoreductases.
Post-translational regulation of enzyme activity. Many enzymes are produced as inactive precursors, or zymogens, which require proteolytic processing for activation. Enzyme activity can be further regulated by interactions with endogenous protein inhibitors. The field of proteomics aims to develop and apply technologies for the characterization of protein function on a global scale. Toward this end, synthetic chemistry has played a major role by providing new reagents to profile segments of the proteome based on activity rather than abundance. Small molecule probes for activity-based protein profiling have been created for more than a dozen enzyme classes and used to discover several enzyme activities elevated in disease states. These innovations have inspired complementary advancements in analytical chemistry, where new platforms have been introduced to augment the information content achievable in chemical proteomics experiments. Here, we will review these analytical platforms and discuss how they have exploited the versatility of chemical probes to gain unprecedented insights into the function of proteins in biological samples of high complexity.
Advanced analytical platforms utilize a range of separation and detection strategies, including LC-MS, CELIF, and antibody microarrays, to achieve an unprecedented breadth and depth of proteome coverage in ABPP investigations. The complementary strengths and weaknesses of each of these methods suggest that the selection of an appropriate analytical platform should be guided by the specific experimental question being addressed. SA Sieber and BF Cravatt. Analytical platforms for activity-based protein profiling – exploiting the versatility of chemistry for functional proteomics. Chem. Commun. 2006, 2311–2319. http://www.rsc.org/chemcomm
Diagnostic Therapeutics in Activity Based Probes Activity-based chemical proteomics-an emerging field involving a combination of organic synthesis, biochemistry, cell biology, biophysics and bioinformatics-allows the detection, visualisation and activity quantification of whole families or selected sub-sets of proteases based upon their substrate specificity. This approach can be applied for drug target/lead identification and validation, the fundamentals of drug discovery. The activity-based probes discussed in this review contain three key features;
a ‘warhead’ (binds irreversibly but selectively to the active site),
a ‘tag’ (allowing enzyme ‘handling’, with a combination of fluorescent, affinity and/or radio labels),
a linker region between warhead and tag.
From the design and synthesis of the linker arise some of the latest developments discussed here; not only can the physical properties (e.g., solubility, localisation) of the probe be tuned, but the inclusion of a cleavable moiety allows selective removal of tagged enzyme from affinity beads etc. Heal WP, Wickramasinghe SR, Tate EW. Activity based chemical proteomics: profiling proteases as drug targets. Curr Drug Discov Technol 2008; 5(3):200-12. PMID: 18690889
The genomic revolution has created a wealth of information regarding the fundamental genetic code that defines the inner workings of a cell. However, it has become clear that analyzing genome sequences alone will not lead to new therapies to fight human disease. Rather, an understanding of protein function within the context of complex cellular networks will be required to facilitate the discovery of novel drug targets and, subsequently, new therapies directed against them. The past ten years has seen a dramatic increase in technologies that allow large-scale, systems-based methods for analysis of global biological processes and disease states.
In the field of proteomics, several well-established methods persist as a means to resolve and analyze complex mixtures of proteins derived from cells and tissues. However, the resolving power of these methods is often challenged by the diverse and dynamic nature of the proteome. The field of activity-based proteomics, or chemical proteomics, has been established in an attempt to focus proteomic efforts on subsets of physiologically important protein targets. This new approach to proteomics is centered around the use of small molecules termed activity-based probes (ABPs) as a means to tag, enrich, and isolate, distinct sets of proteins based on their enzymatic activity. Berger AB, Vitorino PM, Bogyo M. Activity-based protein profiling: applications to biomarker discovery, in vivo imaging and drug discovery. Am J Pharmacogenomics. 2004;4(6):371-81.
Recent advances in global genomic and proteomic methods have led to a greater understanding of how genes and proteins function in complex networks within a cell. One of the major limitations in these methodologies is their inability to provide information on the dynamic, post-translational regulation of enzymatic proteins. In particular proteases are often synthesized as inactive zymogens that need to be activated in order to carry out specific biological processes. Thus, methods that allow direct monitoring of protease activity in the context of a living cell or whole animal will be required to begin to understand the systems-wide functional roles of proteases. In this review, we discuss the development and applications of activity based probes (ABPs) to study proteases and their role in pathological processes. Specifically we focus on application of this technique for biomarker discovery, in vivo imaging and drug screening.
Proteases, in particular, are known for their multilayered post-translational activity regulation that can lead to a significant difference between protease abundance levels and their enzyme activity. To address these issues, the field of activity-based proteomics has been established in order to characterize protein activity and monitor the functional regulation of enzymes in complex proteomes.
As a result of the recent enormous technological progress, experimental structure determination has become an integral part of the development of drugs against disease-related target proteins. The post-translational modification of proteins is an important regulatory process in living organisms; one such example is lytic processing by peptidases. Many different peptidases represent disease targets and are being used in structure-based drug design approaches. The development of drugs such as aliskiren and tipranavir, which inhibit renin and HIV protease, respectively, testifies to the success of this approach.
Presenilin is the catalytic component of γ-secretase, a complex aspartyl protease and a founding member of intramembrane-cleaving proteases. γ-Secretase is involved in the pathogenesis of Alzheimer’s disease and a top target for therapeutic intervention. However, the protease complex processes a variety of transmembrane substrates, including the Notch receptor, raising concerns about toxicity. Nevertheless, γ-secretase inhibitors and modulators have been identified that allow Notch processing and signaling to continue, and promising compounds are entering clinical trials.
Molecular and biochemical studies offer a model for how this protease hydrolyzes transmembrane domains in the confines of the lipid bilayer. Progress has also been made toward structure elucidation of presenilin and the γ-secretase complex by electron microscopy as well as by studying cysteine-mutant presenilins. The signal peptide peptidase (SPP) family of proteases are distantly related to presenilins. However, the SPPs work as single polypeptides without the need for cofactors and otherwise appear to be simple model systems for presenilin in the γ-secretase complex.
Critical clues to the identity of γ-secretase included: (1) Genes encoding the multi-pass membrane proteins presenilin-1 and presenilin-2 are, like APP, associated with familial, early-onset Alzheimer’s disease. The disease-causing missense mutations were found to alter how γ-secretase cuts APP, leading to increased proportions of longer, more aggregation-prone forms of Aβ. (2) Knockout of presenilin genes eliminates γ-secretase cleavage of APP. (3) Peptidomimetics that inhibit γ-secretase contain moieties typically found in aspartyl protease inhibitors. These findings led to the identification of two conserved transmembrane aspartates in the multi-pass presenilins that are critical for γ-secretase cleavage of APP, evidence that presenilins are aspartyl proteases. Presenilin is endoproteolytically cleaved into two polypeptides, an N-terminal fragment (NTF) and a C-terminal fragment (CTF), the formation of which is
regulated
metabolically stable
part of a high-molecular weight complex
suggesting that the NTF-CTF heterodimer is the biologically active form. NTF and CTF each contribute one of the essential and conserved aspartates, and transition-state analogue inhibitors of γ-secretase, compounds designed to interact with the active site of the protease, bind directly to presenilin NTF and CTF. Presenilins are also required for Notch signaling (Levitan and Greenwald, 1995), a pathway essential for cell differentiation during development and beyond.
The highly conserved role of γ-secretase in Notch signalling and its importance in development led to genetic screens in Caenorhabditis elegans that identified three other integral membrane proteins besides presenilin that modify Notch signaling. Designed inhibitors have proven to be useful tools in understanding the mechanism of γ-secretase and substrate recognition – affinity labelling with transition-state analogue inhibitors showed binding at the interface between the presenilin NTF and CTF subunits, consistent with the active site residing at this interface, with each presenilin subunit contributing one of the essential aspartates. The concept of presenilin as the catalytic component for γ-secretase was considerably strengthened when
signal peptide peptidase (SPP) was found to be a similar intramembrane aspartyl protease
SPP is exploited by the hepatitis C virus for the maturation of its core protein, suggesting that this protease may be a suitable target for antiviral therapy
SPP was identified by affinity labeling with a peptidomimetic inhibitor, and the protein sequence displayed similarities with presenilin.
SPP contains two conserved aspartates, each predicted to lie in the middle of a transmembrane domain, and the aspartate-containing sequences resemble those found in presenilins.
SPP appears to be less complicated than γ-secretase.
Expression of human SPP in yeast reconstituted the protease activity, suggesting that the protein has activity on its own and does not require other mammalian protein cofactors.
Aspartyl I-CLiPs are found in all forms of life and play essential roles in biology and disease. How these enzymes carry out hydrolysis in the membrane is a fascinating question that is not entirely resolved, but evidence suggests an initial substrate docking site and a lateral gate into a pore where water and the active site aspartates reside. Designed inhibitors have been critical in elucidating these mechanisms, but inhibitors targeting γ-secretase for the treatment of Alzheimer’s disease must avoid interfering with Notch signaling.
MS Wolfe. Structure, Mechanism and Inhibition of γ-Secretase and Presenilin-Like Proteases. Biol Chem. 2010 August; 391(8): 839–847. doi: 10.1515/BC.2010.086. PMCID: PMC2997569. NIHMSID: NIHMS254540 Study Suggests Expanding the Genetic Alphabet May Be Easier than Previously Thought Genomics Monday, June 4, 2012 A new study led by scientists at The Scripps Research Institute suggests that the replication process for DNA—the genetic instructions for living organisms that is composed of four bases (C, G, A and T)—is more open to unnatural letters than had previously been thought.
An expanded “DNA alphabet” could carry more information than natural DNA, potentially coding for a much wider range of molecules and enabling a variety of powerful applications, from precise molecular probes and nanomachines to useful new life forms. The new study, which appears in the June 3, 2012 issue of Nature Chemical Biology, solves the mystery of how a previously identified pair of artificial DNA bases can go through the DNA replication process almost as efficiently as the four natural bases. “We now know that the efficient replication of our unnatural base pair isn’t a fluke, and also that the replication process is more flexible than had been assumed,” said Floyd E. Romesberg, principal developer of the new DNA bases.
Adding to the DNA Alphabet Romesberg and his lab have been trying to find a way to extend the DNA alphabet since the late 1990s. In 2008, they developed the efficiently replicating bases NaM and 5SICS, which come together as a complementary base pair within the DNA helix, much as, in normal DNA, the base adenine (A) pairs with thymine (T), and cytosine (C) pairs with guanine (G).
The following year, Romesberg and colleagues showed that NaM and 5SICS could be efficiently transcribed into RNA. But these bases’ lack the ability to form the hydrogen bonds that join natural base pairs in DNA. Such bonds had been thought to be an absolute requirement for successful DNA replication‑—a process in which a large enzyme, DNA polymerase, moves along a single, unwrapped DNA strand and stitches together the opposing strand, one complementary base at a time.
An early structural study of a very similar base pair in double-helix DNA added to Romesberg’s concerns. The data strongly suggested that NaM and 5SICS do not even approximate the edge-to-edge geometry of natural base pairs—termed the Watson-Crick geometry, after the co-discoverers of the DNA double-helix. Instead, they join in a looser, overlapping, “intercalated” fashion. “Their pairing resembles a ‘mispair,’ such as two identical bases together, which normally wouldn’t be recognized as a valid base pair by the DNA polymerase.” Yet in test after test, the NaM-5SICS pair was efficiently replicable.
Edge to Edge The NaM-5SICS pair maintain an abnormal, intercalated structure within double-helix DNA—but remarkably adopt the normal, edge-to-edge, “Watson-Crick” positioning when gripped by the polymerase during the crucial moments of DNA replication. “The DNA polymerase apparently induces this unnatural base pair to form a structure that’s virtually indistinguishable from that of a natural base pair.” NaM and 5SICS, lacking hydrogen bonds, are held together in the DNA double-helix by “hydrophobic” forces, which cause certain molecular structures to be repelled by water molecules, and thus to cling together in a watery medium. “It’s very possible that these hydrophobic forces have characteristics that enable the flexibility and thus the replicability of the NaM-5SICS base pair.”
An Arbitrary Choice? The finding suggests that NaM-5SICS and potentially other, hydrophobically bound base pairs could some day be used to extend the DNA alphabet. It also hints that Evolution’s choice of the existing four-letter DNA alphabet—on this planet—may have been somewhat arbitrary. “It seems that life could have been based on many other genetic systems.” Source: The Scripps Research Institute
DNA damage response (DDR) network
Eukaryotic cells have evolved an intricate system to resolve DNA damage to prevent its transmission to daughter cells. This system, collectively known as the DNA damage response (DDR) network, includes many proteins that detect DNA damage, promote repair, and coordinate progression through the cell cycle. Because defects in this network can lead to cancer, this network constitutes a barrier against tumorigenesis. The modular BRCA1 carboxyl-terminal (BRCT) domain is frequently present in proteins involved in the DDR, can exist either as an individual domain or as tandem domains (tBRCT), and can bind phosphorylated peptides. We performed a systematic analysis of protein-protein interactions involving tBRCT in the DDR.
We identified 23 proteins containing conserved BRCT domains and generated a human protein-protein interaction network for seven proteins with tBRCT. This study also revealed previously unknown components in DNA damage signaling, such as COMMD1 and the target of rapamycin complex mTORC2. Additionally, integration of tBRCT domain interactions with DDR phosphoprotein studies and analysis of kinase-substrate interactions revealed signaling subnetworks that may aid in understanding the involvement of tBRCT in disease and DNA repair.
Mitochondria have various essential functions in metabolism and in determining cell fate during apoptosis. In addition, mitochondria are also important nodes in a number of signaling pathways. For example, mitochondria can modulate signals transmitted by second messengers such as calcium. Because mitochondria are also major sources of reactive oxygen species (ROS), they can contribute to redox signaling—for example, by the production of ROS such as hydrogen peroxide that can reversibly modify cysteine residues and thus the activity of target proteins. Mitochondrial ROS production is thought to play a role in hypoxia signaling by stabilizing the oxygen-sensitive transcription factor hypoxia-inducible factor–1α. New evidence has extended the mechanism of mitochondrial redox signaling in cellular responses to hypoxia in interesting and unexpected ways. Hypoxia altered the microtubule-dependent transport of mitochondria so that the organelles accumulated in the perinuclear region, where they increased the intranuclear concentration of ROS. The increased ROS in turn enhanced the expression of hypoxia-sensitive genes such as VEGF (vascular endothelial growth factor) not by reversibly oxidizing a protein, but by oxidizing DNA sequences in the hypoxia response element of the VEGF promoter. This paper and other recent work suggest a new twist on mitochondrial signaling: that the redistribution of mitochondria within the cell can be a component of regulatory pathways.
A challenge in the treatment of lung cancer is the lack of early diagnostics. Here, we describe the application of monoclonal antibody proteomics for discovery of a panel of biomarkers for early detection (stage I) of non-small cell lung cancer (NSCLC). We produced large monoclonal antibody libraries directed against the natural form of protein antigens present in the plasma of NSCLC patients. Plasma biomarkers associated with the presence of lung cancer were detected via high throughput ELISA. Differential profiling of plasma proteomes of four clinical cohorts, totaling 301 patients with lung cancer and 235 healthy controls, identified 13 lung cancer-associated (p < 0.05) monoclonal antibodies. The monoclonal antibodies recognize five different cognate proteins identified using immunoprecipitation followed by mass spectrometry. Four of the five antigens were present in non-small cell lung cancer cells in situ.
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