The authors have declared that no competing interests exist. The authors have filled a PCT Patent Application “PLA2 Activity as a Marker for Ovarian and Other Gynecologic Cancers” in 2011. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.
Conceived and designed the experiments: YX. Performed the experiments: HC. Analyzed the data: HC ZL. Contributed reagents/materials/analysis tools: EGC MVC DKR BWR NMH PJL MLH. Wrote the paper: YX HC.
Only in recent years have phospholipase A2 enzymes (PLA2s) emerged as cancer targets. In this work, we report the first detection of elevated PLA2 activities in plasma from patients with colorectal, lung, pancreatic, and bladder cancers as compared to healthy controls. Independent sets of clinical plasma samples were obtained from two different sites. The first set was from patients with colorectal cancer (CRC; n = 38) and healthy controls (n = 77). The second set was from patients with lung (n = 95), bladder (n = 31), or pancreatic cancers (n = 38), and healthy controls (n = 79). PLA2 activities were analyzed by a validated quantitative fluorescent assay method and subtype PLA2 activities were defined in the presence of selective inhibitors. The natural PLA2 activity, as well as each subtype of PLA2 activity was elevated in each cancer group as compared to healthy controls. PLA2 activities were increased in late stage vs. early stage cases in CRC. PLA2 activities were not influenced by sex, smoking, alcohol consumption, or body-mass index (BMI). Samples from the two independent sites confirmed the results. Plasma PLA2 activities had approximately 70% specificity and sensitivity to detect cancer. The marker and targeting values of PLA2 activity have been suggested.
Cancer is one of the major health burdens worldwide. More than 1.6 million new cancer cases and over 577,000 deaths from cancer are projected to occur in the United States in 2012
Phospholipase A2 enzymes (PLA2s) are the major enzymes producing the cyclooxygenase-2 (COX-2) substrate, arachidonic acid (AA), as well as lysophospholipids. Both of these classes of products are signaling molecules involved in cancers. However, only in recent years have PLA2s emerged as cancer targets
In the current work we have focused on PLA2 activities rather than expression of individual PLA2 enzymes and examined PLA2 activities in blood plasma samples from patients with different cancers, in comparison with those from healthy controls. We used our recently validated quantitative, convenient, highly reproducible PLA2 assay method
Two sets of independently collected clinical plasma samples were obtained for this study. The first set from the Indiana University School of Medicine (IUSM) focused on CRC patients and healthy controls screened by colonoscopy and found negative for adenomatous polyps and CRC. Blood samples collected in the presence of EDTA were centrifuged at 1,750 g for 15 min at 15–24°C, aliquoted in siliconized Eppendorf tubes and stored at −80°C. The second set of samples was from patients with lung, bladder, or pancreatic cancers, as well as healthy controls. These samples were collected by the Hoosier Oncology Group (HOG) in Indianapolis, IN as part of the study entitled, “A Biological Sample Collection Protocol of Patients with and without Metastatic Solid Organ Malignancies: Hoosier Oncology Group Study BANK09-138”. Blood samples were centrifuged at 3,500 rpm for 30 min. The aliquoted samples were stored at −70°C. HOG is a non-profit medical research organization. Although it is a separate entity, IUSM is a supporting organization for board appointments and IRB approvals for HOG. Three separate IRB protocols for collecting and/or use the blood samples related to this work have been approved by the same IUSM Institutional Review Board (Protocol numbers: 0670-81, 0808-24, 0905-20). Written informed consent forms were obtained from all subjects and all clinical investigation had been conducted according to the principles expressed in the Declaration of Helsinki.
The PLA2 substrate 1-O-(6-Dabcyl-Aminohexanoyl)-2-O-(6-(12-BODIPY-Dodecanoyl) Aminohexanoyl)-sn-3-Glyceryl Phosphatidylcholine (DBPC) was from Echelon Bioscience (Salt Lake City, UT, USA). Bromoenol lactone (BEL) and methyl arachidonylfluorophosphonate (MAFP) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
PLA2 activities were analyzed using the fluorescent substrate DBPC, a fluorogenic phosphatidylcholine substrate
Categorical variables were summarized as counts with percentages and continuous variables were summarized as means with standard deviations (SD) across the healthy control, and cancer groups. The Chi-square test was used to test the associations between disease statuses and categorical covariates such as sex, race, smoking, and alcohol drinking. For continuous covariates such as age and BMI, one-way ANOVA was used to test the overall difference and Student
The demographic data for the study participants are summarized in
Age (years) | Sex (%) | Race (%) | |||||||||
No. | Mean | SD | Male | Female | White | African American | Others | ||||
Participants from IUSM | |||||||||||
Healthy | 77 | 51.6 | 13.5 | 40.3 | 59.7 | 89.6 | 9.1 | 1.3 | |||
CRC | 38 | 56.0 | 13.4 | 0.1075 | 44.7 | 55.3 | 0.6470 | 86.8 | 5.3 | 7.9 | 0.4501 |
Participants from HOG | |||||||||||
Healthy | 79 | 42.7 | 12.0 | 77.2 | 22.8 | 89.3 | 2.7 | 8.0 | |||
LC | 95 | 63.2 | 9.5 | <0.0001 | 64.2 | 35.8 | 0.0621 | 89.5 | 8.4 | 2.1 | 0.0654 |
BC | 31 | 70.5 | 10.0 | <0.0001 | 87.1 | 12.9 | 0.2438 | 93.6 | 6.5 | 0.0 | 0.1868 |
PC | 38 | 68.7 | 8.7 | <0.0001 | 63.2 | 36.8 | 0.1102 | 92.1 | 5.3 | 2.6 | 0.4327 |
CRC: colorectal cancer; LC: Lung cancer; BC: Bladder cancer; PC: Pancreatic cancer; SD, standard deviation; HOG: Hoosier Oncology Group.
In the HOG set: • For gender, the overall
We have recently validated the quantitative nature of the DBPC-based PLA2 assays and optimized the conditions for biological fluids and tissues samples
Blood iPLA2 and cPLA2 activities have not been previously reported in human samples. We analyzed the natural PLA2 (defined as the PLA2 activities measured under the conditions without any modifiers, which may be lower than the sum of the subgroup PLA2 activities measured under modified conditions), as well as cPLA2, iPLA2, and sPLA2 activities in plasma samples.
Except for the healthy vs. CRC cPLA2 activity, all other comparisons showed that PLA2 activities were significantly elevated in the cancer groups (
Comparison of PLA2 activities in the healthy control (normal colonoscopy; n = 77) and CRC (n = 38) groups. A. Comparison of PLA2 activities in the healthy (n = 79) and other cancer groups. Lung cancer (LC, n = 95), bladder cancer (BC, n = 31) and pancreatic cancer (PC, n = 38). The distribution of the natural and individual groups of PLA2 activities were analyzed as described in
Since sPLA2 is secreted, it was expected that sPLA2 activity would account for the major portion of the PLA2 activity detected in the blood. However, in our results, high levels of iPLA2 and cPLA2 and low levels of sPLA2 activities were observed in all plasma samples. Blood exosomes may be the carriers of these activities
Interestingly, the natural, iPLA2, and sPLA2 activities in plasma of CRC patients were increased in subjects with late stages (III and IV) as compared to earlier stage (I and II) disease (
The natural and individual groups of PLA2 activities were progressively increased by tumor stage. Stage I and II (n = 7), Stage III and IV (n = 31).
PLA2 | iPLA2 | cPLA2 | sPLA2 | ||||||||||
No. | Mean | SD | Mean | SD | Mean | SD | Mean | SD | |||||
Primary site | 0.6856 |
0.7407 |
0.6419 |
0.3410 |
|||||||||
Colon | 24 | 1412.3 | 473.75 | 1302.8 | 428.78 | 1424.0 | 480.16 | 476.3 | 199.33 | ||||
Rectum | 14 | 1348.0 | 460.33 | 1254.7 | 428.33 | 1347.7 | 489.95 | 408.3 | 195.50 | ||||
Previous treatment | 0.6543 |
0.3081 |
0.3348 |
0.6212 |
|||||||||
Surgery only | 7 | 1340.3 | 382.29 | 1193.1 | 286.55 | 1303.4 | 424.30 | 435.5 | 92.75 | ||||
CT and RT | 3 | 1614.8 | 542.80 | 1599.5 | 626.64 | 1755.4 | 707.50 | 495.9 | 219.34 | ||||
Surgery and CT | 10 | 1496.8 | 596.54 | 1399.5 | 543.97 | 1521.1 | 633.55 | 519.8 | 264.44 | ||||
Surgery, CT and RT | 5 | 1459.0 | 437.46 | 1402.9 | 322.07 | 1511.8 | 322.27 | 472.8 | 200.95 | ||||
Untreated | 13 | 1252.2 | 404.71 | 1128.6 | 341.99 | 1221.8 | 327.50 | 388.5 | 183.68 |
Student
One-way ANOVA.
CT: chemotherapy; RT: radiotherapy.
PLA2 | iPLA2 | cPLA2 | sPLA2 | ||||||||||
No. | Mean | SD | Mean | SD | Mean | SD | Mean | SD | |||||
Lung cancer | |||||||||||||
T stage | 0.4760 |
0.5001 |
0.4960 |
0.6129 |
|||||||||
T1 | 19 | 1714.2 | 548.5 | 1626.6 | 514.2 | 2190.2 | 752.6 | 457.1 | 280.7 | ||||
T2 | 21 | 1569.3 | 671.8 | 1490.6 | 640.6 | 1924.1 | 754.0 | 478.7 | 261.4 | ||||
T3 | 9 | 1437.9 | 479.0 | 1347.4 | 460.7 | 1721.3 | 480.5 | 356.2 | 166.6 | ||||
T4 | 35 | 1803.5 | 878.2 | 1691.8 | 823.9 | 2141.7 | 1105.9 | 471.5 | 228.4 | ||||
N stage | 0.9973 |
0.9685 |
0.9758 |
0.8009 |
|||||||||
N0 | 14 | 1626.7 | 566.2 | 1559.5 | 522.6 | 2061.3 | 584.4 | 386.3 | 143.2 | ||||
N1 | 11 | 1574.8 | 508.1 | 1442.7 | 479.8 | 1960.3 | 671.2 | 404.1 | 200.4 | ||||
N2 | 21 | 1613.9 | 643.7 | 1524.1 | 631.4 | 1961.6 | 883.6 | 434.1 | 222.9 | ||||
N3 | 29 | 1604.2 | 671.7 | 1510.2 | 620.6 | 1944.3 | 888.0 | 454.8 | 270.0 | ||||
Bladder cancer | |||||||||||||
T stage | 0.7482 |
0.7320 |
0.5790 |
0.1793 |
|||||||||
Tis | 2 | 1736.6 | 429.9 | 1568.4 | 406.3 | 2046.9 | 355.0 | 395.2 | 119.1 | ||||
T1 | 4 | 1326.0 | 525.4 | 1306.3 | 517.3 | 1717.4 | 525.6 | 299.2 | 222.9 | ||||
T2 | 10 | 1833.5 | 832.3 | 1710.5 | 759.0 | 2207.7 | 1074.9 | 451.0 | 160.6 | ||||
T3 | 7 | 1812.3 | 522.3 | 1833.4 | 506.3 | 2541.3 | 630.6 | 444.4 | 141.7 | ||||
T4 | 8 | 1800.7 | 582.9 | 1630.9 | 544.2 | 2098.4 | 638.9 | 596.2 | 263.3 | ||||
N stage | 0.8224 |
0.7579 |
0.7786 |
0.1010 |
|||||||||
N0 | 10 | 1665.1 | 479.6 | 1535.2 | 446.2 | 2158.4 | 619.1 | 436.1 | 168.2 | ||||
N1 | 5 | 1602.0 | 482.3 | 1549.8 | 507.6 | 1872.4 | 726.4 | 411.2 | 173.2 | ||||
N2 | 8 | 1813.4 | 964.3 | 1806.0 | 881.1 | 2333.5 | 1107.6 | 400.4 | 180.1 | ||||
N3 | 5 | 1937.5 | 428.0 | 1761.7 | 394.4 | 2135.4 | 234.1 | 671.9 | 287.7 | ||||
Pancreatic cancer | |||||||||||||
T stage | 0.7748 |
0.8479 |
0.8488 |
0.8453 |
|||||||||
T1 | 1 | 1398.3 | 1294.2 | 1400.7 | 419.7 | ||||||||
T2 | 12 | 1823.2 | 708.6 | 1730.9 | 647.8 | 2413.7 | 897.1 | 476.3 | 139.5 | ||||
T3 | 8 | 2031.6 | 1477.7 | 1891.1 | 1421.5 | 2693.9 | 2009.0 | 556.0 | 286.2 | ||||
T4 | 15 | 2279.0 | 1509.8 | 2077.8 | 1423.9 | 2741.7 | 1919.3 | 595.7 | 506.3 | ||||
N stage | 0.1928 |
0.1448 |
0.1243 |
0.0671 |
|||||||||
N0 | 9 | 2663.9 | 1855.8 | 2564.3 | 1749.6 | 3475.3 | 2290.2 | 699.2 | 377.1 | ||||
N1 | 19 | 1756.4 | 837.8 | 1598.2 | 738.6 | 2133.6 | 964.7 | 428.6 | 152.1 |
Student
One-way ANOVA.
We have collected other factors, which might influence PLA2 activities, including smoking, alcohol consumption, and body-mass index (BMI). For the CRC study, from the 80 subjects with available smoking information (27 CRC and 53 healthy cases), the majority (96%) of people did not smoke when the samples were collected, with an average 50.5% people never smoked and 45.7% of past smoker, and only 9.4% and 19.2% people had second hand smoking exposure in each group. The smoking statuses were similarly distributed in the two groups (
PLA2 activities in healthy and CRC samples with different smoking statuses(a)and second hand smoking statuses(b). A. Comparison of PLA2 activities between different smoking statuses(a) and second hand smoking statuses(b) in participants with LC, BC, and PC.
Healthy | CRC | ||||
No. | % | No. | % | ||
Smoking status | |||||
Current smoker | 2 | 3.8 | 1 | 3.7 | 0.4609 |
Past smoker | 18 | 34.0 | 13 | 48.2 | |
Never smoked | 33 | 62.3 | 13 | 48.2 | |
Second hand smoke | 0.2185 | ||||
No | 48 | 90.6 | 21 | 80.8 | |
Yes | 5 | 9.4 | 5 | 19.2 | |
Alcohol | 0.2542 | ||||
No | 6 | 11.3 | 1 | 3.7 | |
Yes | 47 | 88.4 | 26 | 96.3 | |
BMI (Mean ± SD) | 28.2±6.96 | 25.4±4.01 | 0.0272 |
These data are available from a subset of the participants.
Healthy | LC | BC | PC | ||||||||
No. | % | No. | % | No. | % | No. | % | ||||
Smoking status | <0.0001 | 0.0017 | 0.016 | ||||||||
Current smoker | 4 | 5.1 | 29 | 30.5 | 4 | 12.9 | 7 | 18.4 | |||
Past smoker | 25 | 31.6 | 58 | 61.1 | 19 | 61.3 | 16 | 42.1 | |||
Never smoked | 50 | 63.3 | 8 | 8.4 | 8 | 25.8 | 15 | 39.5 | |||
Second hand smoke | <0.0001 | 0.0091 | 0.0741 | ||||||||
No | 32 | 40.5 | 10 | 10.5 | 4 | 13.8 | 9 | 23.7 | |||
Yes | 47 | 59.5 | 85 | 89.5 | 25 | 86.2 | 29 | 76.3 | |||
Alcohol | <0.0001 | 0.0018 | <0.0001 | ||||||||
No | 23 | 29.6 | 67 | 70.5 | 19 | 61.3 | 31 | 81.6 | |||
Yes | 56 | 70.4 | 28 | 29.5 | 12 | 38.7 | 7 | 18.4 | |||
BMI (Mean ± SD) | 28.3±5.55 | 26.5±6.76 | 0.0707 | 28.3±5.01 | 0.9625 | 25.9±6.17 | 0.0374 |
LC: Lung cancer; BC: Bladder cancer; PC: Pancreatic cancer.
All
Alcohol consumption in the control and CRC groups were not significantly different (
Comparison of PLA2 activities between “non-alcohol” and “alcohol” groups with the healthy and CRC participants. A. Comparison of PLA2 activities between “non-alcohol” and “alcohol” groupsin the second set of studies with healthy, LC, BC, and PC participants.
PLA2 activity comparison in males and females in the healthy and the CRC groups. A. PLA2 activity comparison in males and females in the healthy and other cancer groups. Data from all subjects in each set of studies are also presented.
The differences in PLA2 activities between sexes did not reach a statistical significance in the control or any cancer groups in either set of studies (
The subjected in healthy and CRC groups were divided into two groups: BMI 18.5 to <25 (For all subjects, n = 43) and BMI>25 (For all subjects, n = 80). A. The subjected in healthy and other cancer groups were divided into two groups: BMI≤25 (For all subjects, n = 80) and BMI>25 (For all subjects, n = 148).
Age is one of the most clearly identified risk factors for developing CRC and other cancers. The American Cancer Society and the U.S. Preventive Services Task Force recommend that people receive colonoscopy screenings every 10 years beginning at age 50. We thus analyzed the age effects on PLA2 activities by dividing the subjects into two groups (age <50 years, n = 37 and ≥50 years, n = 78). Although there was a trend of increased PLA2 activities in the ≥50 years-old group in the CRC group, the differences were not statistically significant (
The participants in healthy and CRC groups were divided into two groups: age<50 (For all subjects, n = 42) and age ≥50 (For all subjects, n = 153). Student t-test was performed to analyze the differences between these two groups. A. The participants in healthy and other cancer groups were divided into two groups: age<50 (For all subjects, n = 61) and age ≥50 (For all subjects, n = 144). Student t-test was performed to analyze the differences between these two groups. ***P<0.001.
A direct comparison of the two sets of healthy controls showed that although their sex, age, second-hand smoking, and alcohol consumption were different, there were no differences in any PLA2 activity measured (
1st set | 2nd set | ||||
No. | % | No. | % | ||
Gender | <0.0001 | ||||
Male | 31 | 40.3 | 61 | 77.2 | |
Female | 46 | 59.7 | 18 | 22.8 | |
Smoking status | |||||
Current smoker | 2 | 3.8 | 4 | 5.1 | 0.889 |
Past smoker | 19 | 35.8 | 25 | 31.6 | |
Never smoked | 32 | 60.4 | 50 | 63.3 | |
Second hand smoke | <0.0001 | ||||
No | 48 | 90.6 | 32 | 40.5 | |
Yes | 5 | 9.4 | 47 | 59.5 | |
Alcohol | <0.0001 | ||||
No | 6 | 11.3 | 23 | 29.6 | |
Yes | 47 | 88.4 | 56 | 70.4 | |
Age (Mean ± SD) | 51.6±13.5 | 42.7±12.03 | <0.0001 | ||
PLA2 activities(Mean ± SD) | |||||
PLA2 | 1135.9±266.2 | 1156.3±311.8 | 0.660 | ||
iPLA2 | 1082.0±286.1 | 1105.4±301.6 | 0.620 | ||
cPLA2 | 1246.1±318.7 | 1287.8±425.9 | 0.488 | ||
sPLA2 | 299.1±164.7 | 293.9±137.3 | 0.828 |
Logistic regression was used to evaluate the classification performance of PLA2 activities in cancers. All four PLA2 measurements were kept as predictors regardless of their significance. The classification performances were summarized by receiver operating characteristic (ROC) curves (
CRC cases vs. healthy subjects. A. All cancers vs. healthy subjects in the 2nd set. B. Lung cancer vs. healthy subjects. C. Bladder cancer vs. healthy subjects. D. Pancreatic cancer vs. healthy subjects. E. All cancers vs. all healthy subjects with combined two sets.
The prediction formula to separate CRC cases from healthy subjects is:
Since external validations would require an independent study, we instead adopted internal methods to validate the classification performances. Among different internal validation methods, it has been shown that bootstrap outperforms jackknife, cross-validation and data-splitting methods
The prediction formula and the ROC curve to separate all cancer cases from healthy subjects in the second set of study are as follows and shown in
The prediction formulas and the ROC curves for the separate LC, BC, and PC cases from healthy subjects in the second set of study are listed below and
Finally, since we have found that the PLA2 activities were essentially no difference in the two sets of healthy controls, we combined all healthy controls and all cancer cases in both sets of studies and generated a combined formula, with an AUC = 0.8011 (
Without other parameters | With other parameters | |||
Sensitivity | Specificity | Sensitivity | Specificity | |
First set | ||||
CRC vs. Healthy | 60.5% | 77.9% | 63.0% | 75.5% |
Second set | ||||
All cancer vs. healthy | 81.1% | 72.2% | 87.2% | 88.6% |
LC vs. healthy | 82.1% | 67.1% | 85.3% | 91.1% |
BC vs. healthy | 80.6% | 75.9% | 87.1% | 87.3% |
PC vs. healthy | 81.6% | 77.2% | 86.8% | 96.2% |
Combined two sets | ||||
Cancer vs. healthy | 80.2% | 66.7% | 83.2% | 81.1% |
Although most other demographic and/or environmental factors tested were not significantly different in cancer and control groups (
The prediction formula and the ROC curve to separate CRC from healthy subjects in the first set of study are as follows and shown in
CRC cases vs. healthy subjects. A. All cancer cases vs. healthy subjects in the second set. B. Lung cancer vs. healthy subjects. C. Bladder cancer vs. healthy subjects. D. Pancreatic cancer vs. healthy subjects. E. All cancers vs. all healthy subjects with combined two sets.
The prediction formulas and the ROC curves for the separate LC, BC, and PC cases from healthy subjects in the second set of study are listed below and
Finally, since we found that the PLA2 activities were essentially no difference in the two sets of healthy controls, we combined all healthy controls and all cancer cases in both sets of studies and generated a combined formula, with an AUC = 0.9140 (
In this work, we have presented the first measurements of plasma natural and subtype PLA2 activities and their performances as potential markers for four different cancers, CRC, LC, BC, and PC. We have measured the PLA2 activities in more than 20 blood samples with or without adding 1.2 mM calcium (the natural ionized calcium concentration in blood) to the final assay mixtures and did not detect any significant differences. Hence, we define the PLA2 activities obtained without any additives as the “natural” PLA2 activity. We have selected optimal conditions to measure each sub-family of PLA2 activities. These modified conditions do not co-exist in the blood. Therefore, it is not surprising that the sum of the subfamily PLA2 activities is greater than the “natural” PLA2 activity. However, it is important to note that although modified conditions were used to obtain the activities for subfamilies of PLA2s, the final results are from the natural PLA2s present in the blood samples.
While the advantages of using convenient serologic markers are obvious, no reliable blood markers for any of these cancers are currently available. For CRC, developing/validating non-invasive or minimally invasive detection methods is a major focus in the field. Current methods include stool and blood tests, such as fecal immunochemical tests (FITs) and guaiac-based fecal occult blood testing (got). Although the specificity is high (>85%), these tests have a low sensitivity (<23%) for colorectal adenomas and are thus unlikely to be able to increase the early detection of CRC
Is a marker which may detect multiple types of cancers useful? The answer is likely to be yes. Even after decades of efforts, the success rate in finding highly specific markers for specific cancers has been low. Prostate-specific antigen is an exception, but even the value of this marker as a screening tool in prostate cancer has been recently questioned
Another major advantage of the test is that it is reproducible, robotic, and stable. It is well-recognized that many blood markers are sensitive to how blood samples are handled, processed, and stored. It is almost impossible to unify these procedures in the USA or anywhere in the world. Freeze-and-thaw is another well-known factor affecting marker stability. These reasons account, at least in part, for the fact that although thousands of markers have been reported in the past decades, seldom have any of them been cross-validated in different centers and moved to the clinic. We have used two completely independent sets of human plasma samples, which were processed somewhat differently (see
A potential caveat of the clinical usage of PLA2 activity is that these enzymes are known to be involved in inflammation and may be elevated in patients with benign inflammatory diseases. This needs to be experimentally tested in clinical samples. However, there are mounting lines of evidence supporting the strong causal connections between inflammation and cancers
Our results also strongly imply that the PLA2 activities (which may or may not correlate to their RNA or protein expression levels) are potential targets for cancer treatment tested here. Most, if not all previous PLA2 assays conducted in tissues or cell lines mainly focus on their expression levels using PCR and IHC, which are gene/protein specific, time-consuming, costly, and require relatively large amount of samples to cover different isoforms of PLA2s. Since there are more than 30 PLA2s, none of the previous studies provide an overall picture of PLA2s in any cancer. More importantly, their enzymatic activities, but not necessarily their RNA and/or protein expression levels, are directly related to the biological effects, since PLA2 activities are well-known to be regulated post-transcriptionally
We highly appreciate the proof-reading and editing of Dr. Caryl Antalis.