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Accuracy of positron emission tomography/computed tomography and clinical assessment in the detection of complete rectal tumor regression after neoadjuvant chemoradiation†
Long-term results of a prospective trial (National Clinical Trial 00254683)
Article first published online: 15 NOV 2011
Copyright © 2011 American Cancer Society
Volume 118, Issue 14, pages 3501–3511, 15 July 2012
How to Cite
Perez, R. O., Habr-Gama, A., Gama-Rodrigues, J., Proscurshim, I., Julião, G. P. S., Lynn, P., Ono, C. R., Campos, F. G., Silva e Sousa, A. H., Imperiale, A. R., Nahas, S. C. and Buchpiguel, C. A. (2012), Accuracy of positron emission tomography/computed tomography and clinical assessment in the detection of complete rectal tumor regression after neoadjuvant chemoradiation. Cancer, 118: 3501–3511. doi: 10.1002/cncr.26644
All authors were involved in the study design, development and interpretation of results. Drafting and revision of the article was undertaken by all authors. All authors approved the final version of the article. The first author had full access to all data and had final responsibility for the decision to submit the article for publication.
- Issue published online: 2 JUL 2012
- Article first published online: 15 NOV 2011
- Manuscript Accepted: 26 SEP 2011
- Manuscript Revised: 14 SEP 2011
- Manuscript Received: 20 AUG 2011
- rectal cancer;
- neoadjuvant chemoradiation;
- positron emission tomography/computed tomography;
- tumor response;
- complete clinical response
Neoadjuvant chemoradiation (CRT) therapy may result in significant tumor regression in patients with rectal cancer. Patients who develop complete tumor regression have been managed by treatment strategies that are alternatives to standard total mesorectal excision. Therefore, assessment of tumor response with positron emission tomography/computed tomography (PET/CT) after neoadjuvant treatment may offer relevant information for the selection of patients to receive alternative treatment strategies.
Patients with clinical T2 (cT2) through cT4NxM0 rectal adenocarcinoma were included prospectively. Neoadjuvant therapy consisted of 54 grays of radiation and 5-fluorouracil-based chemotherapy. Baseline PET/CT studies were obtained before CRT followed by PET/CT studies at 6 weeks and 12 weeks after the completion of CRT. Clinical assessment was performed at 12 weeks after CRT completion. PET/CT results were compared with clinical and pathologic data.
In total, 99 patients were included in the study. Twenty-three patients were complete responders (16 had a complete clinical response, and 7 had a complete pathologic response). The PET/CT response evaluation at 12 weeks indicated that 18 patients had a complete response, and 81 patients had an incomplete response. There were 5 false-negative and 10 false-positive PET/CT results. PET/CT for the detection of residual cancer had 93% sensitivity, 53% specificity, a 73% negative predictive value, an 87% positive predictive value, and 85% accuracy. Clinical assessment alone resulted in an accuracy of 91%. PET/CT information may have detected misdiagnoses made by clinical assessment alone, improving overall accuracy to 96%.
Assessment of tumor response at 12 weeks after CRT completion with PET/CT imaging may provide a useful additional tool with good overall accuracy for the selection of patients who may avoid unnecessary radical resection after achieving a complete clinical response. Cancer 2012;3501–3511. © 2011 American Cancer Society.
Multimodality therapy has been considered the preferred initial treatment strategy for patients with locally advanced rectal cancer.1, 2 It has been demonstrated that neoadjuvant chemoradiation (CRT) improves local disease control after radical surgery. In addition, neoadjuvant CRT leads to variable degrees of tumor downstaging.3
The effects of radiation and chemotherapy on tumor downstaging have suggested the possibility surgical procedures that may be alternatives to total mesorectal excision. These alternatives are particularly relevant in the management of distal rectal cancers that ultimately will require a major surgical procedure because of the high-risk of developing postoperative urinary, fecal, and sexual dysfunctions.4, 5
Between 5% and 42% of patients who receive neoadjuvant CRT may have a complete pathologic response (pCR).6 These patients with pCR are associated with improved survival and local disease control compared with patients who have residual disease after undergoing radical resection.7 In the absence of residual cancer cells, the role of radical surgery has been challenged by alternative transanal local excision (ypT0) or even observation alone without immediate surgery for patients who have clinical evidence of complete tumor regression (complete clinical response [cCR]).8
Still, the clinical assessment of tumor response reportedly is to be insufficient for the accurate determination of a pCR.9 In addition, standard radiologic studies have failed to provide clinically reliable information for the selection of patients who may be spared from surgical resection.10-13 In this setting, a radiologic tool capable of accurately assessing tumor response to neoadjuvant CRT is highly desirable, because it may allow the proper identification of patients who have complete tumor regression after CRT.
Positron-emission tomography (PET) integrated with computed tomography (CT) may provide additional information regarding metabolic activity in these tumors after neoadjuvant CRT. Several studies have suggested the use of PET/CT in assessing tumor response to CRT with conflicting results.14-20 For these reasons, we decided to perform a prospective trial to study the role of PET/CT in identifying patients who achieve a complete tumor response to neoadjuvant CRT.
MATERIALS AND METHODS
Consecutive patients from the Colorectal Surgery Division, University of Sao Paulo School of Medicine with rectal adenocarcinoma were eligible for the study between January 2005 and February 2009. Patients who had tumors with an inferior tumor border up to 7 cm from the anal verge measured by rigid proctoscopy were enrolled in the study. Staging included abdominal and chest CT scans and pelvic high-resolution MRI studies. Inclusion criteria included clinically staged cT2 to cT4 or cN1 to cN2 rectal cancers. Exclusion criteria included age <18 years, pregnancy, synchronous cancer, and metastatic disease detected.
Initial clinical assessment of patients comprised complete physical examination, digital rectal examination, rigid proctoscopy, carcinoembryonic antigen level determination, and the above-mentioned radiologic studies. Institutional review board approval was obtained in January 2005 before patient accrual, and the trial was registered as National Clinical Trial (NCT) no. NTC00254683.
Neoadjuvant Chemoradiation Therapy
All patients were received long-course neoadjuvant chemoradiation. Radiation therapy consisted of 45 grays (Gy) of radiation delivered by a 3-field approach with daily doses of 1.8 Gy on weekdays to the pelvis followed by a 9-Gy boost to the primary tumor and perirectal tissue (54 Gy total). Concurrently, patients received 5-fluororacil (425 mg/m2 daily) and folinic acid (20 mg/m2 daily) administered intravenously for 5 consecutive days on the first and last days of radiation therapy.
Assessment of Tumor Response
Patients were assessed for tumor response at 12 weeks after the completion of CRT by a single colorectal surgeon who used clinical and endoscopic approaches identical to those used at the initial assessment. A pelvic CT scan or pelvic MRI was used for the assessment of residual mesorectal or pelvic disease. The results of clinical assessment at 12 weeks were recorded as a cCR or an incomplete response (partial or no response), as described elsewhere.21 Management was determined based on clinical and standard radiologic findings without knowledge of PET/CT results. The only exception to this included a finding of unsuspected metastatic disease.
Patients who attained a cCR were offered no immediate radical surgery and were enrolled in a strict follow-up program consisting of monthly clinical reassessments during the first year and radiologic studies repeated after 6 months of initial assessment. All patients were fully informed that radical surgery is the standard of care even for patients who achieve a cCR after neoadjuvant CRT. Patients with local tumor recurrence or regrowth were recommended for immediate radical surgery. Follow-up for the second and third years included visits every 3 months for repeat clinical response assessment.
Patients who attained an incomplete clinical response (iCR) were referred for radical resection, including total mesorectal excision, unless it was contraindicated by existing medical comorbidities or patient refusal. Pathology was performed routinely using current American Joint Committee on Cancer/International Union Against Cancer recommendations for TNM classification. Tumor regression was graded according to the classification of Dworak et al.22
Positron Emission Tomography/Computed Tomography
All patients underwent a baseline PET/CT study to provide confirmation of fluoro-2-deoxy-D-glucose (FDG) uptake by the primary lesion. Patients who had metastatic disease detected by PET/CT were excluded from the study. A second PET/CT study was scheduled 6 weeks after the completion of CRT, and a third PET/CT study was scheduled 12 weeks after completion of CRT, before clinical assessment. In patients who attained a cCR and managed with no immediate surgery, yearly PET/CT studies were obtained in addition to the strict follow-up program outlined above.
All PET/CT scans were obtained after intravenous injection of 370 megabecquerels (MBq) of 2-fluorine-18-FDG under a 12-hour fasting period. Fasting serum glucose levels were measured 10 to 15 minutes before FDG injection according to standardized protocol. Data were acquired using a dedicated LSO-PET/CT scanner (Biograph 16; SIEMENS, Freeburg, Ill). Whole-body imaging was carried out in all patients and covered from the base of the skull to the proximal thigh. Diluted iodine contrast was administered orally to all patients. Two series of images were obtained for every patient enrolled: an early study (approximately 60 minutes after FDG injection) and a late study (approximately 180 minutes after FDG injection). All patients received a rectal iodine contrast enema before late study acquisition.
Imaging was interpreted qualitatively by a single experienced observer (an expert nuclear imaging radiologist) who was unaware of the patients' clinical assessment status (of tumor response) and was recorded either as a complete response (absence of FDG uptake; PET/CT) or as an incomplete response (presence of FDG uptake; positive PET/CT).
An incomplete PET/CT response was considered the presence of visual residual FDG uptake within the rectal wall or perirectal lymph node always compared with previous baseline studies (Fig. 1). A complete PET/CT response was considered the absence of visual abnormal FDG uptake within the rectal wall or perirectal lymph nodes. Standardized uptake value measurements were not used to determine a complete or incomplete response (Fig. 2). Unspecific structural changes observed on CT scans, such as persistent thickening of the rectal wall or visible perirectal lymph nodes (<1 cm in greatest dimension) also were considered a complete response in the absence of FDG uptake. The images obtained with rectal iodine enemas were analyzed systematically to rule out physiologic mucosal uptake.
Qualitative PET/CT results were compared with a clinical assessment of tumor response and final pathology reports for the determination of sensitivity, specificity, negative and positive predictive values, and overall accuracy. Patients who had a cCR (no surgery) and patients who had a complete pathologic response after surgery (including those who had an iCR) were grouped together as complete responders.
Statistical analysis was performed using the SPSS statistical software package (version 13.0; SPSS, Inc., Chicago, Ill). Numerical variables were analyzed with the Student t test, and categorical variables were evaluated using the chi-square test. Comparison of proportions (accuracy) was performed using the Z test (normal). Differences were considered statistically significant for P values < .05.
One hundred forty-one patients were eligible for the study and had baseline PET/CT studies. All tumors revealed FDG uptake on the baseline study. Of these, 22 patients had metastatic disease, 9 patients refused neoadjuvant CRT, and 5 patients refused sequential PET/CT studies, and all of these patients were excluded from the analysis. Six patients died before the completion of neoadjuvant CRT and also were excluded from the analysis. Overall, 99 patients completed neoadjuvant CRT and PET/CT imaging to assess tumor response: 99 patients had baseline studies, 91 patients had 6-week studies, and 99 patients had 12-week studies (Fig. 3). Patient demographics and baseline characteristics are listed in Table 1.
|Characteristic||Mean±SD [Median] or No. (%)|
|Age, y||60.3±12.7 |
|Tumor size, cm||4.3±1.1 [4.0]|
|Distance from anal verge, cm||3.9±2.0 [4.0]|
|CEA before CRT, ng/mL||23.6±105.1 [3.6]|
|CEA after CRT, ng/mL||10.8 ± 50.7 [2.4]|
|Lymph node status|
|Interval between CRT and PET/CT, d|
|At 6 wk||47.5±8.6 |
|At 12 wk||90.2±17.5 |
Complete clinical response
Of all 99 patients, clinical assessment at 12 weeks after the completion of CRT revealed that 16 patients had a cCR (16%). Of these, 3 patients underwent transanal local excision of a residual scar for confirmation of complete response of the primary tumor (ypT0). None of the patients who had a cCR chose radical surgery instead of strict follow-up without immediate surgery.
One patient developed a secondary malignancy after 14 months of follow-up and, despite a sustained cCR, died of metastatic disease from this second neoplasm at 19 months after the completion of CRT. One additional patient developed a local recurrence after 11 months of follow-up (6%). This patient was managed by transanal local excision (ypT2) and currently has no evidence of disease after 32 months of follow-up. None of the 14 remaining patients who attained a cCR developed local recurrence, tumor regrowth, or systemic relapse after a mean follow-up of 42.6 ± 15.6 months (median, 46.5 months). There were no losses to follow-up among these patients. The 3-year overall and disease-specific survival rates were 92% and 88%, respectively.
Complete pathologic response
Of the remaining 83 patients who had an iCR, 7 underwent radical resection (2 abdominoperineal resections (APRs) and 5 low anterior resections) with the finding of a partial clinical response (pCR) (7%). Altogether, 23 patients were grouped as complete responders (cCRs + pCR) for statistical analysis (23%) (Table 2). The 3-year overall and disease-free survival rates of complete responders were 92% and 90%, respectively (Figs. 4, 5).
|Mean±SD [Median] or No. (%)|
|Characteristic||Complete Responders||Incomplete Responders||P|
|Age, y||60.4 ± 14.6 ||60.2 ± 12.2 ||.96|
|Men:women||11:12 (48:52)||36:40 (47:53)||.96|
|Tumor size, cm||4.3 ± 1.1 [4.0]||4.3 ± 1.2 [4.0]||.75|
|Distance from anal verge, cm||3.7 ± 2.1 [3.0]||4.0 ± 2.0 [4.0]||.66|
|CEA before CRT, ng/mL||10.3 ± 20.3 [3.0]||27.7 ± 119.5 [4.3]||.24|
|CEA after CRT, ng/mL||2.9 ± 2.8 [2.5]||13.3 ± 58.0 [2.6]||.14|
|T2||4 (17)||2 (3)|
|T3||18 (78)||69 (91)|
|T4||1 (4)||5 (7)||.033|
|Lymph node status|
|N0||13 (56)||43 (57)|
|N1||10 (43)||33 (43)||.99|
|Interval between CRT and PET/CT, d|
|At 6 wk||49.8 ± 10.1 ||46.8 ± 8.1 ||.21|
|At 12 wk||91.2 ± 26.4 ||89.9 ± 14.1 ||.82|
Seventy-six patients had an incomplete pathologic response 12 weeks after the completion of CRT. Sixty-three patients underwent surgical resection for persistent residual disease. Six patients were considered medically unfit for any surgical procedure and did not undergo surgery. An additional 7 patients refused surgery. All 13 patients who did not undergo surgery did undergo biopsies, which confirmed pathologic evidence of residual disease.
Surgical procedures for 63 patients who had an iCR included total mesorectal excision in 59 patients (33 APRs) and transanal local excision in 4 patients. The final pathologic staging for these patients is provided in Table 3.
|Variable||No. of Patients (%)|
|Final pathologic staging|
|Lymph node statusa|
|Radial margin involvement (≤1 mm)||8 (13)|
|Perineural invasion||28 (44)|
|Lymphovascular invasion||20 (32)|
Positron Emission Tomography/Computed Tomography Results
Of all 99 patients, 18 patients had a complete PET response, whereas 81 patients had an incomplete PET response at 12 weeks. Overall, PET/CT findings compared with clinical and pathologic findings resulted in sensitivity of 93%, specificity of 53%, a negative predictive value of 73%, a positive predictive value of 87%, and overall accuracy of 85% for the detection of persistent residual cancer (incomplete response) (Table 4).
|PET/CT Response Assessment||No. of Complete Clinical or Pathologic Responses||No. of Incomplete Responses (Confirmed by Pathology)||Total No. of Responses|
Only 8 of 18 patients had a PET/CT image that revealed a complete response at 6 weeks. None of the patients who had a complete PET response at 6 weeks regressed to an incomplete response at 12 weeks. Of the 8 patients who had complete PET responses at 6 weeks, 2 were false-negative results, and 6 were true-negative results. Overall, <50% of true complete responders were detected by PET/CT at 6 weeks (46%).
Long-Term Positron Emission Tomography/Computed Tomography Studies
Of the 16 patients who attained a cCR and were managed with no immediate surgery, 1 patient developed a local recurrence at 11 months, refused radical surgery, and was managed by transanal local excision before the first 12-month PET/CT could be obtained. The remaining 15 patients had negative 12-month PET/CT results in accordance with clinical endoscopic findings. Thereafter, 12 patients have completed 2-year follow-up PET/CT studies, 9 have completed the 3-year PET/CT study, 8 have completed the 4-year PET/CT study, and 4 have completed the 5-year PET/CT study. All of these patients had complete PET/CT responses coincident with clinical and endoscopic findings.
Of the 10 patients who had 12-week false-positive PET/CT results (false incomplete PET response), 6 patients (60%) had a cCR and were not managed by surgical resection. In all 6 of these patients, the 1-year PET/CT study was negative. Currently, these patients have a median follow-up of 38.5 months with no local or systemic recurrences.
The remaining 4 of 10 patients who had false-positive PET/CT results had an iCR and underwent radical resection. The final surgical specimen in each patient revealed a pCR.
Of the 5 patients who had false-negative (false complete PET response) 12-week PET/CT results, an iCR indicated immediate radical resection for all. One patient underwent APR, 3 underwent ultralow anterior resection, and 1 other underwent local excision. The final pathologic report revealed ypT3N1 in 1 patient, ypT3N0 in 3 patients, and ypT2Nx in 1 patient.
Added Positron Emission Tomography/Computed Tomography Information Among Misdiagnoses in Clinical Assessment Alone
Clinical assessment alone resulted in 1 “false” cCR in a patient who developed local recurrence or tumor regrowth at 11 months (false-negative result) and 7 “false” iCRs (false-positive results) in patients who ultimately underwent surgery for pCRs. This resulted in an overall accuracy of 91% (Table 5). Although PET/CT and clinical assessment data were blinded, PET/CT data for these particular patients would have indicated an incomplete PET response for the “false cCR” (1 patient) and a complete PET response in 3 of the 7 patients who had a pCR (“false incomplete responders”) (Table 6, Fig. 6).
|Clinical Response Assessment||No. of Complete Clinical or Pathologic Responses||No. of Incomplete Responses (Confirmed by Pathology)||Total No. of Responses|
|Response||No. of Complete Clinical or Pathologic Responses||No. of Incomplete Responses (Confirmed by Pathology)||Total No. of Responses|
|PET or clinical complete responsea||15 (Clinical true-negative results) plus 3 (PET/CT true-negative results)||0 (PET/CT and clinical false-negative results)||18|
|PET or clinical incomplete response||4 (PET/CT and clinical false-positive results)||77||81|
Patients who attained a cCR or a pCR had improved disease-free survival compared with patients who had an incomplete pathologic response (P = .01). A PET complete response was not associated with improved overall or disease-free survival.
The overall immediate postoperative morbidity and mortality rates associated with radical resection for rectal cancer led surgeons to search for less aggressive treatment strategies with acceptable oncologic outcomes. Neoadjuvant CRT may result in significant tumor regression, including a decrease in primary tumor size, in the depth of penetration, and in the risk of perirectal lymph node metastases. In some of these patients, a complete tumor response is observed, frequently after radical resection and without the removal of a single cancer cell. Therefore, patients who experience complete tumor regression would be ideal candidates for less aggressive surgical approaches. It has been suggested that these patients would not benefit from surgery at all.8 However, the identification of patients who attain a complete tumor response is not straightforward and may represent a significant challenge even for experienced colorectal surgeons.21
Although there have been reports that patients who had complete tumor regression were identified accurately by clinical assessment alone, others have reported quite disappointing results. In a retrospective analysis of patients who received neoadjuvant CRT and were assessed between 6 weeks and 8 weeks by clinical assessment alone, almost 80% of patients who had responses that were considered cCRs ultimately had residual cancer in the resected specimen.9 Standard radiologic assessment with CT scans, high-resolution MRI, and endorectal ultrasound have not added significant accuracy in identifying patients with complete tumor regression. Diffusion-weighted MRI may further improve the radiologic detection of pCRs; however, only small studies have addressed this issue to date, and further research is needed.23, 24 For these reasons, radiologic studies that incorporate metabolic information in addition to standard radiologic imaging, such as PET/CT studies, are expected to improve the overall accuracy of detecting patients who have complete tumor regression.
In our current study, PET/CT response was associated with an overall accuracy of 85%. Although PET/CT was only able to detect approximately half of the patients who achieved had complete tumor regression (53%), the negative predictive value was considerably high (73%), and there was a low frequency of false-negative results (5%). Because the standard treatment for patients after neoadjuvant CRT is radical surgery, false-positive results are somewhat less problematic (or dangerous) than false-negative results. In this setting, 5% of false-negative PET results potentially would lead to the consideration of inappropriate treatment strategies in only 5% of patients if only PET/CT was used to assess tumor response. It is noteworthy that the use of PET/CT information among patients misdiagnosed by clinical assessment alone could have slightly improved (although not statistically significant) overall assessment accuracy. However, this information should be considered with caution, because it is impossible to predict upfront when PET/CT results actually will “correct” clinical assessment errors. Because the accuracy of clinical assessment alone was superior to that of PET/CT alone, the information from the latter studies may not be useful in all patients. Still, this may suggest a role for PET/CT information in patients who have an equivocal clinical response to CRT in future studies. Also, it provides radiologic documentation of a complete response to CRT in addition to clinical assessment data.
Several studies have addressed the role of PET/CT imaging in detecting the response of rectal cancer to neoadjuvant CRT. Although some of these studies were able to establish a correlation between PET/CT results after CRT with overall downstaging, the specific identification of patients with who achieve a complete pathologic response has been somewhat disappointing.20, 25, 26
Several factors may have contributed to this limitation. First, studies that used higher radiation doses may have produced more false-positive results because of the effects of radiation within the rectal wall. In addition, an absence of the routine use of rectal iodine contrast may have increased the difficulty in distinguishing between residual cancer and intraluminal or physiologic mucosal activity uptake. Again, the pattern of metabolic abnormality detected on PET studies seems to be important for differentiating inflammation (diffuse uptake) from residual viable tumor (focal and subtle uptake). The considerably small sample sizes also may have influenced the initial experience with PET/CT for response assessment in some studies. Like in any other medical procedure, a learning curve also may be clinically relevant in PET-CT assessment of tumor response to CRT.20 Finally, timing also may have played a role. It has been suggested that rates of complete tumor regression may be significantly higher for longer intervals after the completion of CRT (up to 12 weeks after CRT completion).27 Therefore, assessment of tumor response by PET/CT at 3 to 6 weeks or even at 8 weeks may have underestimated pCR rates in most studies. It is noteworthy that, in our study, PET/CT at 6 weeks was able to detect <50% of actual complete tumor responders to neoadjuvant CRT. Had these patients been assessed for management decision only at 6 weeks, >50% of patients would have been considered incomplete responders and would have undergone potentially unnecessary surgery.
The grouping of patients who achieve cCRs and pCRs together may have constituted a significant limitation of the current study because of the risk of residual microscopic disease in the former group. However, attaining a cCR and avoiding immediate radical surgery probably is the most clinically relevant endpoint to be evaluated in this setting. Also, only 1 of 16 patients who attained a cCR developed local failure after CRT. Therefore, it is unlikely that any of the remaining 15 patients still had residual cancer and mistakenly were considered complete responders.
Surprisingly, a complete PET/CT response was not associated with improved survival in our study, although there were a few false-positive and false-negative results. A small sample size and a rather short follow-up may have contributed to this finding. Another potential limitation of the current study is the inclusion of patients who had T2N0 rectal cancer potentially caused by over treatment from the delivery of unnecessary CRT. However, it has been demonstrated that patients who have distal T2N0 rectal cancer actually may benefit from CRT in terms of both local disease control and sphincter preservation.27-29 In fact, our study suggests that these patients (cT2) are more likely to have a cCR after neoadjuvant CRT and, thus, may represent the majority of patients who can benefit from a nonoperative strategy after attaining a cCR.
In conclusion, to our knowledge, this is the first prospective study to report the accuracy of the clinical assessment of tumor response to neoadjuvant CRT in a rather large cohort of patients for a longer (12-week) interval after completing CRT. The combination of clinical assessment in patients according to stringent criteria and based on PET/CT imaging at 12 weeks after the completion of treatment provides for the appropriate and safe selection of patients for alternative management with no immediate surgery after they attain a cCR to neoadjuvant CRT.
This study was funded in part by the National Research Council (CNPq) and by the São Paulo State Foundation for Research Support (FAPESP) (CNPq 483752/2006-1 and FAPESP 07/51069-01).
CONFLICT OF INTEREST DISCLOSURES
The authors made no disclosures.
- 16The role of dual-time combined 18-fluorodeoxyglucose positron emission tomography and computed tomography in the staging and restaging workup of locally advanced rectal cancer, treated with preoperative chemoradiation therapy and radical surgery. Int J Radiat Oncol Biol Phys. 2009; 74: 1461-1469., , , et al.