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RE: p53 PROTEIN AND GENE ALTERATIONS IN PATHOLOGICAL STAGE C PROSTATE CARCINOMA
1648
LETTERS TO THE EDITOR
Repry by Authors. This letter raises 2 important issues to be addressed in more detail. First, Pantuck calculates a blood loss of 3.5 1. following application of leeches in our case of scrotal hematoma. However, blood hemoglobin concentration decreased only moderately from 11.9 gmldl. before application to 10.6 gmJdl. &r a total of 70 leeches had been applied for 12 days, which was clinically not significant inasmuch as the patient tolerated it well with no demand for transfusion. The difference between our observation and the calculation of Pantuck may be caused by the fact that some leeches, similar to human beings, drink more and some drink less. Additionally, persistent bleeding from leech bites did not seem to be as pronounced as described by Pantuck (approximately 45 ml. per leech). Therefore, the average total blood loss per leech applied may have been about 20 ml. in our case. Some controversy exists in the literature with regard to the prophylactic use of antibiotics during leech therapy. Because such therapy has an inherent risk (0 to 20%) of infection with Aeromonas hydrophila, which is part of the normal gut flora of leeches, many authors recommend aminoglycosides or third generation cephalosporins for prophylaxis.1 Nevertheless, in accordance with other authors: we dispensed with prophylactic antibiotics and no infection was observed during leech therapy.
tumor being positive for p53 was sufficient in their cases to result in p53 positive metastatic lesions. Wide differences in methodology must be addressed and consensus Ireached if p53 or other molecular biomarkers are ever to be used clinically. We are presently conducting a study examining p53 immunohistochemical expression in completely sectioned whole mount radid prostatedomy specimens. The p53 staining pattern is being traced on each slide and reconstructed in 3-dimension using computer software. We believe that this model will address the issue of p53 h e t e n geneity and multifcdity, and further define what amount is clinically relevant. Respectfully, Judd W. Moul, John J . Bauer, Isabel1 A. Sesterhenn, George Bennett Stackhouse and Shiv Srivastava Center for Prostate Disease Research Uniformed Services University Bethesda, Maryland 20814 and Armed Forces Institute of Pathology Washington, D. C. 20307
1. Wells, M. D., Manktelow, R. T., Boyd, J. B. and Bowen, V.: The medical leech: an old treatment revisited. Microsurgery, 14: 183,1993. 2. Rao, P., Bailie, F. B. and Bailey, B. N.: Leechmania in microsurgery. Practitioner, 229: 901, 1985.
1. Bauer, J. J., Sesterhenn, I. A., Mostofi, F. K, McLeod, D. G., Srivastava, S. and Moul, J. W.: p53 nuclear protem expression is an independent prognostic market in clinically localized prostate cancer patients undergoing radical prostatectomy. Clin. Cancer Res., 1: 1295, 1995. 2. Bauer, J. J., Sesterhenn, I. A., Mostofi, F. K., McLeod, D. G., Srivastava, S. and Moul, J. W.: Elevated levels of apoptosis regulator proteins p53 and bcl-2d are independent prognostic biomarkers in surgically treated clinically localized prostate cancer patients. J. Urol., 156: 1511, 1996. 3. Yang, G., Stapleton, A. M. F., Wheeler, T. M., Truong, L. D., Timme. T. L.. Scardino. P. T. and Thompson, T. C.: Clustered p53 immunostaining: a’novel pattern associated with prostate cancer progression. Clin. Cancer Res., 2: 399, 1996. 4. Stapleton, A. M. F., Timme, T. L., Gousse, A. E., Li, Q., Tobon, A. A., Kahan, K. M., Wheeler, T. M., Scardino, R. T. and Thompson, T. C.: Primary human prostate cancer cells harboring p53 mutations are clonally expanded in metastases. Clin. Cancer Res., 3 1389, 1997.
RE: p53 PROTEIN AND GENE ALTERATIONS IN PATHOLOGICAL STAGE C PROSTATE CARCINOMA C . E. Salem, N . A. Tomasic, D. A. Elmajian, D. Esrig, P. W. Nichols, C. R. Taylor, D. G. Skinner, P. Roy-Burman, G. Lieskovsky and R. J . Cote
J. Urol., 188:510-514, 1997 To the Editor. The authors studied 96 pathological stage C radical prostateetomy tissue samples by immunohistochemistry for p53 tumor suppressor gene protein expression, and found only 10 positive cases (10.4%)and no correlation with recurrence. These results differ substantially from our recent work (see table).1.2 The reasons why we found a higher p53 positive rate in association with Gleason sum and recurrence rate are explained by important differences in methodology. In our studies all hematoxylin and eosin stained glass slides for each radical prostatectomy were reviewed, and the correspondmgarchival blocks with the largest tumor number and tumor volume were selected for p53 analysis. Salem et al do not specify how the archival blocks for their study were selected. Regarding p53 nuclear immunohistochemical staining positivity, we included as positive any p53 expression greater than 1%of cancer cell nuclei in a focal area under high power. Salem et al, based on their experience with bladder cancer, required that the tumor harbor more than 10% of p53 staining nuclei to consider the case positive. (We assume this means 10% of the entire malignant tumor volume, although they did not specify.) Yang et al have also recently confirmed the focal clustered nature of p53 positivity and found prognostic importance,3which casts doubt on the methodological extrap olation from bladder to prostate cancer p53 positivity as performed by Salem et al. Furthermore, this focal clustered p53 positivity has recently been confirmed as biologically important. Stapleton et al found identical p53 mutations in these focal areas of the primary and metastatic tumors from the same patient.4 Less than 10% of the
No. p53 PosRotal No.(%I Bauer et a1 Pathological stage C (pT3) Gleason sum: 2-1 5-7
aio
Recurrence: Yes
No
Salem et a1
56/81 (69)
10/96 (10.4)
3/27 (11) 70198 (71) 12114 (86)
0/4 (-1 7/64 (11) 3/28 (11)
39/47(83) 46/92 (50)
3/35 (9) 7/61 (12)
Reply by Authors. We entirely agree that methodological concerns are extremely important in any scientific study. However, to evaluate rationally methodological differences, it is valuable to compare the methods to appropriate benchmarks. In the case of p53 alterations as determined by immunohistochemistry the most appropriate benchmark is the presence or absence of a p53 gene mutation, which is exactly the approach we have taken in our studies of bladder cancer and prostate.1.2 As we reported in our original article, p53 immunohistochemical results were compared to the results of p53 gene analyses and, as we found in bladder cancer, were concordant. Furthermore, our results are entirely consistent with the body of literature that now exists for p53 alterations in prostate carcino1118.319 Indeed, other molecular data clearly show that p53 gene mutations are relatively uncommon in prostate cancer, particularly clinically localized disease.3-5 Immunohistochemical results from other groups are also consistent with our results.”9 Our reported results, that is 10%of prostate carcinomas show p53 nuclear reactivity, were for cases showing any positive nuclear reactivity. Only 6% of the cases showed so-called high level p53 immunoreactivity, that is greater than 10% tumor cells showing p53 immunoreactivity. Furthermore we are well aware of the issue of p53 heterogeneity in prostate cancer, since we published the first report describing this phenomenon.10 Our studies were based on an appraisal of p53 in the most representative sections from a case after a complete review of the radical proctectomy specimen and, when appropriate, multiple sections were tested. The most important methodological difference among our study, other studiessg and those of Moul et a1 is the choice of anti-p53 antibodies. We used the well characterized monoclonal antibody 1801, which has been shown to correlate with gene status by us and others in several tumor systems, including prostate,1-4.11while Moul et a1 used a polyclonal antibody and did not provide characterization (references 1and 2 in letter), which could account, a t least in part, for the discrepancies in results. Methodological differences will not be addressed simply by performing more tests of uncertain characterization. Rather, they will
LETTERS TO THE EDITOR
Repry by Authors. This letter raises 2 important issues to be addressed in more detail. First, Pantuck calculates a blood loss of 3.5 1. following application of leeches in our case of scrotal hematoma. However, blood hemoglobin concentration decreased only moderately from 11.9 gmldl. before application to 10.6 gmJdl. &r a total of 70 leeches had been applied for 12 days, which was clinically not significant inasmuch as the patient tolerated it well with no demand for transfusion. The difference between our observation and the calculation of Pantuck may be caused by the fact that some leeches, similar to human beings, drink more and some drink less. Additionally, persistent bleeding from leech bites did not seem to be as pronounced as described by Pantuck (approximately 45 ml. per leech). Therefore, the average total blood loss per leech applied may have been about 20 ml. in our case. Some controversy exists in the literature with regard to the prophylactic use of antibiotics during leech therapy. Because such therapy has an inherent risk (0 to 20%) of infection with Aeromonas hydrophila, which is part of the normal gut flora of leeches, many authors recommend aminoglycosides or third generation cephalosporins for prophylaxis.1 Nevertheless, in accordance with other authors: we dispensed with prophylactic antibiotics and no infection was observed during leech therapy.
tumor being positive for p53 was sufficient in their cases to result in p53 positive metastatic lesions. Wide differences in methodology must be addressed and consensus Ireached if p53 or other molecular biomarkers are ever to be used clinically. We are presently conducting a study examining p53 immunohistochemical expression in completely sectioned whole mount radid prostatedomy specimens. The p53 staining pattern is being traced on each slide and reconstructed in 3-dimension using computer software. We believe that this model will address the issue of p53 h e t e n geneity and multifcdity, and further define what amount is clinically relevant. Respectfully, Judd W. Moul, John J . Bauer, Isabel1 A. Sesterhenn, George Bennett Stackhouse and Shiv Srivastava Center for Prostate Disease Research Uniformed Services University Bethesda, Maryland 20814 and Armed Forces Institute of Pathology Washington, D. C. 20307
1. Wells, M. D., Manktelow, R. T., Boyd, J. B. and Bowen, V.: The medical leech: an old treatment revisited. Microsurgery, 14: 183,1993. 2. Rao, P., Bailie, F. B. and Bailey, B. N.: Leechmania in microsurgery. Practitioner, 229: 901, 1985.
1. Bauer, J. J., Sesterhenn, I. A., Mostofi, F. K, McLeod, D. G., Srivastava, S. and Moul, J. W.: p53 nuclear protem expression is an independent prognostic market in clinically localized prostate cancer patients undergoing radical prostatectomy. Clin. Cancer Res., 1: 1295, 1995. 2. Bauer, J. J., Sesterhenn, I. A., Mostofi, F. K., McLeod, D. G., Srivastava, S. and Moul, J. W.: Elevated levels of apoptosis regulator proteins p53 and bcl-2d are independent prognostic biomarkers in surgically treated clinically localized prostate cancer patients. J. Urol., 156: 1511, 1996. 3. Yang, G., Stapleton, A. M. F., Wheeler, T. M., Truong, L. D., Timme. T. L.. Scardino. P. T. and Thompson, T. C.: Clustered p53 immunostaining: a’novel pattern associated with prostate cancer progression. Clin. Cancer Res., 2: 399, 1996. 4. Stapleton, A. M. F., Timme, T. L., Gousse, A. E., Li, Q., Tobon, A. A., Kahan, K. M., Wheeler, T. M., Scardino, R. T. and Thompson, T. C.: Primary human prostate cancer cells harboring p53 mutations are clonally expanded in metastases. Clin. Cancer Res., 3 1389, 1997.
RE: p53 PROTEIN AND GENE ALTERATIONS IN PATHOLOGICAL STAGE C PROSTATE CARCINOMA C . E. Salem, N . A. Tomasic, D. A. Elmajian, D. Esrig, P. W. Nichols, C. R. Taylor, D. G. Skinner, P. Roy-Burman, G. Lieskovsky and R. J . Cote
J. Urol., 188:510-514, 1997 To the Editor. The authors studied 96 pathological stage C radical prostateetomy tissue samples by immunohistochemistry for p53 tumor suppressor gene protein expression, and found only 10 positive cases (10.4%)and no correlation with recurrence. These results differ substantially from our recent work (see table).1.2 The reasons why we found a higher p53 positive rate in association with Gleason sum and recurrence rate are explained by important differences in methodology. In our studies all hematoxylin and eosin stained glass slides for each radical prostatectomy were reviewed, and the correspondmgarchival blocks with the largest tumor number and tumor volume were selected for p53 analysis. Salem et al do not specify how the archival blocks for their study were selected. Regarding p53 nuclear immunohistochemical staining positivity, we included as positive any p53 expression greater than 1%of cancer cell nuclei in a focal area under high power. Salem et al, based on their experience with bladder cancer, required that the tumor harbor more than 10% of p53 staining nuclei to consider the case positive. (We assume this means 10% of the entire malignant tumor volume, although they did not specify.) Yang et al have also recently confirmed the focal clustered nature of p53 positivity and found prognostic importance,3which casts doubt on the methodological extrap olation from bladder to prostate cancer p53 positivity as performed by Salem et al. Furthermore, this focal clustered p53 positivity has recently been confirmed as biologically important. Stapleton et al found identical p53 mutations in these focal areas of the primary and metastatic tumors from the same patient.4 Less than 10% of the
No. p53 PosRotal No.(%I Bauer et a1 Pathological stage C (pT3) Gleason sum: 2-1 5-7
aio
Recurrence: Yes
No
Salem et a1
56/81 (69)
10/96 (10.4)
3/27 (11) 70198 (71) 12114 (86)
0/4 (-1 7/64 (11) 3/28 (11)
39/47(83) 46/92 (50)
3/35 (9) 7/61 (12)
Reply by Authors. We entirely agree that methodological concerns are extremely important in any scientific study. However, to evaluate rationally methodological differences, it is valuable to compare the methods to appropriate benchmarks. In the case of p53 alterations as determined by immunohistochemistry the most appropriate benchmark is the presence or absence of a p53 gene mutation, which is exactly the approach we have taken in our studies of bladder cancer and prostate.1.2 As we reported in our original article, p53 immunohistochemical results were compared to the results of p53 gene analyses and, as we found in bladder cancer, were concordant. Furthermore, our results are entirely consistent with the body of literature that now exists for p53 alterations in prostate carcino1118.319 Indeed, other molecular data clearly show that p53 gene mutations are relatively uncommon in prostate cancer, particularly clinically localized disease.3-5 Immunohistochemical results from other groups are also consistent with our results.”9 Our reported results, that is 10%of prostate carcinomas show p53 nuclear reactivity, were for cases showing any positive nuclear reactivity. Only 6% of the cases showed so-called high level p53 immunoreactivity, that is greater than 10% tumor cells showing p53 immunoreactivity. Furthermore we are well aware of the issue of p53 heterogeneity in prostate cancer, since we published the first report describing this phenomenon.10 Our studies were based on an appraisal of p53 in the most representative sections from a case after a complete review of the radical proctectomy specimen and, when appropriate, multiple sections were tested. The most important methodological difference among our study, other studiessg and those of Moul et a1 is the choice of anti-p53 antibodies. We used the well characterized monoclonal antibody 1801, which has been shown to correlate with gene status by us and others in several tumor systems, including prostate,1-4.11while Moul et a1 used a polyclonal antibody and did not provide characterization (references 1and 2 in letter), which could account, a t least in part, for the discrepancies in results. Methodological differences will not be addressed simply by performing more tests of uncertain characterization. Rather, they will