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Occult lymph node metastases in patients with ‘node negative’ breast carcinoma treated with conservation surgery and axillary node sample and who subsequently developed axillary recurrence
The Breast (2002) 11, 249–251 r 2002 Published by Elsevier Science Ltd doi:10.1054/brst.2001.0399, available online at http://www.idealibrary.com on
ORIGINAL ARTICLE
Occult lymph node metastases in patients with ‘node negative’ breast carcinoma treated with conservation surgery and axillary node sample and who subsequently developed axillary recurrence K. L. Murray,1 P. A. Lambah,2 T. J. Anderson1 and J. M. Dixon2 1
Department of Pathology, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG, UK; 2Edinburgh Breast Unit, Western General Hospital, Crewe Road, Edinburgh, EH4 2XU, UK S U M M A R Y . We examined axillary lymph nodes from 26 patients with node-negative breast cancer managed by axillary node sampling and no further axillary treatment, but who subsequently developed axillary recurrence after a mean follow-up of 7 years to determine the incidence of micrometastatic disease in these patients. Twenty-six matched controls with an identical length of follow-up who were node-negative on an axillary node sample, but have not developed axillary recurrence, also underwent node examination and the incidence of metastases in the two groups were compared. Lymph nodes were sectioned at two additional levels 100 mm apart. Sections at each level were stained with haematoxylin and eosin (H&E) and antibodies to PanCK and MUC1 protein. The original H&E section from each node was reviewed and additional sections from each lymph node were examined by a pathologist who was blinded to outcome. Review of the original H&E sections of the nodes revealed metastases that had been overlooked at the time of diagnosis in two (8%) patients from the recurrence group. A further two (8%) patients from the recurrence group and three (12%) from the control group had axillary nodes which contained micrometastases. Immunocytochemistry was important in identifying all micrometastases. There was no significant difference in the incidence of axillary node micrometastases between patients with and without axillary node recurrence. Although the number of cases was small, this study suggests that axillary recurrence following a negative sampling procedure is not commonly due to missed axillary node metastases. r 2002 Published by Elsevier Science Ltd
of detecting the smallest metastases numerous sections have to be examined with both conventional stains and with immunocytochemistry, but this requires considerable resources in terms of time and money, with no clear indication of benefit.5 The aim of this study was to determine whether nodenegative breast cancer patients managed by axillary node sampling alone who subsequently developed axillary recurrences had micrometastatic disease originally undetected by routine histopathological examination.
INTRODUCTION Assessment of axillary lymph node status remains the most important prognostic indicator in women with breast carcinoma. However, 16% of women with nodenegative breast cancer will go on to develop distant metastases.1 The reported incidence and clinical significance of nodal micrometastases continues to be debated.2,3 As yet, there is no consensus on the optimal method of examining axillary nodes, although guidelines for this have been published.4 In order to have the best chance
MATERIALS AND METHODS
Address correspondence to: Dr Kendra Murray, Department of Pathology, Victoria Hospital Kirkcaldy, Hayfield Road, Kirkcaldy, KY2 5AH, UK. Tel.: +44 (0) 1592 643355; Fax: +44 (0) 1592 648055 Received: 11 September 2001 Accepted: 4 October 2001
We examined 133 axillary lymph nodes from 26 nodenegative breast cancer patients, who subsequently 249
250
The Breast
developed axillary recurrence. A further 133 lymph nodes from 26 control patients who had an identical length of follow-up but had not developed axillary recurrence and who were also matched for menopausal status, number of lymph nodes sampled, tumour size, grade and type were also examined. All of these cases were taken from a previously published series of more than 900 patients treated at the Edinburgh Breast Unit by conservation surgery and axillary node sample between 1987 and 1995. The mean follow-up was 7 years.6
recurrence status, and the original H&E stained sections from each lymph node were reviewed. The location and size of each metastasis was documented and the quality and intensity of immunostaining was noted. Statistical analysis was carried out using chi-squared tests to assess the difference in incidence of micrometastases between cases with and without axillary node recurrence.
Examination of lymph nodes
Review of original H&E sections revealed metastases in nodes which had been overlooked at the time of diagnosis in two (8%) cases from the recurrence group (Table 1). Metastases were 1 mm and 3 mm in each case, respectively. A further two (8%) cases from the recurrence group and three (12%) from the control group contained nodal metastases which were intraparenchymal, sub-capsular or both, and were all less than 1 mm in diameter. There was no statistically significant difference between the test and control groups (P=1). In all cases, immunocytochemical staining was required to identify the metastases, although they were all identified on H&E staining retrospectively. The quality and intensity of staining of panCK and MUC1 was similar. All primary breast tumours showed positive immunostaining for MUC1.
Each formalin-fixed, paraffin-embedded node was sectioned at two additional levels 100 mm apart, the optimal suggested method for detecting micrometastases.2 Three sections were taken at each level and one of these was stained with H&E. The remaining sections were stained with antibodies to pan-cytokeratin (MNFl16, Dako, UK) and MUC1 protein (HMFG2, Prof J. TaylorPapadimitrou, ICRF, London, UK), respectively, and a section from each primary tumour was stained with HMFG2 antibody to MUC1 protein using an avidin– biotin complex immuno-peroxidase technique. Sections were de-waxed and rehydrated. Sections for cytokeratin staining were microwaved in vector antigen retrieval solution 3 for 5 min and washed in water. The HMFG2 antibody did not require antigen retrieval. All sections were immersed in a solution of 1.5% hydrogen peroxide in methanol for 10 min then loaded in a Biogenex Optimax staining machine and incubated with 1 : 5 normal rabbit serum diluted in Optimax buffer for 30 min. The MNF 116 and HMFG2 primary antibodies were applied for 60 min at dilutions of 1 : 500 and 1 : 4, respectively, and following a buffer wash, sections were incubated for a further 30 min in a 1 : 400 dilution of biotinylated rabbit anti-mouse antiserum (Dako). After a further buffer wash, sections were incubated in readyto-use ABC (Vector Labs) for 30 min and after a final rinse in buffer solution, sites of peroxidase activity were developed using 3,3-diaminobenzidine (DAB) for 5 min. Histological analysis All samples were examined using a standard microscopic technique with the observer blinded to the
RESULTS
DISCUSSION Assessment of axillary lymph node status remains the most important prognostic indicator in women with breast carcinoma. Over the last few years much attention has been paid to the incidence, significance and methods of detection of nodal micrometastases in pathologically ‘node negative’ breast cancer patients, with estimates of the incidence of micrometastases varying between 7 and 31% of patients.7 The clinical significance of these micrometastases is still controversial with some reports suggesting that their presence results in a poorer disease-free survival and overall survival, whereas other studies have shown no difference in outcome. It has been suggested that these discrepancies could be because some studies have short follow-up
Table 1 Number of metastases identified
Axillary recurrence group Control group
No. of cases
No. of nodes
Metastases overlooked
No. of micrometastases
Total no. of metastases
26 26
133 133
2 (8%) 0
2 (8%) 3 (12%)
4 (16%) 3 (12%)
Occult lymph node metastases in ‘node negative’ breast cancer and small numbers, thus explaining the failure to detect significant differences in outcome.3 Previous studies have investigated the incidence of micrometastases after standard histological node-negative evaluation of breast cancer patients and compared this with clinical outcome.8 In contrast, we have investigated a significant cohort of patients with axillary recurrence following a histologically node-negative axillary node sample to determine the incidence of micrometastases in these patients. The mean follow-up of the patients studied was 7 years. No greater incidence of micrometastases was detected in these patients compared with a series of age-matched node-negative patients without axillary recurrence. The expectation was that there would be more micrometastases in the group who developed axillary recurrence. Although our numbers were small and a larger study is required for confirmation, it does not seem that patients who are node-negative on an axillary node sample who develop recurrence do so because of micrometastatic nodal disease missed by the pathologist. Rather, it suggests either that the sampling procedure is at fault, and involved nodes present at the time of initial surgery were missed by the surgeon, or nodal metastases develop later from residual tumour in lymphatics or as a consequence of local recurrence or new primary breast metastasising to axillary nodes. Sentinel node biopsy, where the first draining lymph node is identified and removed, could reduce the chance of missing an involved axillary node. However, initial experience of the ALMANAC trial is that there is a 7% false negative rate.9 The proportion of cases with ‘occult’ micrometastases in our series is at the lower end of the published range.7 Recent comments addressed the nature of probabilities affecting occult micrometastasis detection10 and the relevance of qualitative differences.11 In all cases, immunocytochemistry was more sensitive than conventional H&E in detecting metastases, but there was no difference in sensitivity between CK and MUC1. MUC1 is a mucin that is commonly found in breast carcinoma and has been reported to be superior to cytokeratin (MNF116 antibody).2 We used the monoclonal antibody HMFG2 to MUC1, which may not share the same epitope as that used by McGuckin and colleagues.2 Immunocytochemistry is not currently recommended for the routine examination of axillary lymph nodes,4 but it has been shown that examining sentinel node biopsies at multiple levels with immunocytochemistry accurately predicts axillary node status.1
251
The current ALMANAC trial has been set up to compare the efficacy of sentinel node biopsy against to the current standard of care, whether it is axillary sampling or clearance. The results of this study are awaited with interest.
Acknowledgements Ethical approval for this study was obtained from the Lothian Research Ethics Committee, Lothian Health Board. Patients in this study were managed by the Edinburgh Breast Unit Team. The two trials of axillary node sampling and clearance were initiated by Professor Sir Patrick Forrest and Mr Udi Chetty. Their role, and that of the patients who willingly participated in these studies, is greatly appreciated. The authors would like to thank the Imperial Cancer Research Fund for the gift of the MUC1 antibody and Mrs Frances Ray and her staff for their technical assistance.
References 1. Rosen P P, Saigo P, Braum D W Jr., Weathers E, Depalo A. Predictors of recurrence in Stage 1 (T1N1M0) breast carcinoma. Ann Surg 1981; 193: 15–25. 2. McGuckin M A, Cummings M C, Walsh M D, Hohn B G, Bennett I C, Wright R G. Occult axillary node metastases in breast cancer: their detection and prognostic significance. Br J Cancer 1996; 73: 88–95. 3. Yeatman T J, Cox C E. The significance of breast cancer lymph node micrometastases. Surg Oncol Clin N AM 1999; 8: 481–496. 4. Sloane J, Anderson T J, Davies J D et al. Guidelines for Breast Pathology Services, 2nd ed. Sheffield: NHSBSP Publications, 1995. 5. Galea M H, Athanassou E, Bell J et al. Occult regional lymph node metastases from breast carcinoma: immunohistological detection with antibodies Cam 5.2 and NCRC-11. J Pathol 1991; 165: 221–227. 6. Dixon J M, Dillon P, Anderson T J, Chetty U. Axillary node sampling in breast cancer: and assessment of its efficacy. Breast 1998; 7: 206–208. 7. Dowlatshahi K, Fan M, Snider H C, Habib F A. Lymph node micrometastases from breast carcinoma. Reviewing the dilemma. Cancer 1997; 80: 1188–1197. 8. International (Ludwig) Breast Cancer Study Group: prognostic importance of occult axillary lymph node micrometastases from breast cancers. Lancet 1990; 335: 1565–1568. 9. Manse1 RE on behalf of ALMANAC Collaborators Group. The Almanac Trial: initial experience. Breast Cancer Res Treat 2000; 64: 36. 10. Anderson T J. The challenge of sentinel lymph node biopsy. Histopathology 1999; 35: 82–84. 11. Hermanek P, Hutter R V P, Sobin L H, Wittekind C. Classification of isolated tumour cells and micrometastasis. Cancer 1999; 86: 2668–2673. 12. Cohen L F, Breslin T M, Kuerer H M, Ross M I, Hunt K K, Sahin A A. Identification and evaluation of axillary sentinel lymph nodes in patients with breast carcinoma treated with neoadjuvant chemotherapy. Am J Surg Path 2000; 24: 1266–1272.
ORIGINAL ARTICLE
Occult lymph node metastases in patients with ‘node negative’ breast carcinoma treated with conservation surgery and axillary node sample and who subsequently developed axillary recurrence K. L. Murray,1 P. A. Lambah,2 T. J. Anderson1 and J. M. Dixon2 1
Department of Pathology, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG, UK; 2Edinburgh Breast Unit, Western General Hospital, Crewe Road, Edinburgh, EH4 2XU, UK S U M M A R Y . We examined axillary lymph nodes from 26 patients with node-negative breast cancer managed by axillary node sampling and no further axillary treatment, but who subsequently developed axillary recurrence after a mean follow-up of 7 years to determine the incidence of micrometastatic disease in these patients. Twenty-six matched controls with an identical length of follow-up who were node-negative on an axillary node sample, but have not developed axillary recurrence, also underwent node examination and the incidence of metastases in the two groups were compared. Lymph nodes were sectioned at two additional levels 100 mm apart. Sections at each level were stained with haematoxylin and eosin (H&E) and antibodies to PanCK and MUC1 protein. The original H&E section from each node was reviewed and additional sections from each lymph node were examined by a pathologist who was blinded to outcome. Review of the original H&E sections of the nodes revealed metastases that had been overlooked at the time of diagnosis in two (8%) patients from the recurrence group. A further two (8%) patients from the recurrence group and three (12%) from the control group had axillary nodes which contained micrometastases. Immunocytochemistry was important in identifying all micrometastases. There was no significant difference in the incidence of axillary node micrometastases between patients with and without axillary node recurrence. Although the number of cases was small, this study suggests that axillary recurrence following a negative sampling procedure is not commonly due to missed axillary node metastases. r 2002 Published by Elsevier Science Ltd
of detecting the smallest metastases numerous sections have to be examined with both conventional stains and with immunocytochemistry, but this requires considerable resources in terms of time and money, with no clear indication of benefit.5 The aim of this study was to determine whether nodenegative breast cancer patients managed by axillary node sampling alone who subsequently developed axillary recurrences had micrometastatic disease originally undetected by routine histopathological examination.
INTRODUCTION Assessment of axillary lymph node status remains the most important prognostic indicator in women with breast carcinoma. However, 16% of women with nodenegative breast cancer will go on to develop distant metastases.1 The reported incidence and clinical significance of nodal micrometastases continues to be debated.2,3 As yet, there is no consensus on the optimal method of examining axillary nodes, although guidelines for this have been published.4 In order to have the best chance
MATERIALS AND METHODS
Address correspondence to: Dr Kendra Murray, Department of Pathology, Victoria Hospital Kirkcaldy, Hayfield Road, Kirkcaldy, KY2 5AH, UK. Tel.: +44 (0) 1592 643355; Fax: +44 (0) 1592 648055 Received: 11 September 2001 Accepted: 4 October 2001
We examined 133 axillary lymph nodes from 26 nodenegative breast cancer patients, who subsequently 249
250
The Breast
developed axillary recurrence. A further 133 lymph nodes from 26 control patients who had an identical length of follow-up but had not developed axillary recurrence and who were also matched for menopausal status, number of lymph nodes sampled, tumour size, grade and type were also examined. All of these cases were taken from a previously published series of more than 900 patients treated at the Edinburgh Breast Unit by conservation surgery and axillary node sample between 1987 and 1995. The mean follow-up was 7 years.6
recurrence status, and the original H&E stained sections from each lymph node were reviewed. The location and size of each metastasis was documented and the quality and intensity of immunostaining was noted. Statistical analysis was carried out using chi-squared tests to assess the difference in incidence of micrometastases between cases with and without axillary node recurrence.
Examination of lymph nodes
Review of original H&E sections revealed metastases in nodes which had been overlooked at the time of diagnosis in two (8%) cases from the recurrence group (Table 1). Metastases were 1 mm and 3 mm in each case, respectively. A further two (8%) cases from the recurrence group and three (12%) from the control group contained nodal metastases which were intraparenchymal, sub-capsular or both, and were all less than 1 mm in diameter. There was no statistically significant difference between the test and control groups (P=1). In all cases, immunocytochemical staining was required to identify the metastases, although they were all identified on H&E staining retrospectively. The quality and intensity of staining of panCK and MUC1 was similar. All primary breast tumours showed positive immunostaining for MUC1.
Each formalin-fixed, paraffin-embedded node was sectioned at two additional levels 100 mm apart, the optimal suggested method for detecting micrometastases.2 Three sections were taken at each level and one of these was stained with H&E. The remaining sections were stained with antibodies to pan-cytokeratin (MNFl16, Dako, UK) and MUC1 protein (HMFG2, Prof J. TaylorPapadimitrou, ICRF, London, UK), respectively, and a section from each primary tumour was stained with HMFG2 antibody to MUC1 protein using an avidin– biotin complex immuno-peroxidase technique. Sections were de-waxed and rehydrated. Sections for cytokeratin staining were microwaved in vector antigen retrieval solution 3 for 5 min and washed in water. The HMFG2 antibody did not require antigen retrieval. All sections were immersed in a solution of 1.5% hydrogen peroxide in methanol for 10 min then loaded in a Biogenex Optimax staining machine and incubated with 1 : 5 normal rabbit serum diluted in Optimax buffer for 30 min. The MNF 116 and HMFG2 primary antibodies were applied for 60 min at dilutions of 1 : 500 and 1 : 4, respectively, and following a buffer wash, sections were incubated for a further 30 min in a 1 : 400 dilution of biotinylated rabbit anti-mouse antiserum (Dako). After a further buffer wash, sections were incubated in readyto-use ABC (Vector Labs) for 30 min and after a final rinse in buffer solution, sites of peroxidase activity were developed using 3,3-diaminobenzidine (DAB) for 5 min. Histological analysis All samples were examined using a standard microscopic technique with the observer blinded to the
RESULTS
DISCUSSION Assessment of axillary lymph node status remains the most important prognostic indicator in women with breast carcinoma. Over the last few years much attention has been paid to the incidence, significance and methods of detection of nodal micrometastases in pathologically ‘node negative’ breast cancer patients, with estimates of the incidence of micrometastases varying between 7 and 31% of patients.7 The clinical significance of these micrometastases is still controversial with some reports suggesting that their presence results in a poorer disease-free survival and overall survival, whereas other studies have shown no difference in outcome. It has been suggested that these discrepancies could be because some studies have short follow-up
Table 1 Number of metastases identified
Axillary recurrence group Control group
No. of cases
No. of nodes
Metastases overlooked
No. of micrometastases
Total no. of metastases
26 26
133 133
2 (8%) 0
2 (8%) 3 (12%)
4 (16%) 3 (12%)
Occult lymph node metastases in ‘node negative’ breast cancer and small numbers, thus explaining the failure to detect significant differences in outcome.3 Previous studies have investigated the incidence of micrometastases after standard histological node-negative evaluation of breast cancer patients and compared this with clinical outcome.8 In contrast, we have investigated a significant cohort of patients with axillary recurrence following a histologically node-negative axillary node sample to determine the incidence of micrometastases in these patients. The mean follow-up of the patients studied was 7 years. No greater incidence of micrometastases was detected in these patients compared with a series of age-matched node-negative patients without axillary recurrence. The expectation was that there would be more micrometastases in the group who developed axillary recurrence. Although our numbers were small and a larger study is required for confirmation, it does not seem that patients who are node-negative on an axillary node sample who develop recurrence do so because of micrometastatic nodal disease missed by the pathologist. Rather, it suggests either that the sampling procedure is at fault, and involved nodes present at the time of initial surgery were missed by the surgeon, or nodal metastases develop later from residual tumour in lymphatics or as a consequence of local recurrence or new primary breast metastasising to axillary nodes. Sentinel node biopsy, where the first draining lymph node is identified and removed, could reduce the chance of missing an involved axillary node. However, initial experience of the ALMANAC trial is that there is a 7% false negative rate.9 The proportion of cases with ‘occult’ micrometastases in our series is at the lower end of the published range.7 Recent comments addressed the nature of probabilities affecting occult micrometastasis detection10 and the relevance of qualitative differences.11 In all cases, immunocytochemistry was more sensitive than conventional H&E in detecting metastases, but there was no difference in sensitivity between CK and MUC1. MUC1 is a mucin that is commonly found in breast carcinoma and has been reported to be superior to cytokeratin (MNF116 antibody).2 We used the monoclonal antibody HMFG2 to MUC1, which may not share the same epitope as that used by McGuckin and colleagues.2 Immunocytochemistry is not currently recommended for the routine examination of axillary lymph nodes,4 but it has been shown that examining sentinel node biopsies at multiple levels with immunocytochemistry accurately predicts axillary node status.1
251
The current ALMANAC trial has been set up to compare the efficacy of sentinel node biopsy against to the current standard of care, whether it is axillary sampling or clearance. The results of this study are awaited with interest.
Acknowledgements Ethical approval for this study was obtained from the Lothian Research Ethics Committee, Lothian Health Board. Patients in this study were managed by the Edinburgh Breast Unit Team. The two trials of axillary node sampling and clearance were initiated by Professor Sir Patrick Forrest and Mr Udi Chetty. Their role, and that of the patients who willingly participated in these studies, is greatly appreciated. The authors would like to thank the Imperial Cancer Research Fund for the gift of the MUC1 antibody and Mrs Frances Ray and her staff for their technical assistance.
References 1. Rosen P P, Saigo P, Braum D W Jr., Weathers E, Depalo A. Predictors of recurrence in Stage 1 (T1N1M0) breast carcinoma. Ann Surg 1981; 193: 15–25. 2. McGuckin M A, Cummings M C, Walsh M D, Hohn B G, Bennett I C, Wright R G. Occult axillary node metastases in breast cancer: their detection and prognostic significance. Br J Cancer 1996; 73: 88–95. 3. Yeatman T J, Cox C E. The significance of breast cancer lymph node micrometastases. Surg Oncol Clin N AM 1999; 8: 481–496. 4. Sloane J, Anderson T J, Davies J D et al. Guidelines for Breast Pathology Services, 2nd ed. Sheffield: NHSBSP Publications, 1995. 5. Galea M H, Athanassou E, Bell J et al. Occult regional lymph node metastases from breast carcinoma: immunohistological detection with antibodies Cam 5.2 and NCRC-11. J Pathol 1991; 165: 221–227. 6. Dixon J M, Dillon P, Anderson T J, Chetty U. Axillary node sampling in breast cancer: and assessment of its efficacy. Breast 1998; 7: 206–208. 7. Dowlatshahi K, Fan M, Snider H C, Habib F A. Lymph node micrometastases from breast carcinoma. Reviewing the dilemma. Cancer 1997; 80: 1188–1197. 8. International (Ludwig) Breast Cancer Study Group: prognostic importance of occult axillary lymph node micrometastases from breast cancers. Lancet 1990; 335: 1565–1568. 9. Manse1 RE on behalf of ALMANAC Collaborators Group. The Almanac Trial: initial experience. Breast Cancer Res Treat 2000; 64: 36. 10. Anderson T J. The challenge of sentinel lymph node biopsy. Histopathology 1999; 35: 82–84. 11. Hermanek P, Hutter R V P, Sobin L H, Wittekind C. Classification of isolated tumour cells and micrometastasis. Cancer 1999; 86: 2668–2673. 12. Cohen L F, Breslin T M, Kuerer H M, Ross M I, Hunt K K, Sahin A A. Identification and evaluation of axillary sentinel lymph nodes in patients with breast carcinoma treated with neoadjuvant chemotherapy. Am J Surg Path 2000; 24: 1266–1272.