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JCP Online First, published on September 12, 2014 as 10.1136/jclinpath-2014-202361 Original article
Assessment of suitability of the one step nucleic acid amplification (OSNA) assay as an intraoperative procedure for detection of metastasis in sentinel lymph nodes of breast cancer Ana Richelia Jara-Lazaro,1 Ilyana Huda Mohamed Hussain,1 Aye Aye Thike,1 Chow Yin Wong,2 Gay Hui Ho,3 Wei Sean Yong,3 Kong Wee Ong,3 Preetha Madhukumar,3 Benita Kiat Tee Tan,2 Chung Lie Oey,2 Jacqueline Siok Gek Hwang,1 Puay Hoon Tan1 1
Department of Pathology, Singapore General Hospital, Singapore, Singapore 2 Department of General Surgery, Singapore General Hospital, Singapore, Singapore 3 Department of Surgical Oncology, National Cancer Center, Singapore, Singapore Correspondence to Dr Puay Hoon Tan, Department of Pathology, Singapore General Hospital, 20 College Road, Academia, Level 7, Diagnostics Tower, Singapore 169856, Singapore; [email protected] Received 16 April 2014 Revised 18 August 2014 Accepted 25 August 2014
ABSTRACT Aim We aimed to assess the one step nucleic acid amplification (OSNA) assay as an intraoperative method in comparison with frozen sections (FS) for detection of metastasis in sentinel lymph nodes (SLNs) of breast cancer. Method 100 SLNs from patients with breast carcinoma were enrolled within a 3-month period. Alternate 2 mm node slices were subjected to routine FS, and later to permanent histology, and the rest for automated molecular detection of CK19 mRNA using OSNA. FS and OSNA findings were compared with permanent histology results. Difference in turnaround time was also noted. Results With permanent histology as gold standard, OSNA was discrepant in 8 of 98 (3 false negative, 5 false positive) included SLNs whereas FS had 2 false negative cases. FS had higher sensitivity (89%, p=2 mm were categorised as macrometastases, between >0.2 mm and ≤2 mm were categorised as micrometastases, and ≤0.2 mm were categorised as isolated tumour cells (ITCs). In accordance with the UICC staging system, macrometastases and micrometastases were judged as positive and the others were judged as negative. (B) Permanent histology: Pieces subjected as FS were later formalin fixed and submitted for paraffinised tissue processing. Three levels of permanent H&E-stained sections were evaluated as per routine reporting, and the findings compared with the FS and OSNA assay results. (C) OSNA assay: The alternate slices of the excised SLN were combined together and used for a single assay if the weight was less than 600 mg. If the weight was more than 600 mg, the pieces were separated and processed in 2 assays. The pieces were placed in 4 mL of Lynorhag (0.2 M glycin-HCl buffer ( pH 3.5) containing 5% of surfactant Brij35 and 20% of DMSO), and the solution was homogenised to prepare a lymph node lysate. After centrifuging, the resultant supernatant was diluted 10 times with Lynorhag. This solution (sample (1)) was further diluted another 10 times (sample (2)). Each of the prepared samples (1) and (2) and LYNOAMP BC ( positive control, negative control, CK19 primer solution and enzyme solution) were set on RD100i, an RT-LAMP instrument being required for this
procedure. To 2 μL of each sample, 20 μL of CK19 primer solution and 3 μL of enzyme solution were added, and an isothermal reaction was performed at 65°C. For each sample, the time required for magnesium pyrophosphate that deposited along with the progress of amplification reaction to reach a certain turbidity (wavelength 465 nm) was determined (rise time). Based on this rise time, the number of CK19 mRNA copies in each sample was determined using a previously constructed CK19 mRNA calibration curve. The OSNA result categories were: ‘++’, ‘+’, ‘+i’, ‘−’ or ‘ND’ (not detected). Based on the cut-off levels determined by Tsujimoto et al,7 ‘++’ and ‘+’ were equivalent to macrometastasis and micrometastasis, respectively, ‘+i’ ( positive but with reaction inhibited) was equivalent to either macrometastasis or micrometastasis, and both ‘−’ and ‘ND’ were equivalent to negative for metastasis. Interpretation of OSNA results is summarised in table 1. The sensitivity, specificity and concordance rates of results using FS and OSNA techniques in comparison with the paraffinised histology findings were compared. The turnaround time of both FS and OSNA procedures was also compared. Turnaround time was defined as the period of arrival of SLNs for intraoperative referral up to the time the full FS or OSNA results were obtained. (D) Further work-up of discrepant results: In cases where the OSNA result was discrepant with permanent histology, the following procedures were performed: 1. OSNA negative but positive for metastasis on permanent histology: Paraffinised sections were further subjected to immunohistochemical staining with CK19 to rule out the possibility of CK19 negative metastatic carcinoma. 2. OSNA positive but negative for metastasis on permanent histology: six deeper levels from the paraffinised blocks were further obtained and microscopically examined.
Statistical analysis The Statistical Package for Social Sciences (SPSS) for Windows, V18 was used for statistical analysis. The κ statistic measured agreement between OSNA/FS results with the permanent histology findings, with the following interpretation of κ values: 0– 0.2 were regarded as no agreement, 0.21–0.4 as fair agreement, 0.41–0.6 as moderate agreement, 0.61–0.8 as substantial agreement and 0.81–1 as almost perfect agreement.13
Table 1 results
Interpretation of one step nucleic acid amplification
Results interpreted as positive for metastasis: ++ CK19 mRNA (copy number) in sample (1) was ≥5000 copies/μL + CK19 mRNA (copy number) in sample (1) was
JCP Online First, published on September 12, 2014 as 10.1136/jclinpath-2014-202361 Original article
Assessment of suitability of the one step nucleic acid amplification (OSNA) assay as an intraoperative procedure for detection of metastasis in sentinel lymph nodes of breast cancer Ana Richelia Jara-Lazaro,1 Ilyana Huda Mohamed Hussain,1 Aye Aye Thike,1 Chow Yin Wong,2 Gay Hui Ho,3 Wei Sean Yong,3 Kong Wee Ong,3 Preetha Madhukumar,3 Benita Kiat Tee Tan,2 Chung Lie Oey,2 Jacqueline Siok Gek Hwang,1 Puay Hoon Tan1 1
Department of Pathology, Singapore General Hospital, Singapore, Singapore 2 Department of General Surgery, Singapore General Hospital, Singapore, Singapore 3 Department of Surgical Oncology, National Cancer Center, Singapore, Singapore Correspondence to Dr Puay Hoon Tan, Department of Pathology, Singapore General Hospital, 20 College Road, Academia, Level 7, Diagnostics Tower, Singapore 169856, Singapore; [email protected] Received 16 April 2014 Revised 18 August 2014 Accepted 25 August 2014
ABSTRACT Aim We aimed to assess the one step nucleic acid amplification (OSNA) assay as an intraoperative method in comparison with frozen sections (FS) for detection of metastasis in sentinel lymph nodes (SLNs) of breast cancer. Method 100 SLNs from patients with breast carcinoma were enrolled within a 3-month period. Alternate 2 mm node slices were subjected to routine FS, and later to permanent histology, and the rest for automated molecular detection of CK19 mRNA using OSNA. FS and OSNA findings were compared with permanent histology results. Difference in turnaround time was also noted. Results With permanent histology as gold standard, OSNA was discrepant in 8 of 98 (3 false negative, 5 false positive) included SLNs whereas FS had 2 false negative cases. FS had higher sensitivity (89%, p=2 mm were categorised as macrometastases, between >0.2 mm and ≤2 mm were categorised as micrometastases, and ≤0.2 mm were categorised as isolated tumour cells (ITCs). In accordance with the UICC staging system, macrometastases and micrometastases were judged as positive and the others were judged as negative. (B) Permanent histology: Pieces subjected as FS were later formalin fixed and submitted for paraffinised tissue processing. Three levels of permanent H&E-stained sections were evaluated as per routine reporting, and the findings compared with the FS and OSNA assay results. (C) OSNA assay: The alternate slices of the excised SLN were combined together and used for a single assay if the weight was less than 600 mg. If the weight was more than 600 mg, the pieces were separated and processed in 2 assays. The pieces were placed in 4 mL of Lynorhag (0.2 M glycin-HCl buffer ( pH 3.5) containing 5% of surfactant Brij35 and 20% of DMSO), and the solution was homogenised to prepare a lymph node lysate. After centrifuging, the resultant supernatant was diluted 10 times with Lynorhag. This solution (sample (1)) was further diluted another 10 times (sample (2)). Each of the prepared samples (1) and (2) and LYNOAMP BC ( positive control, negative control, CK19 primer solution and enzyme solution) were set on RD100i, an RT-LAMP instrument being required for this
procedure. To 2 μL of each sample, 20 μL of CK19 primer solution and 3 μL of enzyme solution were added, and an isothermal reaction was performed at 65°C. For each sample, the time required for magnesium pyrophosphate that deposited along with the progress of amplification reaction to reach a certain turbidity (wavelength 465 nm) was determined (rise time). Based on this rise time, the number of CK19 mRNA copies in each sample was determined using a previously constructed CK19 mRNA calibration curve. The OSNA result categories were: ‘++’, ‘+’, ‘+i’, ‘−’ or ‘ND’ (not detected). Based on the cut-off levels determined by Tsujimoto et al,7 ‘++’ and ‘+’ were equivalent to macrometastasis and micrometastasis, respectively, ‘+i’ ( positive but with reaction inhibited) was equivalent to either macrometastasis or micrometastasis, and both ‘−’ and ‘ND’ were equivalent to negative for metastasis. Interpretation of OSNA results is summarised in table 1. The sensitivity, specificity and concordance rates of results using FS and OSNA techniques in comparison with the paraffinised histology findings were compared. The turnaround time of both FS and OSNA procedures was also compared. Turnaround time was defined as the period of arrival of SLNs for intraoperative referral up to the time the full FS or OSNA results were obtained. (D) Further work-up of discrepant results: In cases where the OSNA result was discrepant with permanent histology, the following procedures were performed: 1. OSNA negative but positive for metastasis on permanent histology: Paraffinised sections were further subjected to immunohistochemical staining with CK19 to rule out the possibility of CK19 negative metastatic carcinoma. 2. OSNA positive but negative for metastasis on permanent histology: six deeper levels from the paraffinised blocks were further obtained and microscopically examined.
Statistical analysis The Statistical Package for Social Sciences (SPSS) for Windows, V18 was used for statistical analysis. The κ statistic measured agreement between OSNA/FS results with the permanent histology findings, with the following interpretation of κ values: 0– 0.2 were regarded as no agreement, 0.21–0.4 as fair agreement, 0.41–0.6 as moderate agreement, 0.61–0.8 as substantial agreement and 0.81–1 as almost perfect agreement.13
Table 1 results
Interpretation of one step nucleic acid amplification
Results interpreted as positive for metastasis: ++ CK19 mRNA (copy number) in sample (1) was ≥5000 copies/μL + CK19 mRNA (copy number) in sample (1) was
