Blood and Lymphatic Vessel Invasion in Colorectal Cancer
Blood and Lymphatic Vessel Invasion in Colorectal Cancer
A total of 1450 patients with CRC who underwent surgical resection in 2003 from eight institutions under the Japanese Society for Cancer of the Colon and Rectum were reviewed. Clinicopathological factors including the TNM stage according to the fifth edition of TNM classification, the presence of BLI, number of paraffin blocks taken to examine primary tumours, use of megablock, tangential tissue sectioning, histochemical staining and immunohistochemical staining were reviewed and compared. The range of histochemical staining and antibody used for immunohistochemical staining were also reviewed.
Eighty consecutive, surgically resected specimens of Stage II CRC according to the seventh edition of TNM classification between 2003 and 2005 from the National Cancer Center Hospital East were used for the interobserver study. Eight pathologists from eight institutions assessed the slides. Specimens for pathological assessment were divided into six cohorts as follows (Table 1) and concordance of diagnosis was reviewed. Cohort 1: H&E-stained slides without any guiding criteria. Cohort 2: H&E-stained slides without any criteria, but focus on designated area of lesion. Assessment was later checked to see which histological findings associated with BLI had good agreement. Concordance of assessment for designated area was also reviewed (figure 1). Cohort 3: H&E-stained, elastica-stained and D2-40-stained slides. Histochemical and immunohistochemical staining without any guiding criteria. Cohort 4: H&E-stained, elastica-stained and D2-40-stained slides. Histochemical and immunohistochemical staining without any guiding criteria but focus on designated area of lesion (figure 1). Observation was later checked to determine which slides of H&E, histochemical or immunohistochemical staining associated with BLI diagnosis had good concordance. Concordance of assessment for designated area of lesion was also reviewed. Cohort 5: H&E-stained, elastic-stained and D2-40-stained slides. Histochemical and immunohistochemical staining and our new criterion were used for assessment. Finally, for Cohort 6, the same H&E-stained, elastic-stained and D2-40-stained slides used in Cohort 3 were assessed by the pathologists (who were unaware of reviewing the same slides) to check diagnostic agreement of histochemical and immunohistochemical staining using our new criterion. Cohorts 1, 3, 5 and 6 each consisted of 20 CRC specimens and Cohorts 2 and 4 each consisted of 10 CRC specimens. H&E-stained slides and slides of largest slice from blocks of specimen including the deepest invasive area of tumour were used in Cohorts 1, 3, 5 and 6, while one representative slide of the tumour was used in Cohorts 2 and 4. In Cohorts 2 and 4, assessment of designated area was reviewed to evaluate the agreement of BLI diagnosis. Three areas of lesion containing histological tumour cluster surrounded by some space or fibrous rim-like vascular structure were chosen randomly and marked with ink. Since the designated area was very small, virtual slides were used to indicate with an arrow where assessment should be made (figure 1). In Cohorts 1, 3, 5 and 6 the 'presence' or 'absence' of BLI was reported. In Cohorts 2 and 4, assessment of designated area was made as 'blood vessel invasion', 'lymphatic vessel invasion' or 'neither'. In Cohorts 2 and 4, histological findings associated with the diagnosis of BLI were collected. This was reviewed thoroughly in a meeting and was recorded in the survey sheet, as either 'present' or 'absent' (Table 2).
(Enlarge Image)
Figure 1.
(A–D) A case from Cohort 2. In Cohorts 2, 4, three regions of interest within the histological tumour cluster with surrounding space or fibrous rim-like vascular structure were chosen randomly and marked with ink near the lesion (A). The lesion to be reviewed was indicated with an arrow on the virtual slides (B–D). Eight pathologists reviewed the slides. The assessment for each of the indicated lesions was reported as 'blood vessel invasion', 'lymphatic vessel invasion' or 'neither'. Furthermore, pathological findings associated with the diagnosis of blood and lymphatic vessel invasion were studied. Reviewers recorded their interpretations of the indicated lesion using the query sheet, answering the questions as 'present' or 'absent'.
Four rounds of consensus meetings, participated by eight pathologists were held as shown in figure 2. Before the meeting, a survey on histological, histochemical and immunohistochemical diagnostic criteria of BLI was prepared. All pathologists were requested to answer the survey anonymously and send it by mail after the meeting. There were a total of 34 questions: 2 on the definition of BLI, 7 on the assessment of BLI, 4 on the use of histochemical and immunohistochemical staining, 8 on assessment of blood vessel invasion and 13 on the assessment of lymphatic vessel invasion (Table 3). Scoring was based on 1 to 6 Likert scale (1=strong disagreement, 2=moderate disagreement, 3=some disagreement, 4=some agreement, 5=moderate agreement, 6=strong agreement), maximum score being 6 points. Scores of 5 and 6 were regarded as 'agreement'. Consensus was considered to be achieved when over 80% of the participants' scores resulted in 'agreement', based on the previously described scoring method. Four rounds of meetings with active discussion took place and surveys were conducted three times, after the second and third rounds of meetings, as shown in figure 2. At the beginning of the second and third rounds of meetings, interim results of survey were reported to the participants to facilitate building consensus on key histological findings with high concordance. For findings that failed to present immediate agreement, further discussion took place and the next vote was performed. Consistent with the Delphi method, some questions in the survey were modified to enable building general agreement.After the third round of meetings, the findings for which consensus had been reached were summarised and new diagnostic criterion was developed.
(Enlarge Image)
Figure 2.
Time flow for consensus development.
The concordance reached by pathologists on rating tumour invasion was evaluated using κ coefficients. Using %mκ SAS macro, we estimated the Fleiss type multi-rater κ coefficient and corresponding 95% CI. All statistical analyses was performed with SAS Release V.9.3 (SAS Institute, Inc, Cary, North Carolina, USA).
Materials and Methods
Multicentre, Retrospective Review of Pathological Assessment at Different Departments of Pathology
A total of 1450 patients with CRC who underwent surgical resection in 2003 from eight institutions under the Japanese Society for Cancer of the Colon and Rectum were reviewed. Clinicopathological factors including the TNM stage according to the fifth edition of TNM classification, the presence of BLI, number of paraffin blocks taken to examine primary tumours, use of megablock, tangential tissue sectioning, histochemical staining and immunohistochemical staining were reviewed and compared. The range of histochemical staining and antibody used for immunohistochemical staining were also reviewed.
Interobserver Study
Eighty consecutive, surgically resected specimens of Stage II CRC according to the seventh edition of TNM classification between 2003 and 2005 from the National Cancer Center Hospital East were used for the interobserver study. Eight pathologists from eight institutions assessed the slides. Specimens for pathological assessment were divided into six cohorts as follows (Table 1) and concordance of diagnosis was reviewed. Cohort 1: H&E-stained slides without any guiding criteria. Cohort 2: H&E-stained slides without any criteria, but focus on designated area of lesion. Assessment was later checked to see which histological findings associated with BLI had good agreement. Concordance of assessment for designated area was also reviewed (figure 1). Cohort 3: H&E-stained, elastica-stained and D2-40-stained slides. Histochemical and immunohistochemical staining without any guiding criteria. Cohort 4: H&E-stained, elastica-stained and D2-40-stained slides. Histochemical and immunohistochemical staining without any guiding criteria but focus on designated area of lesion (figure 1). Observation was later checked to determine which slides of H&E, histochemical or immunohistochemical staining associated with BLI diagnosis had good concordance. Concordance of assessment for designated area of lesion was also reviewed. Cohort 5: H&E-stained, elastic-stained and D2-40-stained slides. Histochemical and immunohistochemical staining and our new criterion were used for assessment. Finally, for Cohort 6, the same H&E-stained, elastic-stained and D2-40-stained slides used in Cohort 3 were assessed by the pathologists (who were unaware of reviewing the same slides) to check diagnostic agreement of histochemical and immunohistochemical staining using our new criterion. Cohorts 1, 3, 5 and 6 each consisted of 20 CRC specimens and Cohorts 2 and 4 each consisted of 10 CRC specimens. H&E-stained slides and slides of largest slice from blocks of specimen including the deepest invasive area of tumour were used in Cohorts 1, 3, 5 and 6, while one representative slide of the tumour was used in Cohorts 2 and 4. In Cohorts 2 and 4, assessment of designated area was reviewed to evaluate the agreement of BLI diagnosis. Three areas of lesion containing histological tumour cluster surrounded by some space or fibrous rim-like vascular structure were chosen randomly and marked with ink. Since the designated area was very small, virtual slides were used to indicate with an arrow where assessment should be made (figure 1). In Cohorts 1, 3, 5 and 6 the 'presence' or 'absence' of BLI was reported. In Cohorts 2 and 4, assessment of designated area was made as 'blood vessel invasion', 'lymphatic vessel invasion' or 'neither'. In Cohorts 2 and 4, histological findings associated with the diagnosis of BLI were collected. This was reviewed thoroughly in a meeting and was recorded in the survey sheet, as either 'present' or 'absent' (Table 2).
(Enlarge Image)
Figure 1.
(A–D) A case from Cohort 2. In Cohorts 2, 4, three regions of interest within the histological tumour cluster with surrounding space or fibrous rim-like vascular structure were chosen randomly and marked with ink near the lesion (A). The lesion to be reviewed was indicated with an arrow on the virtual slides (B–D). Eight pathologists reviewed the slides. The assessment for each of the indicated lesions was reported as 'blood vessel invasion', 'lymphatic vessel invasion' or 'neither'. Furthermore, pathological findings associated with the diagnosis of blood and lymphatic vessel invasion were studied. Reviewers recorded their interpretations of the indicated lesion using the query sheet, answering the questions as 'present' or 'absent'.
Developing Diagnostic Criteria Using the Delphi Method
Four rounds of consensus meetings, participated by eight pathologists were held as shown in figure 2. Before the meeting, a survey on histological, histochemical and immunohistochemical diagnostic criteria of BLI was prepared. All pathologists were requested to answer the survey anonymously and send it by mail after the meeting. There were a total of 34 questions: 2 on the definition of BLI, 7 on the assessment of BLI, 4 on the use of histochemical and immunohistochemical staining, 8 on assessment of blood vessel invasion and 13 on the assessment of lymphatic vessel invasion (Table 3). Scoring was based on 1 to 6 Likert scale (1=strong disagreement, 2=moderate disagreement, 3=some disagreement, 4=some agreement, 5=moderate agreement, 6=strong agreement), maximum score being 6 points. Scores of 5 and 6 were regarded as 'agreement'. Consensus was considered to be achieved when over 80% of the participants' scores resulted in 'agreement', based on the previously described scoring method. Four rounds of meetings with active discussion took place and surveys were conducted three times, after the second and third rounds of meetings, as shown in figure 2. At the beginning of the second and third rounds of meetings, interim results of survey were reported to the participants to facilitate building consensus on key histological findings with high concordance. For findings that failed to present immediate agreement, further discussion took place and the next vote was performed. Consistent with the Delphi method, some questions in the survey were modified to enable building general agreement.After the third round of meetings, the findings for which consensus had been reached were summarised and new diagnostic criterion was developed.
(Enlarge Image)
Figure 2.
Time flow for consensus development.
Statistical Analysis
The concordance reached by pathologists on rating tumour invasion was evaluated using κ coefficients. Using %mκ SAS macro, we estimated the Fleiss type multi-rater κ coefficient and corresponding 95% CI. All statistical analyses was performed with SAS Release V.9.3 (SAS Institute, Inc, Cary, North Carolina, USA).