1. Pathology Journal Reading
Presented by Intern 曾德朋

2. Objective
To identify the role of cytokeratins in distinguishing intraductal papilloma from papillary ductal carcinoma in situ

3. Introduction Papillary breast tumors:
Proliferated mammary epithelium projects into duct lumen
Intraductal papilloma
Preinvasive papillary ductal carcinoma in situ (DCIS)
Sometimes difficult to distinguish: overlapping microscopic appearances

4. The need to separate them…
Distinct biologic behavior
Papillary DCIS potentially progressing to invasive cancer
Surgical extirpation
Benign papillomas
Discharged from clinical follow up

5. Cytokeratins (CK) Fundamental markers of epithelial differentiation
Cell type & differentiation status
Previous studies:
CK5/CK6, 34BE12 and CK14 in distinguishing usual epithelial hyperplasia from atypical ductal hyperplasia (ADH) and DCIS
Presence of myoepithelial cells
Preserved in benign papillomas, scant in papillary DCIS
Muscle actin and 34BE12 (less specific)

6. In this study… Detail the expression of three CK antibody preparation
34BE12 (recognizing CKs 1, 5, 10 and 14)

7. Aim of this study…
determining their role (CKs) in differentiating the benign papilloma from malignant in situ papillary carcinoma
Scant literature that specifically addresses papillary lesions
previous work focusing on proliferative breast lesions as a generic group versus DCIS
Semiquantitative criteria of immunoscores to evaluate the CKs
Ascertain the findings by extrapolating to a separate group

8. Materials and Methods Patients and tumors
50 excision biopsies of papillary breast lesions (25 intraductal papillomas and 25 papillary DCIS)
Department of Pathology, Singapore General Hospital, 2002~2003
Variously reported by 14 general surgical pathologists - initially
Diagnostic review and assignment to papilloma and papillary DCIS by Pathologist P.H.T
Results were all in concordance with initial diagnosis

9. Materials and Methods…
Cont…
Intraductal papillomas (25 cases)
Age: mean 44.1 y/o (22-78 y/o)
Epithelial hyperplasia of mild to florid degree
No atypical ductal hyperplasia
Papillary DCIS (25 cases)
Age: mean 57.4 y/o (35-78 y/o)
No invasive elements present

10. Materials and Methods…
Cont…
43 Hong Kong cases
As to confirm the result with a separate distinct cohort
Initial diagnosis: 10 general surgical pathologists
Histologically reviewed by pathologist G.M.T
Cases: age: mean 52.5 y/o
1993 ~ 2001
Excision biopsies 36 cases
Core biopsies 7 cases
Submitted for CK immunohistochemical staining at SGH, without prior discussion

11. Materials and Methods…
Cont…
Immunohistochemistry
Human tonsil, squamous cell carcinoma and prostate: positive controls for CK5/6, CK14, and 34BE12, respectively
Normal ducts and ductules in the breast tissues: internal control

12. Materials and Methods…
Cont…
Scoring of sections
Staining intensity: 0, no staining; 1+, weak; 2+, moderate; 3+, strong
Quantification of positivity (0%~100%)
Estimate of the percentage of stained tumor cells in the lesion
Immunoscores: multiplying the staining intensity with percentage positivity (0~300)
Negative or low (0~50); Moderate (51~100); High (101~200); Very high (201~300)

13. Materials and Methods…
Cont…
Confocal microscopy
Statistical analysis
Two-tailed t test: differences in immunoreaction between the two sample groups
Positive predictive value: CK immunoscore of <50
Papillary DCIS
Negaive predictive value: CK immunoscore of >50
Papilloma
Reevaluate for cases with discrepancies

14. Results: Cytokeratin expression in a normal breast ductule

15. Immunoreactions Intraductal Papillomas and Papillary DCIS (SGH cases)

16. Immunoreactions Intraductal Papillomas and Papillary DCIS (SGH cases)
CK5/CK6
Papillomas
72% : moderate to high immunoscores
Papillary DCIS
All: low immunoscores with 10 being completely negative
CK14
84% high to very high immunoscores
16% moderate to low immunoscores
84% low immunoscores
16% moderate immunoscores
34BE12
56% high to very high immunoscores
44% low to moderate
80% low
20% moderate (1 case: high positivity)

17. Immunoreactions Intraductal Papillomas and Papillary DCIS (SGH cases)
t test
Staining intensity, precetage positivity, immunoscores for each CK:
all three parameters showed significantly higher in papillomas than DCIS

18. CK5/CK6
CK5/CK6 expression in an intraductal papilloma (left panel) and
Papillary DCIS (right panel)
About half of the tumor cells in the papilloma were stained, whereas
tumor cells in DCIS were nonreactive

19. CK14 CK14 expression in an intraductal papilloma (left panel) and
Papillary DCIS (right panel)
More than half of the tumor cells in the papilloma were stained,
whereas tumor cells in DCIS were nonreactive

20. 34BE12 34BE12 expression in an intraductal papilloma (left panel) and
Papillary DCIS (right panel)
Approximately half of the papilloma tumor cells were stained,
some DCIS tumor cells were also decorated

21. Confocal microscopy of CKs

22. Results… Hong Kong cases
Immunoscores objectively determined by PHT (SGH)
CK5/CK6 corroborated
Papilloma 89.3%
Papillary DCIS 86.7%
CK14
Papilloma 92.9%
34BE12
Papilloma 96.4%
Papillary DCIS 33.3%

23. Discordant cases

24. SGH and HK cases

25. Statistic values…

26. Discussion Cytoskeleton
Microtubules, microfilaments and intermediate filaments (CKs belong to one of 5 classes of intermediate filaments)
CKs: cytoplasmic scaffold
Sustain mechanical and nonmechanical stresses
Participation in the response to stress, cell signaling and apoptosis
To date: 20 CKs (12: acidic type I; 8 neutral-basic type II)

27. Discussion Normal resting mammary gland Epithelium lining
Inner luminal epithelial (LE) cells
CKs 7, 8, 18, 19
Outer myoepithelial (ME) cells
CKs 5, 14, 17

28. Discussion Previous studies
Monoclonal antibodies specific against simple and/or basal type CKs
Benign and malignant intraepithelial proliferations of breast
Atypical proliferations
Invasive breast carcinomas
No study compares the expression of basal-type CKs in papillary tumors

29. Discussion Our results
CK14 stained a significantly larger percentage of tumor cells in papilloma
Breast epithelium of proliferating mammary gland
3 types of cells: immature precursor (CK5/CK6)
intermediate (CK5/CK6, CK 8, 14, 18, 19), fully mature (CK14, 18, 19)
Intraductal papilloma
Larger proportion of fully mature cells

30. Discussion 34BE12 Stained more DCIS tumor cells
Recognizes CK1, 10, 5, 14
CK10 expressed in some LE cells in breast cancers (previous studies)

31. Discussion
Results applied to a separate distinct cohort of HK cases, confirmation of
Benign papilloma diagnosis 89.3% (CK5/CK6) to 96.4% (34BE12)
Discordant cases: small or core biopsy
ADH case: necessity for microscopic reevaluation
Papillary DCIS (result at odds)
Core biopsy
Invasive component (detect LE as well)
Not as reliable or sensitive as the other 2 CKs
Apocrine nature

32. In summary The three CKs can serve as helpful adjunctive markers
Particularly combination of CK5/CK6 and CK14
34BE12: low detection rate
Especially in delineation of papillary DCIS
Use of CKs on small lesions, core biopsies, apocrine morphology, and associated invasive cancer need further evaluation

33. Thank you for your attention