Dr Madiha JC β-catenin, Pax2, and Pten Panel.pptx

beta catenin pax2

1. β-catenin, Pax2, and Pten Panel Identifies Precancers Among Histologically Subdiagnostic Endometrial Lesions Mitzi Aguilar, Hao Chen, Subhransu S. Sahoo , Wenxin Zheng , Jessica Grubman , Jeffrey A, Elena Lucas, Diego H. Castrillon . American Journal of Surgical Pathology Volume 47, Number 5, May 2023. Dr Syeda Madiha 23/03/2024

2. Introduction The diagnosis of Endometrial precancers poses everyday problems in pathology practice. Endometrial biopsies may be limited or highly fragmented. C yclical variations or exogenous hormones can confound histologic features of endometrial precancers [ Atypical hyperplasia(AH) or endometrioid intraepithelial neoplasia , ( EIN)]

3. S ome AH/EIN appear as tight clusters of crowded glands that are well-demarcated and distinctive cytologically or architecturally from adjacent “normal” glands but other AH/EIN can be more diffuse, with a continuum of architectural features that can make it difficult to clearly delineate a lesion and confidently classify it. In such cases, distinction from adjacent normal glands ( to assess atypia ) is not always possible. Gland crowding leading to a reduction in stromal volume (gland area> stromal area, or > 50% gland area) is a useful histologic criteria.

4. B enign endometria such as secretory endometrium, or with extensive areas of cystically dilated glands associated with atrophy , can exceed 50% gland area . Hence a panel of 3 immunohistochemical (IHC) markers was defined comprising of β-catenin , Pax2, and Pten and its practical utility in the diagnosis of AH/EIN was evaluated.

5. Arid1a and Mlh1 are sometimes lost in AH/EIN and are reliably scored, but are rarely found if they are the sole aberrant markers , hence they were not included in the core panel. β-catenin , Pax2, and Pten are each aberrant in more than half of AH/EIN; Hence with a panel of all 3, at least 1 is aberrant in 93% of cases .

6. The 3 Marker panel application was studied in a spectrum of lesions including Disordered P roliferative E ndometrium (DPE) and N on-Atypical H yperplasia (NAH). These results were correlated to clinical disease progression and next-generation sequencing (NGS) analyses.

7. Materials and Methods Cases were retrospectively identified via text searches with a final diagnosis of Non-Atypical H yperplasia (NAH) or Disordered P roliferative E ndometrium (DPE) admitted between 2010 and 2021 at the UT Southwestern Clements University Hospital. Exclusion criteria were a prior diagnosis of AH/EIN or uterine malignancy. A diagnosis of cancer in an unrelated organ system was not an exclusion criterion.

8. T he original hematoxylin and eosin (H&E) slides were reviewed by a panel of 3 specialty pathologists to reach a consensus diagnosis, blinded to the original diagnosis, and before the performance of the IHC panel. Standard histologic diagnostic criteria were used for the initial diagnosis of AH/EIN i.e , Gland:Stroma ratio > 1. Overt nuclear atypia / cytologic demarcation from background endometrium, and Exclusion of mimics .

9. D isordered P roliferative E ndometrium (DPE) DPE was defined by the presence of well-spaced, cystically dilated/enlarged glands without cribriforming or epithelial stratification , resembling the pattern that is typical in the perimenopausal state.

10. Non-Atypical Hyperplasia (NAH) NAH was defined as the presence of more significant and/or diffuse gland crowding but falling short of the criteria for AH/EIN , with no cribriforming , epithelial stratification , or definitive cytologic distinctiveness/ atypia .

11. Hence most of the cases could be placed into a consensus category of P roliferative endometrium [PE], DPE, NAH, AH/EIN. B ut those that could not and raised suspicion for AH/EIN were labeled as Ambiguous/Difficult.

12. Initially 92 Cases were identified and cases of Definitive AH/EIN or Carcinoma in the index biopsy or Reclassified as normal in the expert pathology review were excluded. i .e , 2 from the initial pool of 92 cases were excluded, leaving 90 cases.

13. Patient records for all 90 cases were then systematically reviewed for any follow-up endometrial tissue samples (biopsies and hysterectomies) after the index case. For patients who developed subsequent AH/EIN or carcinoma ( i.e , Progressors ) T he access date for the first tissue specimen harboring AH/EIN or carcinoma was used for the progression-free survival (PFS) analysis.

14. Immunochemistry For Pax2, Pten , β-catenin , and Mlh1 staining protocols previously validated for clinical testing were performed on 4 μm sections in the clinical IHC laboratory on a DAKO Autostainer Link 48 instrument . The following primary antibodies were used: Mlh1 ( clone ES05), β- catenin (clone β- catenin-1), Pax2 (clone EP235), and Pten (clone 6H2.1 ) .

15. With antigen retrieval performed in low pH (6.0) for β- catenin and high pH (9.0) Tris /EDTA solution for the other markers at 97°C for 20 minutes. FLEX peroxidase block was performed for 10 minutes for β- catenin and 5 minutes for other markers. Primary antibody incubation time was 20 minutes for β- catenin , and 40 minutes for Pax2, Pten , and Mlh1.

16. Arid1a IHC was performed on a DAKO Autostainer Link 48 instrument in a research core facility (1:200 dilution, clone D2A8U) with low pH (6.0) Tris /EDTA solution for 20 minutes at 97°C. Primary and secondary incubation times were 20 minutes each. For all antibodies, the enzymatic conversion of the 3,3′-diaminobenzidine tetrahydrochloride chromogen was performed for 10 minutes at room temperature.

17. Scoring of the 3 Markers by IHC β- catenin Aberrancy is manifested as nuclear localization versus its normal membranous/cytoplasmic localization , often associated with overexpression. Strong nuclear β-catenin is scored as aberrant, even if focal. Low levels of nuclear β-catenin are normal ; the criterion for strong nuclear expression is nuclear staining clearly greater than that of the lateral cell membranes .

18. Pax2 Pax2 is a nuclear transcription factor expressed within the uterus only in endometrial epithelium, and scoring aberrancy requires complete loss of expression within all of the nuclei in an entire gland in cross-section . Decreased expression is not scored as aberrant. In most specimens , residual normal glands that retain Pax2 serve as internal controls for Pax2 IHC.

19. Pten Pten is ubiquitously expressed in endometrial glands, stroma , and leukocytes , and true loss within the endometrial glandular epithelium gives rise to a “ punched-out” appearance of glands relative to the surrounding stroma . Focal loss of Pax2 and Pten in individual glands or small clusters of glands can occur in normal endometria , with >10% loss across the glands in the entire specimen is used as the cutoff for aberrancy . However , most cases of AH/EIN exhibit Pax2 or Pten loss in > 25% of glands , making scoring more straightforward.

20. Results A total of n = 92 cases were identified. The only exclusion criteria were a prior diagnosis of any uterine/ ovarian neoplasia including AH/EIN . All original H&E slides for each case were reviewed to reach a consensus diagnosis and final placement into 3 diagnostic categories: DPE , NAH , and Ambiguous/Difficult .

21. Cases that after review were diagnosed as PE ( ie , no meaningful architectural abnormalities) or AH/EIN (definitive premalignancy ) and thus did not fall into the diagnostic spectrum between normalcy and AH/EIN were not included in the study. This included 1 case rediagnosed as PE, and 1 as AH/EIN, leaving a set of n =90 new cases for further investigation .

22. An additional n =180 previously reported PE and definitive AH/EIN subjected to the 3-marker panel were included for comparative purposes.

23. One block from these 90 index cases representing the most prominent architectural abnormalities was selected for immunostaining with 3-marker panel. The 270( 90+180) IHC slides were scored as per previously described criteria.

24. The fraction of cases aberrant for ≥1 marker was 18.2% for DPE 47.5% for NAH 50.0% for ambiguous/difficult cases. This difference in marker aberrancy between DPE and NAH was statistically significant (P = 0.0052)

25. F raction of cases aberrant for ≥1 marker

26. In NAH, the 3 markers were aberrant in roughly equal proportions, whereas in DPE, Pax2 aberrancy was much more common .

27. To summarise , in contrast to PE, the 3-marker panel discriminated among endometria across the diagnostic categories, with some cases exhibiting marker aberrancy for 1 or more marker . Hence it may be unexpected because current histopathologic diagnostic scheme classify DPE and NAH as unequivocally benign entities.

28. Association of 3-marker Aberrancy With Risk of Disease Progression The medical records for the n= 90 women were searched for subsequent tissue specimens for formal analysis of endometrial disease progression.(To look for Progression free Survival PFS). Overall , 52/90 patients (58%) had a subsequent endometrial tissue sample in hospital records. The tissue follow-up intervals ranged from 1 to 9 years .

29. Women with subsequent tissue-based diagnoses of AH/EIN or cancer (in all cases FIGO 1 endometrioid adenocarcinoma ) were categorized as Progressors . The original H&Es for these subsequent specimens were reviewed to confirm progression. In 1 case, the original diagnosis of AH/EIN (in a hysterectomy) was revised to grade 1 adenocarcinoma based on small foci of malignant epithelium without intervening stroma .

30. Formal PFS analysis of patients with Marker nonaberrant versus Marker-aberrant index cases showed that the Marker Aberrant cases were more likely to Progress. Only 1/60 marker nonaberrant cases progressed (1.6%) , whereas 8/30 marker-aberrant cases progressed (27%) (P =0.0005) .

31. Patterns of Biomarker Aberrancy in Progressors H istologic features and associated patterns of biomarker aberrancy were analyzed and are described below for several illustrative cases.

32. Patient 9 (DPE) The histologic features of the index case for this patient [ the sole N onaberrant P rogressor ] included : C ystically dilated glands without significant crowding or cribriforming ; AH/EIN was diagnosed 4 years and 5 months after the initial diagnosis of DPE ( progressor specimen ).

33. Since the DPE and subsequent AH/EIN were nonaberrant for the 3 markers, the additional markers Arid1a and Mlh1 were performed. Arid1a and Mlh1 were also N onaberrant in both the Index and P rogressor specimens. Lack of marker aberrancy in the AH/EIN and the preceding DPE in this patient is concordant with prior observations that 7% of AH/EIN are marker nonaberrant , resulting in some “false -negative ” cases with the 3-marker panel.

34. Patient 1 (NAH) The histologic features of this case included: Diffuse ( nonfocal ) gland crowding ( 33% gland area in the representative area shown) without definitive cytologic distinctiveness. T he lesion also showed widespread Pax2 and Pten loss (> 50% each).

35. One notable feature was the presence of areas of degeneration/necrosis, but such areas were not surrounded by more concerning features or increased gland crowding. A hysterectomy performed 9 months later for persistent abnormal uterine bleeding and thickened endometrial stripe showed FIGO 1 endometrioid adenocarcinoma that was similarly Pax2 and Pten aberrant.

36. Patient 2 (NAH) The index case showed NAH with diffuse but Mild gland crowding ( 21% gland density) and lack of cytologic distinctiveness . Pax2 was retained but Pten showed >50% loss . The subsequent FIGO 1 adenocarcinoma (diagnosed 9 years later) showed Pax2 and Pten loss.

37. This case illustrates that additional markers can be lost during disease progression, as would be expected, although sampling issues cannot be entirely excluded. Also, marker aberrancy does not occur in a determinate order: in this case, Pten loss was the initial event,followed by Pax2.

38. Patient 6 (DPE) This aberrant progressor had an index case with features of DPE, with cystically dilated glands and no areas of significant gland crowding or cytologic distinctiveness . The endometrial fragments were aberrant for both Pax2 and β-catenin .

39. The ensuing AH/EIN showed morphologically distinct areas with significant gland crowding and abundant squamous metaplasia, and other areas with crowding but no overt squamous metaplasia. The AH/EIN was aberrant for Pax2 and β-catenin, with β-catenin being distinctly nuclear and overexpressed even in areas without squamous metaplasia, underscoring the utility of β-catenin as an informative marker even in cases or areas without diffuse squamous metaplasia.

40. Mutation Detection by Cancer Gene Panel Rationalizes Biomarker Aberrancy in Progressors Although Pax2 loss has not been associated with gene mutations, β-catenin, Pten , and Arid1a aberrancy are often associated with gene mutations in the CTNNB1, PTEN , and ARID1A genes. AH/EIN and FIGO 1 endometrioid adenocarcinoma progressor cases (patients 1 to 9) were analyzed with a comprehensive clinical NGS gene panel, which was designed for mutation detection in samples with high cellularity such as the progressor cases .

41. The progressor specimens exhibited mutational spectrum expected for endometrioid adenocarcinomas , with mutations in KRAS, FBXW7, FGFR2, PIK3CA, and PIK3R1, among other endometrial cancer genes

42. Discussion There are well-known challenges in diagnosing endometrial pre-cancers (atypical hyperplasia/ endometrioid intraepithelial neoplasia or AH/EIN) based solely on histologic examination in routine clinical practice. Despite ongoing refinements to the morphologic criteria over the years, pathologists frequently encounter ambiguous or borderline cases that create diagnostic uncertainty and can lead to suboptimal patient management decisions.

43. Some of the major hurdles in diagnosis include : Small , limited, or fragmented biopsy specimens Cyclic hormonal variations that can alter endometrial architecture. Effects of exogenous hormone exposures confounding morphologic interpretation. Lack of definitive cytologic atypia or architectural complexity in some precursor lesions. Need to compare lesional tissue to adjacent normal glands to assess for a distinct population. Subjective estimation of quantitative parameters like gland crowding/gland percentage.

44. Hence studies have consistently documented suboptimal interobserver reproducibility in the diagnosis of AH/EIN among pathologists. This imprecision can negatively impact clinical management .  

45. The study defined an immunohistochemical panel of 3 markers ( β- catenin , Pax2, Pten ) with practical utility as an adjunct tool to aid in the diagnosis of AH/EIN. Because the previous validation studies focused only on definitively benign endometria versus definitive AH/EIN cases, leaving unanswered how the panel would perform on more ambiguous or borderline endometrial proliferations.

46. To address this gap, the current study evaluated a spectrum of endometrial samples that exhibited some architectural abnormalities but fell short of the accepted morphologic criteria for diagnosing AH/EIN. This included cases diagnosed as : Disordered proliferative endometrium (DPE) Non-atypical hyperplasia (NAH) Ambiguous lesions raising suspicion for but not definitively classifiable as AH/EIN

47. On using the previously established scoring criteria for each marker, it was found that a substantial proportion of these " subdiagnostic " endometrial samples exhibited aberrant expression of one or more markers i.e ,: 18 % of DPE 48 % of NAH 50 % of ambiguous cases The rate of marker aberrancy increased significantly across the spectrum from DPE to NAH (p=0.0052), suggesting a commensurate rise in the frequency of true neoplastic precursor lesions despite not meeting the classic histologic thresholds.

48. It was found that cases exhibiting marker aberrancy on the initial biopsy were significantly more likely to subsequently progress to AH/EIN or endometrioid carcinoma on follow-up compared to cases lacking marker aberrancy (p=0.0002). Specifically: Only 1 of 60 marker non-aberrant cases progressed (1.6 %) In contrast, 8 of 30 marker-aberrant cases progressed (27%) The progression-free survival curves were also significantly different between the two groups (p=0.0002)

49. On Analysis of individual cases , there was persistence in the specific pattern of marker aberrancy between the original ambiguous precursor lesion and the higher-grade progressor lesion from the same patient . For example : If the initial DPE or NAH showed Pten loss, the subsequent AH/EIN or cancer also exhibited Pten loss .

50. This concordance suggested the initial lesion, despite ambiguous morphology, represented a clonal neoplastic precursor population that underwent progression over time The next-generation sequencing analysis of the progressor AH/EIN and carcinoma cases frequently detected mutations in the genes corresponding to the immunohistochemically aberrant protein markers, such as : CTNNB1 mutations in cases with β-catenin aberrancy PTEN mutations in cases with Pten protein loss ARID1A mutations in 1 case with Arid1a protein loss

51. There were some exceptions where genomic aberrations did not fully explain the protein expression patterns, likely reflecting other mechanisms like epigenetic silencing or limitations of sequencing for detecting large genomic events. These results "unequivocally demonstrate" that a subset of endometrial lesions not meeting current stringent histologic criteria for AH/EIN represent bonafide neoplastic precursor lesions harboring clinically-relevant driver genetic alterations. The 3-marker immunohistochemical panel can reliably detect these precursor lesions that may be missed or under-diagnosed based on morphology alone .

52. Conclusion This study challenges the prevailing dogma that DPE and NAH are uniformly benign entities. By combining immunohistochemical markers with morphology, study showed compelling evidence that at least some cases contain clonal neoplastic precursor populations detectable at an earlier stage before progressing to higher-grade lesions meeting the classic criteria for AH/EIN or endometrial carcinoma . Incorporating this biomarker panel into clinical practice may allow for more precise risk stratification and prevention of missed or delayed diagnoses of precancerous endometrial disease.

53. Abstract Despite refinements in histologic criteria for the diagnosis of endometrioid precancers , many challenging cases are encountered in daily practice, creating diagnostic uncertainty and suboptimal patient management. Recently, an immunohistochemical 3-marker panel consisting of β- catenin , Pax2, and Pten was identified as a useful diagnostic adjunct.

54. However, previous studies focused either on cancers or diagnostically unambiguous precancers , leaving questions about the applicability and utility of the panel in endometria with architectural features near or below the threshold of accepted histologic criteria for endometrioid precancers . In this retrospective study of 90 patients,performance of the 3-marker panel was evaluated.

55. T he panel detected a subset of D isordered Proliferative E ndometria (8/44, 18%), N onatypical H yperplasias (19/ 40, 48%), and Cases with Ambiguous features (3/6, 50%) with aberrancy for ≥1 markers. Marker-aberrant cases were more likely to progress to endometrioid precancer or cancer (P=0.0002).

56. Patterns of marker aberrancy in the index and progressor cases from individual patients provided evidence for origin in a common precursor, and next-generation sequencing of the progressor cases rationalized marker aberrancy for β- catenin and Pten . The results unequivocally demonstrate that some lesions that do not approach current histologic thresholds are bonafide neoplastic precursors with clinically-relevant driver events that can be detected by the 3-marker panel.

57. The findings provide further validation for the diagnostic utility of the panel in clinical practice and its application in difficult or ambiguous cases.

58. Thank You