Template for Reporting Results of Biomarker Testing of Specimens

Template for Reporting Results of Biomarker Testing of

Specimens From Patients With Carcinoma of Gynecologic Origin

Version: 1.0.0.0

Protocol Posting Date: December 2022

The use of this protocol is recommended for clinical care purposes but is not required for accreditation

purposes.

Authors

Barbara A. Crothers, DO*; Lara R. Harik, MD, FCAP*; Angela N. Bartley, MD, PhD, FCAP; Russell R.

Broaddus, MD, PhD, FCAP; Linus T. Chuang, MD; Michael B. Cohen, MD; Patricia Salter Jamieson,

CTR; Elke A . Jarboe, MD; Teri A. Longacre, MD; George L. Mutter, MD; Christopher N. Otis, MD; Patrick

L. Fitzgibbons, MD, FCAP; Richard Zaino, MD; Gulisa Turashvili, MD, PhD; Krisztina Hanley, MD.

With guidance from the CAP Cancer and CAP Pathology Electronic Reporting Committees.

* Denotes primary author.

Accreditation Requirements

Completion of the template is the responsibility of the laboratory performing the biomarker testing and/or

providing the interpretation. When both testing and interpretation are performed elsewhere (eg, a

reference laboratory), synoptic reporting of the results by the laboratory submitting the tissue for testing is

also encouraged to ensure that all information is included in the patient’s medical record and thus readily

available to the treating clinical team. This template is not required for accreditation purposes.

Summary of Changes

v 1.0.0.0

• New Protocol

CAP Approved

Gynecologic.Bmk_1.0.0.0.REL_CAPCP

Reporting Template

Protocol Posting Date: December 2022

Select a single response unless otherwise indicated.

CASE SUMMARY: (Gynecologic Biomarker Reporting Template)

TEST(S) PERFORMED

Completion of the template is the responsibility of the laboratory performing the biomarker testing and / or providing the

interpretation. When both testing and interpretation are performed elsewhere (e.g., a reference laboratory), synoptic reporting of the

results by the laboratory submitting the tissue for testing is also encouraged to ensure that all information is included in the patient’s

medical record and thus readily available to the treating clinical team.

Gene names should follow recommendations of The Human Genome Organisation (HUGO) Nomenclature Committee

(www.genenames.org; accessed October 12, 2022).

All reported gene sequence variations should be identified following the recommendations of the Human Genome Variation Society

(http://varnomen.hgvs.org/; accessed October 12, 2022).

+Testing Performed on Block Number(s) (specify): _________________

+Specimen Type

___ Biopsy / curettage

___ Resection

___ Other (specify): _________________

+Appropriate Controls Verified

___ Yes

___ No

___ Other (specify): _________________

Immunohistochemical Tests Performed (Note A

) (select all that apply)

___ Estrogen Receptor (ER) Status (Note B)

Estrogen Receptor (ER) Status

___ Positive

Percentage of Cells with Nuclear Positivity: _________________ %

Average Intensity of Staining

___ Weak

___ Moderate

___ Strong

___ Negative (less than 1%)

___ Progesterone Receptor (PgR) Status (Note B

)

Progesterone Receptor (PgR) Status

___ Positive

Percentage of Cells with Nuclear Positivity: _________________ %

Average Intensity of Staining

___ Weak

___ Moderate

___ Strong

___ Negative (less than 1%)

___ HER2 Status (for uterine serous carcinoma only) (Note C

)

CAP Approved

Gynecologic.Bmk_1.0.0.0.REL_CAPCP

HER2 Status

# No staining in tumor cells

___ Negative (score 0)#

## Faint / Barely perceptible, incomplete membrane staining in any proportion, or weak complete staining in less

than 10% of tumor cells

___ Negative (score 1+)##

### Strong complete or basolateral / lateral membrane staining in less than or equal to 30%, or weak to moderate

staining in greater than or equal to 10% of tumor cells

___ Equivocal (score 2+)###

#### Strong complete or basolateral / lateral membrane staining in greater than 30% of tumor cells

___ Positive (3+)####

___ Cannot be determined (indeterminate) (explain): _________________

___ Mismatch Repair (MMR) Protein Status (Note D

)

Mismatch Repair (MMR) Protein Status (select all that apply)

___ MLH1

Nuclear MLH1 Expression

___ Intact

___ Loss

___ Other (specify): _________________

___ PMS2

Nuclear PMS2 Expression

___ Intact

___ Loss

___ Other (specify): _________________

___ MSH2

Nuclear MSH2 Expression

___ Intact

___ Loss

___ Other (specify): _________________

___ MSH6

Nuclear MSH6 Expression

___ Intact

___ Loss

___ Other (specify): _________________

___ Background non-neoplastic tissue / internal control shows intact nuclear expression

+Additional Comment (specify percentage loss or other): _________________

Immunohistochemistry (IHC) Interpretation for Mismatch Repair (MMR) Proteins# (Note D

)

# There are exceptions to these IHC interpretations. These results should not be considered in isolation, and

clinical correlation with genetic counseling is recommended to assess the need for germline testing.

___ No loss of nuclear expression of MMR proteins: low probability of microsatellite instability-high

(MSI-H) phenotype#

___ Loss of nuclear expression of MLH1 and PMS2: testing for methylation of the MLH1 promoter is

indicated (the presence of MLH1 methylation suggests that the tumor is sporadic and germline

evaluation is probably not indicated; absence of MLH1 promoter methylation suggests the possibility of

Lynch syndrome, and sequencing and / or large deletion / duplication testing of germline MLH1 is

indicated)#

___ Loss of nuclear expression of MSH2 and MSH6: high probability of Lynch syndrome, genetic

counseling is recommended#

___ Loss of nuclear expression of MSH6 only: high probability of Lynch syndrome, genetic counseling

CAP Approved

Gynecologic.Bmk_1.0.0.0.REL_CAPCP

is recommended#

___ Loss of nuclear expression of PMS2 only: high probability of Lynch syndrome, genetic counseling

is recommended#

___ Other (specify): _________________

___ p53 Status (Note E

)

p53 Status

___ Normal expression (wild type)

___ Abnormal expression (mutated)

___ Overexpression (strong, diffuse basilar nuclear expression in greater than 90% of cells)

___ Null (lack of nuclear or cytoplasmic expression)

___ Cytoplasmic staining, diffuse (with or without nuclear expression)

ADDITIONAL TESTS PERFORMED

+HER2 by in situ Hybridization (Note C

)

"Number of Observers" and "Number of Invasive Tumor Cells Counted" are required only when Negative or Positive is selected.

___ Negative (not amplified)

___ Positive (amplified)

___ Cannot be determined (indeterminate) (explain): _________________

___ Not performed

___ Pending

Number of Observers: _________________

Number of Invasive Tumor Cells Counted: _________________ cells

Method (select all that apply)

___ Not applicable (not performed)

___ Dual probe assay

+Average Number of HER2 Signals per Cell: _________________

+Average Number of CEP17 Signals per Cell: _________________

+HER2 / CEP17 Ratio: _________________

___ Single probe assay

+Average Number of HER2 Signals per Cell: _________________

+Aneusomy (as defined by vendor kit used)

___ Not identified

___ Present (explain): _________________

+Heterogeneous Signals

___ Not identified

___ Present

+Percentage of Cells with Amplified HER2 Signals

___ Specify percentage: _________________ %

___ Other (specify): _________________

___ Cannot be determined

CAP Approved

Gynecologic.Bmk_1.0.0.0.REL_CAPCP

+Microsatellite Instability (MSI) Interpretation (Note F)

The presence of MSI-H / deficient mismatch repair may also be an indication for additional testing for Lynch syndrome and genetic

counselling.

___ MSI-Stable (MSS)

___ MSI-Low (MSI-L)

+___ 1-29% of the markers exhibit instability

+___ 1 of the 5 NCI or mononucleotide markers exhibits instability

+___ Other (specify): _________________

___ MSI-High (MSI-H)

+___ Greater than or equal to 30% of the markers exhibit instability

+___ 2 or more of the 5 NCI or mononucleotide markers exhibit instability

+___ Other (specify): _________________

___ MSI-Cannot be determined (explain): _________________

___ Pending

+MLH1 Promoter Methylation Analysis (Note G

)

___ MLH1 promoter methylation present

___ MLH1 promoter methylation absent

___ Cannot be determined (explain): _________________

___ Pending

+Image Analysis

___ Not performed

___ Performed

+Specify Method: _________________

+Biomarkers Scored by Image Analysis (select all that apply)

___ ER

___ PgR

___ HER2 by IHC

___ HER2 by ISH

___ Other (specify): _________________

CAP Approved

Gynecologic.Bmk_1.0.0.0.REL_CAPCP

Explanatory Notes

A. Biomarker Testing in Gynecologic Carcinoma

Biomarker testing in gynecologic malignancies is an evolving practice with numerous candidates under

investigation for targeted therapies.

1,2,3,4

Only a few of these tests have status in clinical guidelines or

recommendations. Many of these markers have a major role in the diagnostic assessment of tumor type.

American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP)

guidelines for breast cancer tissue ischemia and fixation for testing ER, PgR, and HER2 have been

proposed for gynecologic tumors and may be adopted but are not currently required.

5,6

References

1. Jiang P, Jia M, Hu J, Huang Z, Deng Y, Hu Z. A nomogram model involving

immunohistochemical markers for predicting the recurrence of stage I-II endometrial cancer.

Front Oncol. 2021; 10:586081.

2. Gülseren V, Kocaer M, Özdemir IA, Çakır I, Sanci M, Güngördük K. Do estrogen, progesterone,

p53, and Ki67 receptor ratios determined from curettage materials in endometrioid-type

endometrial carcinoma predict lymph node metastasis? Curr Probl Cancer. 2020; 44(10);100498.

3. Vrede SW, van Weelden WJ, Visser NCM, et al. Immunohistochemical biomarkers are prognostic

relevant in addition to the ESMO-ESGO-ESTRO risk classification in endometrial cancer. Gynec

Oncol. 2021; 161;787-794.

4. Ramon-Patino JL, Ruz-Caracuel I, Heredia-Soto V, et al. Prognosis stratification tools in early-

stage endometrial cancer: could we improve their accuracy? Cancers. 2022; 14(4):912.

5. Allison KH, Hammond MEH, Dowsett M, et al. Estrogen and progesterone receptor testing in

breast cancer: American Society of Clinical Oncology/College of American Pathologists guideline

update. Arch Pathol Lab Med. 2020;144(5):545-563.

6. Wolff AC, Hammond MEH, Allision KH, et al. HER2 testing in breast cancer: American Society of

Clinical Oncology/College of American Pathologists clinical practice guideline focused update.

Arch Pathol Lab Med. 2018; 142:1364–1382.

B. ER and PgR Immunohistochemistry

Hormone receptor expression is occasionally assessed on primary invasive endometrial carcinomas at

the request of the treating clinician in order to predict response to endocrine therapy.

1,2

Guidelines for

reporting results of hormone receptor testing in breast carcinomas published by the American Society of

Clinical Oncology (ASCO) and the College of American Pathologists (CAP) require recording specific

preanalytic and analytic variables that can affect test results.

This information has not been required for

endometrial carcinomas and is optional. However, appropriate positive and negative controls should be

used and evaluated.

Hormone receptor status is typically performed in formalin-fixed, paraffin-embedded tissue sections by

immunohistochemistry (IHC). Only nuclear staining is considered positive. There are many tissue and

technical variables that can affect test results, and the assays must be validated to ensure their accuracy.

In the endometrium, benign endometrial glands, endometrial stroma, or myometrium may serve as

internal control tissue. If internal controls are not present, consider repeating the test on another

specimen with internal controls (if available). Reasons for false-negative results include the following:

• Exposure of tumor cells to heat (e.g., carcinomas transected by using cautery during surgery)

• Prolonged cold ischemic time, which may result in antigenic degradation

CAP Approved

Gynecologic.Bmk_1.0.0.0.REL_CAPCP

• Under- or over-fixation; fixation for at least 6 hours in buffered formalin is recommended;

prolonged fixation can also diminish immunoreactivity

• Type of fixative: ER is degraded in acidic fixatives such as Bouin’s and B-5; formalin should be

buffered to ensure pH range between 7.0 and 7.4

• Decalcification, which may result in loss of immunoreactivity

• Non-optimized antigen retrieval or use of (weeks) old tissue sections

• Type of antibody

• Dark hematoxylin counterstain obscuring faintly positive diaminobenzidine (DAB) staining

False-positive results occur less frequently. Rare reasons would be the use of an impure antibody that

cross-reacts with another antigen or misinterpretation of entrapped normal or hyperplastic cells as

invasive carcinoma. False-positive tests can also be generated by image analysis devices that mistakenly

count overstained nuclei.

Reporting Guidelines

There are currently no outcome-driven consensus opinions that have been developed for the reporting of

the results of immunohistochemical assays for ER and PgR for endometrial cancer. In absence of robust

data, the CAP recommends using a modified reporting format similar to that used for reporting the results

of immunohistochemical assays for ER and PgR for breast cancer (Table 1).

As there is a wide range of receptor levels in individual cancers, a uniform reporting scheme using the

proportion of positive cells as well as the intensity of immunoreactivity is recommended:

• Number of positive cells: The number of positive cells can be reported as a percentage or within

discrete categories (e.g., 10-20%).

• Intensity: Refers to degree of nuclear positivity (i.e., pale to dark). The intensity can be affected

by the amount of protein present, as well as the antibody used and the antigen retrieval system.

In most cancers, there is heterogeneous immunoreactivity with pale to darkly positive cells

present.

Table 1. Reporting Results of Estrogen Receptor (ER) and Progesterone Receptor (PgR) Testing

Result

Criteria

Comments

Positive

Immunoreactive tumor

cells present

(greater

than or equal to 1%

)

The percentage of immunoreactive cells may

determined by visual estimation or

quant

itation.

Quant

itation should be provided by reporting the

percentag

of positive cells in the entire section. If there is significant

regional variation, that should also be reported.

Negative

Less than

1% immunoreactive

tumor cells

presen

References

1. Guan J, Xie L, Luo X, et al. The prognostic significance of estrogen and progesterone receptors

in grade I and II endometrioid endometrial adenocarcinoma: hormone receptors in risk

stratification. J Gynecol Oncol. 2019; 30(1):e13.

2. Jrezak KJ, Duska L, MacKay HJ. Endocrine therapy in endometrial cancer: an old dog with new

tricks. Gynecol Oncol. 2019; 153(1):175-183.

CAP Approved

Gynecologic.Bmk_1.0.0.0.REL_CAPCP

3. Allison KH, Hammond MEH, Dowsett M, et al. Estrogen and progesterone receptor testing in

breast cancer: American Society of Clinical Oncology/College of American Pathologists guideline

update. Arch Pathol Lab Med. 2020;144(5):545-563.

4. Fitzgibbons PL, Dillon DA, Alsabeh R, et al. Template for reporting results of biomarker testing of

specimens from patients with carcinoma of the breast. Arch Pathol Lab Med. 2014;138(5):595-

601.

C. HER2 (ERBB2) Testing

The HER2 (ERBB2) gene is located on chromosome 17 and codes for a tyrosine kinase receptor from the

epidermal growth factor receptor (EGFR) family. This protein is critical in signaling pathways that regulate

cell division, proliferation, differentiation, and apoptosis. There are currently no consensus guidelines that

have been developed for reporting of the results of HER2 testing in endometrioid carcinoma, but HER2

has been proposed as a biomarker, and testing is recommended by the National Comprehensive Cancer

Network (NCCN) guidelines for advanced or recurrent uterine serous carcinoma.

1,2,3

Heterogeneity of

expression within a tumor is a common problem. Unlike the patterns seen in breast carcinoma, staining is

usually lateral or basolateral and spares the apical portion of cells (U-shaped membranous

pattern).

Approximately 25-30% of uterine serous carcinoma will show HER2 overexpression and/or

gene amplification, but divergent results between the two testing methods are common and concordance

may be less than 50%.

4,5,6

Although there is an increased progression-free survival in patients with

homozygous HER2 protein expression treated with targeted chemotherapy, the overall response rate is

low, and most uterine serous carcinomas tend to show heterogeneous HER2 expression. This has

implications for treatment and negative HER2 results on small samples might erroneously exclude

patients from HER2 targeted therapy.

In the absence of conclusive data, CAP suggests using a similar reporting format as that used for

reporting the results of HER2 testing for breast cancer, with modifications below.

4,5,8,9

Table 2. Reporting Results of HER2 Testing by Immunohistochemistry for Endometrial Serous

Carcinoma

Result

Criteria

Negative (Score 0)

No staining observed

Negative (Score 1+)

Incomplete membrane staining that is faint/barely perceptible in any proportion of cells or

Weak complete staining in less than 10% of tumor cells*

Equivocal (Score 2+)

†

Intense complete or basolateral/lateral membrane staining in 30% or less tumor cells*or

Weak to moderate staining in greater than or equal to 10% of tumor cells*

Positive (Score 3+)

Intense complete or basolateral/lateral membrane staining in over 30% of tumor cells*

* Readily appreciated using a low-power objective and observed within a homogeneous and contiguous population of

invasive tumor cells.

† Must order reflex test (same specimen using ISH) or order a new test (new specimen if available, using IHC or

ISH).

HER2 Testing by In Situ Hybridization

The propensity for HER2 heterogeneity in uterine serous carcinoma is mirrored by intratumoral HER2

heterogeneity in fluorescent in situ hybridization (FISH) studies. FISH is performed on tumors with a 2+

IHC score, using the largest tumor area with HER2 immunoreactivity for testing.

Currently, there are no

recommendations to perform FISH in lieu of IHC testing. Based on a large clinical trial, only HER2/CEP17

ratios of greater than or equal to 2.0 are considered amplified and serve as baseline guidance for

CAP Approved

Gynecologic.Bmk_1.0.0.0.REL_CAPCP

reporting pending further studies.

Several unanswered questions remain, such as whether endometrial

biopsy or hysterectomy is the optimal specimen, whether multiple specimens should be tested, whether

metastases should be tested, and if so, how to clinically manage discordant results.

Table 3. Reporting Results of HER2 Testing by Fluorescent In Situ Hybridization (single-probe

assay)

Result

Criteria (single-probe assay)

Negative

· FISH HER2/CEP17 ratio less than 2.0 and

· Average HER2 copy number less than 6 per nucleus

Positive

· FISH HER2/CEP17 ratio greater than or equal to 2.0 or

· FISH HER2/CEP17 ratio less than 2.0, copy number equal to or greater than 6 per nucleus

References

1. Saito A, Yoshida H, Nishikawa T, Yonemon K. Human epidermal growth factor receptor 2

targeted therapy in endometrial cancer: clinical and pathological perspectives. World J Clin

Oncol. 2021; 12(10):868-881.

2. National Comprehensive Cancer Network. NCCN Clinical Practice Guidelines in Oncology

Uterine Neoplasms. Version 1. 2021 [cited 14 December 2021] Available from:

https://www.nccn.org/professionals/physician_gls/pdf/uterine.pdf

3. Buza N, English DP, Santin AD, Hui P. Toward standard HER2 testing of endometrial serous

carcinoma: 4-year experience at a large academic center and recommendations for clinical

practice. Mod Pathol. 2012; 26(12):1605-12.

4. Buza N. HER2 testing in endometrial serous carcinoma: time for standardized pathology practice

to meet the clinical demand. Arch Pathol Lab Med. 2021; 145:687-691.

5. Buza N. HER2 testing and reporting in endometrial serous carcinoma: practical recommendations

for HER2 immunohistochemistry and fluorescent in situ hybridization: proceedings of the ISGyP

companion society session at the 2020 USCAP annual meeting. Int J Gynecol Pathol. 2020;

40(1):17-23.

6. Rottmann D, Assem H, Matsumoto N, Wong S, Hui P, Buza N. Does specimen type have an

impact on HER2 status in endometrial serous carcinoma? Discordant HER2 status of paired

endometrial biopsy and hysterectomy specimens in the presence of frequent intratumoral

heterogeneity. Int J Gynecol Pathol. 2020; 40:263-271.

7. Banet N, Shahi M, Batista D, et al. HER-2 amplification in uterine serous carcinoma and serous

endometrial intraepithelial carcinoma. Am J Surg Pathol. 2021; 45(5):708-715.

8. Wolff AC, Hammond MEH, Allision KH, et al. HER2 testing in breast cancer: American Society of

Clinical Oncology/College of American Pathologists clinical practice guideline focused update.

Arch Pathol Lab Med. 2018; 142:1364–1382.

9. Buza N. Immunohistochemistry in gynecologic carcinomas: practical update with diagnostic and

clinical considerations based on the 2020 WHO classification. Semin Diagn Pathol. 2022;

39(1):58-77.

D. Mismatch Repair (MMR) Immunohistochemistry Testing

Screening for the autosomal dominant Lynch syndrome can be performed by universal MMR testing of

endometrioid carcinoma, one of the most common presentations. Immunohistochemical (IHC) testing for

CAP Approved

Gynecologic.Bmk_1.0.0.0.REL_CAPCP

DNA MMR protein expression (i.e., MLH1, MSH2, MSH6, and PMS2 expression) is performed on

formalin-fixed, paraffin-embedded tissue.

MMR testing in endometrioid carcinoma has been included in the molecular classification system for

endometrioid carcinoma proposed by the World Health Organization.

MMR-deficient patients are eligible

for immune checkpoint inhibitors, although in many cases, treatment is currently restricted to clinically

challenging cases.

If the results of DNA MMR IHC and MSI (microsatellite instability) testing are discordant (e.g., MSI-H

phenotype with normal IHC or abnormal IHC with MSS phenotype), then the laboratory should make sure

that the same sample was used for MSI and IHC testing and that there was no sample mix-up.

Another

possible source of discordance is low tumor volume in the MSI sample. Note that loss of MSH6 protein

expression may occur in absence of MSI-H phenotype.

3,4

Microsatellite instability is a primary pathway of

endometrial carcinogenesis and should be considered when all MMR protein expression is intact.

Any positive reaction in the nuclei of tumor cells is considered as intact expression (normal), and it is

common for intact staining to be somewhat patchy. An interpretation of expression loss in tumor cells

should be made only if a positive reaction is seen in internal control cells, such as the nuclei of stromal,

inflammatory, or non-neoplastic epithelial cells.

4,5

Loss of expression of MLH1 may be due to Lynch

syndrome or methylation of the MLH1 promoter region (as occurs in sporadic MSI carcinoma).

Patients with patterns reflecting a high likelihood of Lynch syndrome should be referred for consultation

with a geneticist. No loss of nuclear expression of MMR proteins indicates a low probability of

microsatellite instability-high (MSI-H). Loss of DNA MMR protein expression is likely to be due to mutation

(either genetic or somatic) in one of the mismatch repair genes.

6,7,8

This information will help identify the

gene that is most likely to have a mutation (e.g., a patient whose tumor shows loss of MSH2 and MSH6

expression, but retention of MLH1 and PMS2 expression, may have an MSH2 germline mutation). Loss of

nuclear expression of MHL1 and PMS2 should be triaged for MLH1 methylation studies. The presence of

MLH1 methylation suggests a sporadic tumor rather than a germline mutation, and further germline

testing is likely not indicated. Absence of MLH1 methylation suggests Lynch syndrome and sequencing

and/or large deletion/duplication testing of germline MLH1 is indicated. Loss of nuclear expression of

MSH2 and MSH6, loss of MSH6 only, or loss of PMS2 only all have a high probability of Lynch syndrome

and genetic counseling should be considered.

References

1. Matias-Guiu X, Oliva E, McCluggage WG, et al. Tumours of the uterine corpus. In: WHO

Classification of Tumours Editorial Board. Female genital tumours [Internet]. Lyon (France):

International Agency for Research on Cancer; 2020 [cited 2020 Nov 20]. (WHO classification of

tumours series, 5th ed.; vol. 4).

Available from: https://tumourclassification.iarc.who.int/chapters/34

2. Casey L, Singh N. POLE, MMR, and MSI testing in endometrial cancer: proceedings of the

ISGyP Companion Society session at the USCAP 2020 annual meeting. Int J Gynecol Pathol.

2020; 40(1):5-16.

3. McConechy MK, Talhouk A, Li-Chang HH, et al. Detection of DNA mismatch repair (MMR)

deficiencies by immunohistochemistry can effectively diagnose the microsatellite instability (MSI)

henotype in endometrial carcinomas. Gynecol Oncol. 2015; 137(2):306-310.

CAP Approved

Gynecologic.Bmk_1.0.0.0.REL_CAPCP

4. Stelloo E, Jansen AML, Osse EM, et al. Practical guidance for mismatch repair-deficiency testing

in endometrial cancer. Ann Oncol. 2017; 28:96-102.

5. Watkins JC, Nucci MR, Ritterhouse LL, Howitt BE, Sholl LM. Unusual mismatch repair

immunohistochemical patterns in endometrial carcinoma. Am J Surg Pathol. 2016; 40(7):909-916.

6. Haraldsdottir S, Hampel H, Tomsic J, et al. Colon and endometrial cancers with mismatch repair

deficiency can arise from somatic, rather than germline, mutations. Gastroenterology. 2014;

147(6):1308-1316.

7. Ligtenberg MJ, Kuiper RP, Chan TL, et al. Heritable somatic methylation and inactivation of

MSH2 in families with Lynch syndrome due to deletion of the 3’ exons of TACSTD1. Nat Genet.

2009; 41(1):112-117.

8. Geurts-Giele WR, Leenen CH, Dubbink HJ, et al. Somatic aberrations of mismatch repair genes

as a cause of microsatellite-unstable cancers. J Pathol. 2014; 234(4):548-559.

E. p53 Expression

p53 is a tumor suppressor protein that induces expression of p21, a cyclin-dependent kinase inhibitor that

is involved in the arrest of cellular proliferation at the G1 phase. Essentially, p53 regulates cell

proliferation, DNA repair, apoptosis, and genetic stability. Inactivation of p53 occurs through mutations of

TP53 or inactivation of p53 through binding proteins, resulting in dysregulated growth. Mutations result in

abnormal cellular expression of the protein (overexpression or lack of expression) that can be detected by

immunohistochemical methods. In gynecologic malignancies, p53 expression is frequently used as a

diagnostic tool but can be employed as a marker for targeted chemotherapy. Mutations of TP53 occur

more commonly (~90%) in serous carcinoma than in endometrioid carcinoma (~10-40%) and are

associated with significantly poorer outcomes.

1,2

Recently the WHO has included TP53 evaluation into its molecular classification of endometrial

carcinoma.

The vast majority of serous-type endometrial carcinomas exhibit mutations in TP53. While

most low-grade endometrioid endometrial tumors are not associated with TP53 mutations, a significant

subset of high-grade endometrioid tumors are; thus, any ancillary testing for the presence of a TP53

mutation should be performed with an awareness of the limitations of the IHC result with respect to

providing a conclusive answer as to classification.

4,5

On occasion, p53 testing may be requested for

treatment purposes, but sequencing of the TP53 gene may be more appropriate to select patients for

targeted therapy.

Extent of p53 specific nuclear immunostaining can be used to assess TP53 gene integrity in endometrial

carcinoma. Normal endometrial glands with an intact TP53 gene express the protein at low levels,

reaching a threshold of immunohistochemical detection (positive staining) in only a small percentage of

cells. Generally, this is 1% to 5% of nuclei but may increase under conditions of cellular damage or repair.

Two different staining patterns are each considered diagnostic of abnormalities of the TP53 gene itself.

Most common are mutations resulting in a qualitatively abnormal p53 protein that stabilizes the p53

complex, resulting in intense nuclear staining in >90% of affected cells. In most cases that harbor

mutations in TP53 that are associated with overexpression, intense nuclear staining is present in over

90% of affected cells. Second is genomic damage causing loss of expression, with a complete absence

of protein in all affected cells. The latter “null” phenotype must be distinguished from a failed stain. Low

levels of expression within internal control tissues (stroma, or nonmalignant epithelium) can be used for

this purpose. It should be noted that p53 expression is significantly affected by non-optimized antigen

retrieval or use of archival (weeks old) tissue sections.

CAP Approved

Gynecologic.Bmk_1.0.0.0.REL_CAPCP

References

1. Nakamura M, Obata T, Daikoku T, Fujiwara H. The association and significance of p53 in

gynecologic cancers: the potential of targeted therapy. Int J Mol Sci. 2019; 20:5482.

2. Zheng W, Xiang L, Fadare O, Kong B. A proposed model for endometrial serous carcinogenesis.

Am J Surg Pathol. 2011; 35:1-14.

3. Matias-Guiu X, Oliva E, McCluggage WG, et al. Tumours of the uterine corpus. In: WHO

Classification of Tumours Editorial Board. Female genital tumours [Internet]. Lyon (France):

International Agency for Research on Cancer; 2020 [cited 2020 Nov 20]. (WHO classification of

tumours series, 5th ed.; vol. 4).

Available from: https://tumourclassification.iarc.who.int/chapters/34

4. Cancer Genome Atlas Research Network. Integrated genomic characterization of endometrial

carcinoma. Nature. 2013;497(7447):67-73.

5. Hoang LN, McConechy MK, Kobel M, et al. Histotype-genotype correlation in 36 high-grade

endometrial carcinomas. Am J Surg Pathol. 2013;37(9):1421-1432.

6. Beinse G, Rance B, Just PA, et al. Identification of TP53 mutated group using a molecular and

immunohistochemical classification of endometrial carcinoma to improve prognostic evaluation for

adjuvant treatments. Int J Gynecol Cancer. 2020; 30(5):640-647.

F. Microsatellite Instability Testing

Detection of hereditary defective mismatch repair has clinical implications for treatment of the affected

patient and family members. Patients with a microsatellite instability-high (MSI-H) phenotype in their

cancer tissues may have a germline mutation in one of several DNA mismatch repair (MMR) genes (e.g.,

MLH1, MSH2, MSH6, or PMS2) or an altered EPCAM (TACSTD1) gene.

1,2,3

After appropriate genetic

counseling, patients may want to consider testing to identify the causative heritable abnormality. An MSI-

H phenotype is more frequently observed in sporadic endometrioid carcinoma (about 15% of cases) due

to somatic abnormalities, usually hypermethylation of the MLH1 gene promoter.

MSI testing protocols are similar to those developed for colon cancer. These are briefly summarized

here, but more complete details are available in the separately issued “Template for Reporting Results of

Biomarker Testing of Specimens From Patients With Carcinoma of the Colon and Rectum”.

Testing is

generally performed with at least 5 microsatellite markers, generally mononucleotide or dinucleotide

repeat markers. In 1998, a National Institutes of Health consensus panel proposed that laboratories use a

5-marker panel consisting of 3 dinucleotide and 2 mononucleotide repeats for MSI testing. Recent data

suggest that dinucleotide repeats may have lower sensitivity and specificity for identifying tumors with an

MSI-H phenotype. As a consequence, there has been a move towards including more mononucleotides

and fewer dinucleotides in MSI testing panels. Many laboratories now use a commercially available kit for

MSI testing that utilizes 5 mononucleotide markers.

If DNA MMR IHC has not been performed, this testing should be recommended for any case that shows

an MSI-H phenotype, because this information will help identify the gene that is most likely to have a

germline (or somatic) mutation.

References

1. Haraldsdottir S, Hampel H, Tomsic J, et al. Colon and endometrial cancers with mismatch repair

deficiency can arise from somatic, rather than germline, mutations. Gastroenterology.

2014;147(6):1308-1316.

CAP Approved

Gynecologic.Bmk_1.0.0.0.REL_CAPCP

2. Ligtenberg MJ, Kuiper RP, Chan TL, et al. Heritable somatic methylation and inactivation of

MSH2 in families with Lynch syndrome due to deletion of the 3’ exons of TACSTD1. Nat Genet.

2009;41(1):112-117.

3. Geurts-Giele WR, Leenen CH, Dubbink HJ, et al. Somatic aberrations of mismatch repair genes

as a cause of microsatellite-unstable cancers. J Pathol. 2014;234(4):548-559.

4. Burgart LJ, Chopp WV, Jain D, Bellizzi AM, Fitzgibbons PL. Template for Reporting Results of

Biomarker Testing of Specimens From Patients With Carcinoma of the Colon and Rectum.

https://documents.cap.org/protocols/ColoRectal.Bmk_1.3.0.0.REL_CAPCP.pdf Published June

2021. Accessed October 12, 2022.

5. Casey L, Singh N. POLE, MMR, and MSI testing in endometrial cancer: proceedings of the

ISGyP Companion Society session at the USCAP 2020 annual meeting. Int J Gynecol Pathol.

2020; 40(1):5-16.

G. MLH1 Promoter Methylation Analysis

Defective mismatch repair in sporadic endometrial cancer is most often due to inactivation of the MLH1

gene promoter by methylation (epigenetic silencing). Most laboratories utilize a methylation-specific real-

time polymerase chain reaction (PCR) assay to determine the presence of methylation.