IGCS Pathology Corner

A guide to interpretation of commonly used immunohistochemical stains in

gynecological oncology.

Part I.

Dr Rachael van der Griend

MBChB FRCPA

Anatomical Pathologist

Canterbury Health Labs, New Zealand

What is an immunohistochemical (IHC) stain?

• Technically not a stain, although this term has become common usage.

• A stain adheres to physical characteristics of the tissue e.g. haematoxylin and eosin (H&E), where haematoxylin stains nucleic acids blue-purple and eosin stains proteins pink.

• Immunohistochemistry is a way to visualise the location and distribution of antigens in tissue.

• Antibodies are labelled with a coloured tag; either a coloured dye (usually a red or brown chromogen) or a fluorescent label.

• You can label the antibody directly or use a secondary antibody carrying the colour tag or an enzyme converter (indirect).

What is IHC used for?

• To characterise the tissue you are looking at or improve reliability/reproducibility – Diagnosis

• To give information about the behaviour of the tumour – Prognosis

• To predict treatment outcomes e.g. HER2, PDL-1

• To identify possible genetic syndromes e.g. Lynch, HLRCC etc.

• To identify organisms e.g. CMV, herpes virus inclusions etc.

Drawbacks

• No antibody is perfect; each IHC has its own specificity, sensitivity and staining peculiarities.

• The more antibodies you use for a case the more likely you are to get an aberrant staining pattern.

• Many tumors show overlapping immunophenotypes i.e. different tumors can show similar patterns of immuno “staining”

• Best practice is to use a carefully selected panel of antibodies and always refer back to the H&E appearances.

Common useful antibodiesp16, p53 and Mismatch repair (MMR) proteins.

p53

• Good surrogate for TP53 mutation; if the p53 IHC staining pattern is abnormal there is almost certainly an underlying TP53 mutation

• Four different patterns of expression

• Should not be reported as simply positive or negative as this can be misleading• i.e. using the term NEGATIVE does not distinguish between no

mutation (wild-type) and complete absence of staining (mutated)

Normal/wild-type p53 expressionAn admixture of staining patterns; some cells are negative, some show weak nuclear positivity and some show strong nuclear positivity.

p53 x 100. FIGO Grade 1 endometrioid adenocarcinoma showing wild-type pattern of p53 immunoreactivity.

Abnormal/overexpression of p53Defined as diffuse strong positive nuclear staining in >80% of tumor cells but, in practice, usually closer to 100%. Associated with TP53 nonsynonymous missense mutation.

Fig. A. HE x200. Peritoneal biopsy with infiltrating high-grade tubo-ovarian serous cancer. Fig. B. p53 x200. Strong positive nuclear expression of p53 in almost every tumor cell.

A B

Abnormal/complete absence of p53Associated with TP53 loss of function mutation. Often called “null phenotype” to avoid use of the term negative.

Fig. A. HE x200. Peritoneal biopsy with infiltrating high-grade tubo-ovarian serous cancer. Fig. B. p53 x200. Complete absence of staining with scattered lymphocytes in the background providing internal positive control.

A B

Abnormal/cytoplasmic expression of p53Rare and should only be interpreted as such with an optimized protocol and appropriate internal positive and negative controls. Associated with TP53 loss of function mutation disrupting nuclear localization domain.

Fig. A. HE x200. Core biopsy of omentum with infiltrating high-grade tubo-ovarian serous cancer. Fig. B. p53 x400. Cytoplasmic expression of p53 in tumor cells.Fig. C. A different example showing strong cytoplasmic and nuclear expression of p53 in tumor cells. Photomicrograph courtesy of Prof. Glenn McCluggage.A

B C

Practical applications… Ovary and FT

• Can be used to confirm areas of serous tubal intraepithelial carcinoma (STIC) of fallopian tube

• Can be helpful to distinguish between between low-grade and high-grade serous carcinoma of tube/ovary/peritoneum where the histomorphology is ambiguous• TP53 mutation is almost universal in high grade serous cancer of tubo-ovarian

origin, almost never seen in low-grade serous cancer

• Use in conjunction with proliferation marker Ki67

Practical applications… Ovary and FTCan confirm areas of serous tubal intraepithelial carcinoma of fallopian tube (STIC)

Fig. A. HE x100. Fallopian tube with short segment of STIC overlying a focus of invasive high-grade serous carcinoma. Fig. B. p53 x100. Strong nuclear staining (mutant/overexpression phenotype).

A B

Practical applications… UterusCan be helpful to distinguish between FIGO Grade 1 or 2 endometrioid carcinoma (wild-type) and serous carcinoma of endometrium (mutated).

Endometrial curettage initially reported as FIGO Grade 2 endometrioid endometrial adenocarcinoma. Nuclear atypia was more apparent on hysterectomy specimen and immunohistochemistry was performed. Fig. A. HE x100. Uterine tumor. Fig. B. p53 x100. Strong nuclear staining (mutant/overexpression phenotype). Final diagnosis: Serous endometrial carcinoma

A B

Practical applications… Uterus

• p53 has prognostic significance in endometrial tumors that may be difficult to classify; tumors with mutant p53 expression show a more aggressive clinical course than those without*

• Can be helpful in the case of small crushed biopsies, or those endometrial carcinomas that have overlapping histologic features of serous and endometrioid carcinoma

• “Morphologically ambiguous high-grade endometrial carcinoma with aberrant p53 expression” can be a clinically useful diagnosis when precise morphologic subtyping is not possible

*Garg K, Leitao MM, Wynveen CA, Sica GL, Shia J, Shi W, Soslow RA. p53 overexpression in morphologically ambiguous endometrial carcinomas correlates with adverse clinical outcomes. Modern Pathology (2010) 23, 80 - 92

Practical applications… Uterus

• Grade 3 endometrioid carcinomas with p53 IHC mutation pattern have worse prognosis than those Grade 3 tumors with wild-type expression *

• p53 IHC mutation pattern makes undifferentiated/dedifferentiated endometrial carcinoma unlikely.

• p53 IHC mutation pattern makes clear cell carcinoma less likely than serous carcinoma with cleared cytoplasm.

*Kobel M, Ronnett B, Singh N, Soslow RA, Gilks CB, McCluggage WG. Interpretation of p53 immunohistochemistry in endometrial carcinomas: toward increased reproducibility. Int J Gyn Path 38:S123-S131 doi: 10.1097/PGP.0000000000000488.

Practical applications… Vulva

• p53 in normal vulvar squamous mucosa is confined to the basal layer.

• In differentiated vulvar intraepithelial neoplasia (d-VIN) abnormal expression of p53 may be seen in cells extending up toward the surface. p16 is usually absent or shows wild-type expression

• In VIN of usual type (HPV-related or warty VIN) the inverse pattern is seen: basal p53 with strong diffuse block-like (mutant) p16 reactivity.

p16

• A tumor suppressor protein which is a cell cycle disruptor

• In the cervix is a surrogate marker for high-risk (oncogenic) HPV infection

• p16 also often shows mutant-type staining in other high-grade tumors - which is not related to HPV - e.g. serous and endometrial cancer but also breast, colorectal and pancreatic carcinoma (p16 acts in the same pathway as p53).

• As for p53, the pattern and intensity of staining is important and the terms positive or negative should be avoided in favor of mutant or wild-type.

Normal/wild-type p16 expressionEither absence of reactivity, or patchy expression with some cells very strong and intense and others weaker.

Fig. A. HE x200. Biopsy from cervical transformation zone showing immature squamous metaplasia. Fig. B. p16 x200. Weak and patchy expression in immature squamous epithelium (wild-type pattern).

A B

Mutant/overexpression of p16Diffuse strong nuclear reactivity, with or without cytoplasmic reactivity, in >90% of tumour cells.

Fig. A. HE x200. Peritoneal biopsy with infiltrating high-grade tubo-ovarian serous cancer. Fig. B. p16 x200. Strong positive nuclear expression of p16 in almost every tumor cell (mutant phenotype).

A B

Mutant/overexpression of p16In squamous epithelium a “block-like” distribution is required:

• strong nuclear +/- cytoplasmic staining of the basal layer and

• extends upwards at least the one third of the epithelium and

• extends laterally for at least 10-20 continuous cells

Fig. A. HE x 100. Cervical transformation zone with a high-grade squamous intraepithelial lesion (HSIL). Fig. B. p16 x 100. “Block-like” expression of p16 (mutant phenotype) involving basal half of the epithelium.

A B

Wild-type p16 expressionStrong but patchy distribution not mutant phenotype. Debate over significance: may indicate presence of HPV but not integrated, may indicate HPV types not 16 or 18.

Biopsy of cervical transformation zone with low-grade SIL. p16 shows patchy (wild-type) expression. Photomicrograph courtesy of Dr Diane Kenwright

Practical applications… Vulva/Vagina/Cervix

• Usually mutant expression in all HPV-related high-grade anogenital precursor lesions e.g. cervix HSIL, AIS, SMILE, usual-type VIN and VAIN.• Useful to distinguish between high-grade squamous intraepithelial lesions (HSIL) and mimics

such as atrophy*• Useful to identify endocervical adenocarcinoma in-situ (AIS) but beware tubal metaplasia

which is often strongly p16 positive.

• Useful to distinguish between usual-type endocervical adenocarcinoma (p16 mutated) and endometrial low-grade adenocarcinoma (p16 wild-type)

* see also recommendations of the LAST project (Lower Anogenital Squamous Terminology Standardization Project for HPV-Associated Lesions: background and consensus recommendations from the College of American Pathologists and the American Society for Colposcopy and Cervical Pathology), link in references.

Practical applications… Vulva/Vagina/Cervix

Fig A. HE x200. Cervical transformation zone showing stratified mucin-producing intraepithelial lesion (SMILE), a high-grade HPV-related premalignant condition. Fig B. p16 x100. Strong diffuse positivity (mutant expression).

A B

Practical applications… Uterus

• Useful to distinguish between endocervical adenocarcinoma (usually p16 mutated) and endometrial low-grade adenocarcinoma (p16 wild-type)

• But beware many high-grade tumors are positive, including high-grade endometrial carcinomas, serous carcinoma, and some carcinosarcomas and leiomyosarcomas.

Practical applications… Ovary and FT

Fig. A. HE x20 Fimbria of fallopian tube with metastatic endocervical adenocarcinoma. Fig. B. p16 x20. Strong diffuse mutant p16 expression in keeping with HPV-related endocervical adenocarcinoma. Fig. C. p53 x20. Wild-type p53 expression rules out STIC or high grade serous cancer of tube.

A

B

C

Mismatch repair (MMR) proteins

• Lynch Syndrome (LS) is due to a germline mutation of MMR genes which predisposes to cancers of colon, rectum, endometrium, ovaries, stomach, pancreas and brain

• Autosomal dominant inheritance; 1:250

• Lifetime risk for endometrial and colon cancer is approximately 60%

• Women are more likely to develop endometrial cancer than colon cancer and endometrial cancer is the first malignancy diagnosed in >50% of LS women

MMR IHC

• IHC can be a useful screening test for Lynch Syndrome.

• There is ongoing debate about who to screen. Family history and histopathologic criteria will miss many cases of LS, particularly in women, and most well-resourced institutes have moved to a Universal Screening approach.

• In lower resource settings this may need to be modified and there is no point in screening without access to appropriate follow up and a clinical genetics service.

MMR IHC

• The most commonly used screening platform for Lynch Syndrome is a combination of 4 antibodies (MLH1, PMS2, MSH6 and MSH2).

• As these proteins form functional dimers, a more cost-effective 2 antibody model (MSH6, PMS2) may be used in some centres.

MMR IHC

• Loss of MMR protein expression is defined as absence of immunoreactivity within all tumor nuclei (with intact nuclear expression in nontumor cells providing an internal positive control).

• Avoid use of terms positive and negative as a positive stain actually implies a negative result (i.e. normal or no mutation).

• Preserved (MMRp) or deficient (MMRd) are better descriptors, commonly used in the Southern Hemisphere

• The College of American Pathologists recommends the terminology intactMMR vs loss of nuclear expression

Endometrial adenocarcinoma with MMRd pattern, with loss of expression of MLH1 and PMS2. Note preserved internal positive control (stromal cells and lymphocytes). Magnification x100.

MSH2

MSH6

PMS2

MLH1

MMRd does not equal Lynch Syndrome

• Inherited (Lynch Syndrome)• Mutation in one of four MMR genes: MLH1, MSH2, MSH6 or PMS2

• Germline mutation in one allele

• Second allele lost as somatic event

• Sporadic • MLH1 promoter methylation

• Somatic mutation

• Acquired, may or may not be driver mutation

Methylation

• In colon cancer BRAF IHC is a good surrogate for tumor methylation but not in endometrial cancer for poorly understood reasons

• Tumor MLH1 promoter hypermethylation currently must be assessed by PCR or other more expensive and time consuming techniques

• In low resource settings it may be reasonable to omit this step as several studies have shown high rate of somatic hypermethylation(95%) in tumors with loss of staining for MLH1 and PMS2

• Cases demonstrating PMS2, MSH6, MSH2/MSH6 or unmethylatedMLH1/PMS2 deficiency require referral of the patient for genetic counselling and germline testing

Practical applications…

• Endometrial cancer in a woman with Lynch syndrome is a potentially curable sentinel event. Surveillance of her and her family can lead to reduction in incidence of malignancy and substantial reduction in mortality

• Testing at biopsy stage allows stratification of patients with low-grade endometrial tumors into those suitable for conservative management (e.g. wanting to conserve fertility or with multiple comorbid conditions) vs those not (Lynch syndrome)

• Currently used to screen endometrial tumors, of all histotypes, and endometrioid and clear cell carcinomas of ovary

Future possibilities...

• MMR status is one of the cornerstones of the TCGA molecular classification which is becoming important for prognosis

• As antiPD-1 and antiPD-L1 therapies become available MMR status will be used to predict treatment response

Selected references Kobel M, Ronnett B, Singh N, Soslow RA, Gilks CB, McCluggage WG. Interpretation of p53 immunohistochemistry in endometrial carcinomas: toward increased reproducibility. Int J Gyn Path 38:S123-S131 doi: 10.1097/PGP.0000000000000488.

Garg K, Leitao MM, Wynveen CA, Sica GL, Shia J, Shi W, Soslow RA. p53 overexpression in morphologically ambiguous endometrial carcinomas correlates with adverse clinical outcomes. Modern Pathology (2010) 23, 80 - 92

Liu Y, Alqatari M, Sultan K, Ye F, Gao D, Sigel K, Zhang D, Kalir T. Using p16 immunohistochemistry to classify morphologic cervical intraepithelial neoplasia 2: correlation of ambiguous staining patterns with HPV subtypes and clinical outcome. Hum Pathol 2017 August; 66: 144 – 151. doi:10.1016/j.humpath.2017.06.014

Lower Anogenital Squamous Terminology Standardization Project for HPV-Associated Lesions: background and consensus recommendations from the College of American Pathologists and the American Society for Colposcopy and Cervical Pathology. Arch Pathol Lab Med 2012 Oct; 136(10): 1266 – 97. doi: 10.5858/arpa.LGT200570

Murali R, Grisham RN, Soslow RA. The roles of pathology in targeted therapy of women with gynecologic cancers. Gynecologic Oncology 2018 148, 213-221DOI: 10.1016/j.ygyno.2017.11.020

Watkins JC, Yang EJ, Muto MG, Feltmate CM, Berkowitz RS, Horowitz NS, Syngal S, Yurgelun MB, Chittendon A, Hornick JL, Crum CP, Sholl LM, Howitt BE. Universal screening for mismatch-repair deficiency in endometrial cancers to identify patients with Lynch syndrome and Lynch-like syndrome. International Journal of Gynaecological Pathology 36:115-127. doi:10.1097/PGP.0000000000000312

Najdawi F, Crook A, Maidens J, McEvoy C, Fellowes A, Pickett J, Ho M, Nevell D, McIlroy K, Sheen A, Sioson L, Ahadi M, Turchini J, Clarkson A, Hogg R, Valmadre S, Gard G, Dooley SJ, Scott RJ, Fox SB, Field M, Gill AJ. Lessons learned from implementation of a Lynch syndrome screening program for patients with gynaecological malignancy. Pathology (August 2017) 49(5) 457-464. doi:10.1016/j.pathol.2017.05.004