Roche BenchMark XT/LT Interpretation Guide

Brand: Roche

Model: BenchMark XT/LT

Type: Interpretation Guide

This manual is also suitable for

BenchMark ULTRA

VENTANA MMR RxDx Panel

Interpretation Guide for Endometrial

Carcinoma (EC)

VENTANA anti-MLH1 (M1) Mouse Monoclonal Primary Antibody

VENTANA anti-PMS2 (A16-4) Mouse Monoclonal Primary Antibody

VENTANA anti-MSH2 (G219-1129) Mouse Monoclonal Primary Antibody

VENTANA anti-MSH6 (SP93) Rabbit Monoclonal Primary Antibody

Table of Contents

Introduction 1

Intended Use 3

Intended Use of Product 3

Purpose of Interpretation Guide 3

Clinical Evaluation 4

Staining Characteristics 4

Morphology and Background Acceptability Criteria 5

Case Criteria for Clinical Evaluation 5

Scoring Algorithm 6

Controls 8

Internal Positive Controls 8

Specimen Workﬂow 10

Reference Images 12

Proﬁcient Cases 12

Deﬁcient Cases 14

Challenging Cases 18

Impact of Pre-analytical Conditions on VENTANA MMR RxDx Panel 28

Acceptable Fixation Conditions to Achieve Optimal Staining Results 28

Cut Slide Stability 32

Tissue Thickness 32

References 33

DNA mismatch repair (MMR) is a conserved molecular

mechanism that functions to correct the improper base

substitutions that spontaneously occur during DNA replication.

Polymerase chain reaction (PCR) based methods have shown

that MMR failure frequently leads to microsatellite instability

(MSI), a condition in which short, tandem nucleotide repeats are

inserted into the DNA. When the number of repeats is equal to

or greater than 30% of the examined microsatellite loci, MSI can

be further characterized as MSI-High (MSI-H). Defects in the

MMR machinery have been attributed to mutations in the MMR

proteins, most commonly MLH1, PMS2, MSH2, and MSH6.

The MLH1 and PMS2 proteins normally function together

in a heterodimeric complex, as do the MSH2 and MSH6

proteins. When MMR is functioning normally, the MSH6/MSH2

heterodimer binds to mismatched DNA. This binding induces a

conformational change that allows the MLH1/PMS2 heterodimer

to bind the DNA-bound MSH6/MSH2 complex, resulting in

excision repair of the affected DNA. Mutations or deﬁciencies

in these proteins result in frequent MSI and somatic mutation due

to replication error. MMR immunohistochemistry (IHC) testing

can be shown to be useful in identifying MMR genes likely to

contain germline or a somatic alterations.

Traditionally, treatment regimens for cancer patients have

been dictated by the speciﬁc malignancy. However, emerging

immunotherapies, particularly those that modify cellular

pathways involving the programmed death 1 (PD-1) or

programmed death ligand 1 (PD-L1) proteins, are reshaping

clinicians’ therapeutic strategies. PD-1 is an inhibitory receptor

expressed on T cells after T-cell activation, which is sustained

in states of chronic stimulation such as in chronic infection or

cancer. PD-L1 expression has been observed in immune cells

and malignant cells, and aberrant expression of PD-L1 on tumor

cells (TC) has been reported to impede anti-tumor immunity,

resulting in immune evasion. Therefore, interruption of the PD-

L1/PD-1 pathway represents an attractive strategy to reinvigorate

tumor-speciﬁc T cell immunity.

MMR proteins (MLH1, PMS2, MSH2, and MSH6) are ubiquitously

expressed in proliferating normal and malignant cells. Many

common cancers are MMR deﬁcient (dMMR) and exhibit MSI.

Multiple studies have demonstrated that MMR deﬁciency

correlate with higher expression of PD-1 or PD-L1 proteins.

Thus, MMR proteins may be useful as predictive biomarkers for

PD-1 targeted therapy; speciﬁcally, a loss of expression of one

or more MMR proteins might predict an increased likelihood of

response to such therapy. PD-1 inhibitors can be beneﬁcial in

many common cancers with a high frequency of MMR deﬁciency

and /or MSI-H regardless of the cancers’ tissue of origin.

Hence, patients considering PD-1 targeted therapy will beneﬁt

from a companion diagnostic assay to identify the patient

population with MMR deﬁciency.

Endometrial carcinoma (EC) is one of the most common

gynecologic malignancies.

It is frequently noted to have many

genetic alterations including MSI.

It has also been suggested

that MSI EC may have increased PD-L1 protein expression as

compared to the microsatellite stable (MSS) EC.

While the

treatment of EC varies depending on the grade, histology and

stage of disease,

evaluation of the MMR status of EC tumors is

helpful for prognosis and guiding treatment

. Therefore, patients

considering PD-1 targeted therapy will beneﬁt from a companion

diagnostic assay to identify MMR status.

A loss of expression of any of the essential MMR proteins,

including MLH1, PMS2, MSH2, or MSH6, causes MMR

deﬁciency. Presence of staining for all four MMR protein markers

in the tumor indicates that the case is proﬁcient for VENTANA

MMR IHC status. Absence of staining in any one of the MMR

protein markers of VENTANA MMR RxDx Panel indicates a case

is deﬁcient for VENTANA MMR IHC status.

Introduction

VENTANA MMR RxDx Panel for Endometrial Carcinoma 1

2 VENTANA MMR RxDx Panel for Endometrial Carcinoma

Intended Use

Intended Use of Product

Refer to the corresponding VENTANA MMR RxDx Panel package

inserts for the detailed intended use of this product including

associated indications.

Purpose of Interpretation Guide

This guide is intended to:

• Provide pathologists with a tool to facilitate the clinical

evaluation of formalin-ﬁxed, parafﬁn-embedded (FFPE)

tumor sections stained with VENTANA MMR RxDx Panel in

accordance with the proposed product labeling.

• Provide photographic images that illustrate the staining

patterns that may result from staining of tumor tissues with the

VENTANA MMR RxDx Panel.

• Provide example images of challenging cases to provide

guidance in their evaluation.

• Provide guidance in using MMR protein-positive normal tissue

elements, which serve as a positive internal control when

stained with the VENTANA MMR RxDx Panel.

VENTANA MMR RxDx Panel for Endometrial Carcinoma 3

MMR Intact and Loss Staining Status: In these endometrial carcinoma cases, nuclei show unequivocal staining, which is the

predominant staining pattern seen in the majority of tumor tissues in MSH6 Intact Status (left panel). Absence of any nuclear

staining in tumor tissues represent MSH6 Loss Status (right panel). Unequivocal nuclear staining in internal control cells (i.e.

lymphocytes) in the vicinity of viable tumors serve as acceptable internal positive controls.

Staining Characteristics

Clinical Evaluation

Cells labeled with each assay in the VENTANA MMR RxDx

Panel are evaluated for the presence or absence of the

diaminobenzidine (DAB) signal. VENTANA MMR RxDx Panel

staining in MMR proteins follows a nuclear pattern. Loss of any

one of the MMR proteins detected by the VENTANA MMR RxDx

Panel is observed in the nuclei of tumor cells. The MMR status is

classiﬁed as intact or loss based on unequivocal nuclear staining

only.

• Intact Status is characterized by tumor cells that exhibit

unequivocal staining of equal to or greater than the intensity of

internal controls above background.

• Loss Status is characterized by an absence of any detectable

signal. Loss cases may still exhibit pale grey nuclear

discoloration.

The signal may be distributed homogeneously, having a uniform

level of intensity throughout the neoplastic portions of the tumor

or distributed heterogeneously having more than one intensity

level. It should be easily detectable at low power magniﬁcations

such as 2x or 4x. A graphical representation of VENTANA MMR

RxDx Panel-labeled neoplastic cell type(s)/staining elements is

provided in the Reference Images section of this interpretation

guide. The species-matched negative reagent control (NRC) is

used to evaluate the presence of background in test samples

and establish a staining intensity baseline. Morphology and

background acceptability criteria are provided in Table 1. Case

criteria for evaluating MMR status of neoplastic tissue with any of

the four assays in the VENTANA MMR RxDx Panel are provided

in Table 2.

Absence of nuclear

staining in tumor cells

Unequivocal nuclear

staining in tumor cells

Strong nuclear staining

in lymphocytes

4 VENTANA MMR RxDx Panel for Endometrial Carcinoma

MSH6 10x MSH6 10x

Morphology and Background Acceptability Criteria

For each case stained with VENTANA MMR RxDx Panel, tissue morphology and background acceptability are assessed using the

criteria described in Table 1.

Case Criteria for Clinical Evaluation

Table 2 summarizes the criteria for a slide with neoplastic tissue to be evaluated for MMR status with any of the assays in the

VENTANA MMR RxDx Panel.

Criteria Acceptable Unacceptable

Morphology Cellular elements of interest are visualized,

allowing clinical interpretation of speciﬁc

staining

Cellular elements of interest are not visualized,

compromising clinical interpretation of speciﬁc

staining

Background Background does not interfere with clinical

interpretation of speciﬁc staining

Background interferes with ability to interpret

speciﬁc staining

Table 1: Morphology and Background Acceptability Criteria

Clinical Interpretation Staining Pattern Criteria

Evaluable (all must be true)

1. H&E has ≥ 50 viable tumor cells

2. Negative Reagent Control Slide is acceptable

3. Morphology is acceptable

4. Background is acceptable

5. Internal positive controls have unequivocal nuclear staining

Not Evaluable (if one criteria in this

section is true the slide staining should

be repeated or the case rejected, where

applicable)

1. H&E has < 50 viable tumor cells

2. Negative Reagent Control Slide is unacceptable

3. Morphology is unacceptable

4. Background is unacceptable

5. Internal positive controls do not have unequivocal nuclear staining

6. Interpretation is not possible due to tissue loss, tumor absence, artifacts

and/or edge artifacts

Table 2: Evaluable and Non-evaluable Criteria

VENTANA MMR RxDx Panel for Endometrial Carcinoma 5

VENTANA MMR RxDx Panel comprises four assays: VENTANA

anti-MLH1 (M1) Mouse Monoclonal Primary Antibody,

VENTANA anti-PMS2 (A16-4) Mouse Monoclonal Primary

Antibody, VENTANA anti-MSH2 (G219-1129) Mouse Monoclonal

Primary Antibody, and VENTANA anti-MSH6 (SP93) Rabbit

Monoclonal Primary Antibody. A serial tissue section of the test

specimen is needed for staining with each of these antibodies;

additional serial tissue sections of the test specimen are

needed for staining with hematoxylin and eosin (H&E) and with

NRC antibody corresponding to the species of the respective

VENTANA MMR RxDx Panel antibody (Negative Control

(Monoclonal) (mNRC) or Rabbit Monoclonal Negative Control Ig

(rNRC)).

Neoplastic cells stained with VENTANA MMR RxDx Panel are

evaluated for the presence or absence of the DAB signal. The

matched NRC-stained slide is used to assess non-speciﬁc

background staining and degree of background staining known

to occur due to speciﬁc tissue elements (refer to proﬁcient and

deﬁcient case images).

If the H&E evaluation indicates that the patient specimen is

inadequate (for example, if fewer than 50 viable tumor cells are

present), then a new specimen should be obtained.

Repeat staining of a specimen for a particular MMR RxDx

assay within the panel (e.g. VENTANA anti-MLH1 (M1) Mouse

Monoclonal Primary Antibody) should be carried out on

unstained slides if (1) the case slide stained with the appropriate

NRC for that assay does not exhibit acceptable staining, or (2)

if the case slide stained with that particular MMR RxDx assay

(e.g. VENTANA anti-MLH1 (M1) Mouse Monoclonal Primary

Antibody) is not evaluable. If any of the slides is not interpretable

due to lack of staining in the internal positive control cells, the

presence of artifacts, edge effects necrosis, lack of tissue or any

other reason, the slide cannot be evaluated. If control staining is

acceptable and the VENTANA MMR RxDx Panel-stained slides

are evaluable, the slides can be evaluated by a trained pathologist

as described in the Scoring Algorithm.

NOTE: OptiView DAB IHC Detection Kit and OptiView

Ampliﬁcation Kit are the only detection reagents that are

recommended for use with the VENTANA MMR RxDx Panel.

The interpretations for protein expression and MMR Status for

the VENTANA MMR RxDx Panel are provided below in Tables

3 and 4. Representative cases are discussed in the Reference

Images section.

Evaluating VENTANA MMR RxDx Panel:

Scoring Algorithm

Table 4: VENTANA MMR RxDx Panel Interpretation for MMR Status

Proﬁcient Deﬁcient

All four proteins (MLH1, PMS2, MSH2, and MSH6) in the

panel exhibit intact expression

At least one protein (MLH1, PMS2, MSH2, and MSH6) in the panel

exhibit loss of expression

Table 3: VENTANA MMR RxDx Panel Interpretation of Protein Expression

Intact Protein Expression Loss of Protein Expression

Unequivocal nuclear staining in viable tumor cells in the

presence of acceptable internal positive controls (e.g.

nuclear staining in lymphocytes, ﬁbroblasts or normal

epithelium in the vicinity of the tumor)

Unequivocal loss of nuclear staining or focal weak equivocal

nuclear staining in the viable tumor cells in the presence of

acceptable internal positive controls. Punctate nuclear staining is

considered negative

6 VENTANA MMR RxDx Panel for Endometrial Carcinoma