Roche BenchMark ULTRA Interpretation Guide

Type
Interpretation Guide

This manual is also suitable for

Roche BenchMark ULTRA, your newly acquired laboratory companion, offers state-of-the-art automated staining for histology and cytology samples. Experience efficiency like never before with its continuous loading capability, allowing you to load slides while the instrument is running. ULTRA's flexible design accommodates a wide range of staining protocols, including special stains and rapid staining options. Get ready to enhance your laboratory workflow and produce high-quality stained slides consistently.

Roche BenchMark ULTRA, your newly acquired laboratory companion, offers state-of-the-art automated staining for histology and cytology samples. Experience efficiency like never before with its continuous loading capability, allowing you to load slides while the instrument is running. ULTRA's flexible design accommodates a wide range of staining protocols, including special stains and rapid staining options. Get ready to enhance your laboratory workflow and produce high-quality stained slides consistently.

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.
12
It is frequently noted to have many
genetic alterations including MSI.
12
It has also been suggested
that MSI EC may have increased PD-L1 protein expression as
compared to the microsatellite stable (MSS) EC.
16
While the
treatment of EC varies depending on the grade, histology and
stage of disease,
17
evaluation of the MMR status of EC tumors is
helpful for prognosis and guiding treatment
12
. 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
Endometrial Carcinoma-Clinical Diagnosis Proficient
MSH2 Intact Status: Strong nuclear
staining in the presence of acceptable
internal positive controls
PMS2 Intact Status: Strong nuclear
staining in the presence of acceptable
internal positive controls
MLH1 Intact Status: Strong nuclear
staining in the presence of acceptable
internal positive controls
Endometrial Carcinoma-Clinical Diagnosis Deficient
MLH1 Loss Status: Absence of nuclear
staining in the presence of acceptable
internal positive controls
MSH6 Loss Status: Absence of nuclear
staining in the presence of acceptable
internal positive controls
MSH2 Loss Status: Absence of nuclear
staining in the presence of acceptable
internal positive controls
MLH1 20x MSH6 20x MSH2 20x
MSH2 20x
PMS2 20x
MLH1 20x
MSH2-
positive
nuclei
PMS2-
positive
nuclei
MLH1-
positive
nuclei
VENTANA MMR RxDx Panel for Endometrial Carcinoma 7
Controls
When stained with VENTANA MMR RxDx Panel, normal
tissue elements, such as lymphocytes, fibroblasts or normal
epithelium, in the vicinity of the tumor show nuclear staining for
MMR proteins. These tissue elements serve as internal positive
controls and must be acceptable to evaluate the stained slide
for each assay in the panel. Internal controls must be evaluated
for each assay and must meet the staining criteria for a panel
interpretation of MMR status. In the absence of unequivocal
nuclear staining in lymphocytes, fibroblasts or normal
epithelium in the vicinity of the tumor, the assay stained slide
shall be deemed “not evaluable.”
Table 5 describes the criteria for acceptable internal control
staining.
Evaluable Not Evaluable
Presence of unequivocal nuclear staining in normal
tissue elements (e.g. lymphocytes, fibroblasts or normal
epithelium) in the vicinity of the tumor
Absence of unequivocal nuclear staining in normal tissue
elements (e.g. lymphocytes, fibroblasts or normal epithelium) in
the vicinity of the tumor
Table 5: Internal Control Staining Criteria
Internal Positive Control Case 1: Endometrial carcinoma exhibits no internal control staining in the vicinity of the tumor cells and
thus the carcinoma cannot be evaluated. The acceptable Negative Reagent Control depicted is mouse NRC (mNRC).
Internal Positive Controls
Representative images of the internal control for the VENTANA MMR RxDx Panel are shown below.
8 VENTANA MMR RxDx Panel for Endometrial Carcinoma
H&E 40x mNRC 40x MLH1 40x
Internal Positive Control Case 2: Strong internal control cell staining is shown in the tumor vicinity of an endometrial carcinoma
MLH1 Loss Status case and a MSH2 Intact Status case. The acceptable Negative Reagent Control is mouse NRC (mNRC).
MLH1 20x
Internal Control Cell staining Internal Control Cell staining
VENTANA MMR RxDx Panel for Endometrial Carcinoma 9
H&E 20x mNRC 20x
MSH2 20x
Specimen Workflow
Testing with VENTANA MMR RxDx Panel requires several serial
tissue sections of each test specimen. One section each is
needed for staining with each of the 4 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. In addition, sections of each test specimen
are needed for staining with H&E, and with NRC antibody
corresponding to the species of the respective VENTANA MMR
RxDx Panel antibody (mNRC or rNRC).
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 negative
reagent control 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 no staining in the internal positive control cells, artifacts,
edge effects, necrosis, lack of tissue, or any other reason, the
slide cannot be evaluated. If controls are 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.
10 VENTANA MMR RxDx Panel for Endometrial Carcinoma
Specimen Flow
VENTANA MMR RxDx Panel for Endometrial Carcinoma 11
4 m thick sections are mounted
on positively-charged slides. (6 or 7
serial sections needed)
Repeat staining with new
patient specimen*
No Yes
Is the H&E slide
acceptable (≥ 50
viable cells)?
One section is stained with each assay in VENTANA MMR RxDx Panel.
Additionally, each assay condition is stained with species matched negative
reagent control, Negative Control (Monoclonal) (mNRC), or Rabbit Monoclonal
Negative Control Ig (rNRC) as applicable on the same instrument run.
The MMR (MLH1, PMS2, MSH2, MSH6) result is determined
by a trained pathologist according to the VENTANA MMR
RxDx Panel Scoring Algorithm.
No
No
No
Yes
Yes
Yes
Is the negative
reagent control
stained specimen
slide acceptable?
Does each stained
specimen slide
exhibit acceptable
internal controls?
Are the VENTANA
MMR RxDx Panel-
stained specimen
slides acceptable?
Tumor H&E
Tumor NRC
*Repeat can be on the same patient block or another tissue
block from the same patient, as applicable.
(All Tumor images at 20x magnification)
Internal control
Tumor IHC
Tumor tissue sample is fixed in
10% neutral buffered formalin for
6-72 hours.
One section is stained with H&E.
Repeat staining run
Repeat staining of case*
Repeat staining of case*
12 VENTANA MMR RxDx Panel for Endometrial Carcinoma
Reference Images
Proficient Case 1: MLH1, PMS2, MSH2 and MSH6 Intact Status in endometrial carcinoma exhibiting strong nuclear staining in
tumor cells in the presence of appropriately stained internal controls. The acceptable Negative Reagent Controls depicted are mouse
NRC (mNRC) and rabbit NRC (rNRC).
Proficient Cases
mNRC 20x
rNRC 20x
MLH1 20x PMS2 20x
MSH2 20x MSH6 20x
H&E 20x
VENTANA MMR RxDx Panel for Endometrial Carcinoma 13
Proficient Case 2: MLH1, PMS2, MSH2 and MSH6 Intact Status in endometrial carcinoma exhibiting strong nuclear staining
in tumor cells in the presence of appropriately stained internal controls. The acceptable Negative Reagent Controls are mouse NRC
(mNRC) and rabbit NRC (rNRC).
H&E 20x
mNRC 20x rNRC 20x
MLH1 20x PMS2 20x
MSH2 20x MSH6 20x
14 VENTANA MMR RxDx Panel for Endometrial Carcinoma
Deficient Case 1: MLH1 and PMS2 Loss Status in endometrial carcinoma exhibiting no nuclear staining in tumor cells in the
presence of appropriately stained internal controls. MSH2 and MSH6 exhibit Intact Status. Cytoplasmic staining is disregarded for
interpretation of status. The acceptable Negative Reagent Controls are mouse NRC (mNRC) and rabbit NRC (rNRC).
Deficient Cases
H&E 20x
mNRC 20x rNRC 20x
MLH1 20x PMS2 20x
MSH2 20x MSH6 20x
VENTANA MMR RxDx Panel for Endometrial Carcinoma 15
Deficient Case 2: MLH1 and PMS2 Loss Status in endometrial carcinoma exhibiting no nuclear staining in tumor cells in the
presence of appropriately stained internal controls. MSH2 and MSH6 exhibit Intact Status. Cytoplasmic staining is disregarded for
interpretation of status. The acceptable Negative Reagent Controls are mouse NRC (mNRC) and rabbit NRC (rNRC).
H&E 20x
mNRC 20x rNRC 20x
MLH1 20x PMS2 20x
MSH2 20x MSH6 20x
16 VENTANA MMR RxDx Panel for Endometrial Carcinoma
Deficient Case 3: MSH2 and MSH6 Loss Status in endometrial carcinoma exhibiting no nuclear staining in tumor cells in the
presence of appropriately stained internal controls. MLH1 and PMS2 exhibit Intact Status. Cytoplasmic staining is disregarded for
interpretation of status. The acceptable Negative Reagent Controls are mouse NRC (mNRC) and rabbit NRC (rNRC).
H&E 20x
mNRC 20x rNRC 20x
MLH1 20x PMS2 20x
MSH2 20x MSH6 20x
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Roche BenchMark ULTRA Interpretation Guide

Type
Interpretation Guide
This manual is also suitable for

Roche BenchMark ULTRA, your newly acquired laboratory companion, offers state-of-the-art automated staining for histology and cytology samples. Experience efficiency like never before with its continuous loading capability, allowing you to load slides while the instrument is running. ULTRA's flexible design accommodates a wide range of staining protocols, including special stains and rapid staining options. Get ready to enhance your laboratory workflow and produce high-quality stained slides consistently.

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