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Evolution of Biomarker Validation

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Most recently, an international team of experts from the International Society for Immunohistochemistry and Molecular Morphology (ISIMM) as well as the International Network for Quality in Pathology (IQN Path) developed new concepts and a new framework for biomarker validation in immunohistochemistry (IHC). Validation is ‘‘confirmation, through the provision of objective evidence, that requirements for a specific intended use or application have been fulfilled’’ (ISO 9000).“Intended use” or “intended application” are synonymous with “purpose” meaning that validation will result in a test that is fit-for-purpose. Provision of “objective evidence” is fulfilled by collecting evidence that relevant test performance characteristics have been identified and assessed. Validation provides a “high degree of assurance” that these needs will be met. This presentation will explore how these three components of validation (intended use, objective evidence, and high degree of assurance) can be incorporated into the validation of biomarkers for in-situ cell-based assays such as IHC.

Learning Objectives:

  1. Overview of terminology and definitions relevant to biomarker validation for in situ, cell-based assays.
  2. Describe components and processes for IHC biomarker validation.
  3. Describe tools required for biomarker validation.

Webinar Transcription

In this presentation, we will briefly review the terminology and definitions that are critical for conceptualizing and conducting validation of biomarkers, as applied to cell-based in situ environments, such as immunohistochemistry.

     Validation of immunohistochemistry biomarkers has several important components and processes.  They will be described here today.

     Finally, as we all know, any trade requires good tools.  We will briefly review the different possible choices that are currently available when choosing the right tools for immunohistochemistry biomarker validation.

I will use this slide to illustrate why immunohistochemistry is not a quantitative assay, but rather, a descriptive assay.

The fundamental design of the immunohistochemistry reaction is shown in this diagram.  What we see is that immunohistochemistry is an in situ immunoassay.  Their specific primary antibody binds to its target isotype or antigen, if you will, on an unstained section.

The word “immunohistochemistry” has three components.  “Immuno”, referring to antibody-antigen reaction; “histo”, when histology tissue sections have been used; and “chemistry”, which consists of developing the chemical reaction at the end of the process.

In most immunohistochemistry tests, we would not be able to see any signals coming from the primary antibody without using amplification. 

Amplification means that once the reaction between the primary antibody and antigen have occurred, we make it visible by light microscopy using various molecules that are bound to the secondary antibody, as depicted here.

Therefore, the chromatin that will develop at the site of the reaction is not directly linked to the primary antibody.  This can be achieved in several ways.

Avidin-biotin-based systems were popular in the past.  But the fact that human tissue contains endogenous biotin was problematic.  Other systems were developed in order to increase the specificity of the reaction so that we do not get false-positive results in cells that contain endogenous biotin.

Today, there are several different types of amplification systems, also called detection systems, which are based on synthetic polymers and monomers.Such polymers do not exist in human tissues. 

And because of that, it is easy to ensure that our immunohistochemistry reactions will be specific, and that the brown signal deposited in the tissue really reflects through specific binding of the primary antibody, which started antigen in the cells.

Once the brown, or let’s say some other signal, but let’s refer to DAB because it’s the most commonly used brown signal in this type of reaction.  It can be further emphasized and made darker.  This step is called enhancement.  And it’s used at the end of immunohistochemistry reaction.   

Many times, enhancement is achieved by using copper sulfate.  In the past, we used an osmium-base compound.  And now, we have the amplification step that occurs after the reaction between the primary antibody and the target antigen.  Immunohistochemistry is, in some ways, actually very similar to basic PCR reaction.

The purpose of this very lengthy description of an immunohistochemistry assay is to emphasize that the intensity of signal at the end of the immunohistochemistry assay, most of the time, does not reflect the amount of antigen present in the cells.

The intensity of the signal definitely relates, in one way or another, to the amount of expressed protein.  But it is only one of the variables.  And how much it contributes to the final staining result is, most of the time, unknown.  And it is not intuitive.

The second component is the composition of the amplification system.  Amplification may have one or more steps.  More steps will lead to more amplification.

After that, the next component is the time we allow for the chromatin to develop.  If this time is very short, then the intensity may be weaker than if we allow several minutes for this process to occur.

Finally, we can significantly change the intensity of signal if we add an enhancement with copper sulfate, which will make the DAB appear very dark brown or almost black.

We have now established that the immunohistochemistry assays generate results that lack proportionality to the amount of protein that we are detecting in this assay.

Therefore, we can say with certainty that immunohistochemistry is a descriptive assay, because the definition of a descriptive assay is that “They generate practical data that lack proportionality to the amount of analyte in a sample.”

Immunohistochemistry is a cell-based, in situ immunological assay.  But let us remember that it is not the only immunological assay that is cell-based.  Flow cytometry is cell-based, but it is not in situ.  ELISA is an immunological assay, but it’s neither cell-based or in situ.

Some in situ assays are not immunological.  For example, DNA and RNA in situ hybridizations are cell-based and in situ, but are not immunological in nature.

This image illustrates that even if you could somehow make immunohistochemistry more quantitative, meaning that a test would be more proportional to the amount of expressed protein.  There is a descriptive component that we cannot remove from immunohistochemistry assay.

Immunohistochemistry is always descriptive, because pathologists interpret cells as positive only if the signal localization is appropriate. 

This Hodgkin lymphoma shows that the Hodgkin cells are negative for BCL6 and that many cells in the - - are positive for BCL6.  We do not use any type of points for this readout.  We simply look at Hodgkin’s cells, which we can recognize by size and shape, and read out absence of brown signals in all nuclei of the malignant cells.

However, readouts would not be any more correct if we were to use any quote, unquote “cutoff” for the number of positive cells, even if all cells in the background are positive, which may account for 90% of cells.  We still read this result of negative since the signal is not where it should be.

This in situ nature of immunohistochemistry assay should never be neglected.

Let’s go back to enhancement and examine what it does to immunohistochemistry results.  Question for the audience.  Do you use enhancements of the immunohistochemistry reaction at the end of your protocol? 

Do you know that intensity of staining in general, on average, could be approximately double of what it would be without the enhancement?

The immunohistochemistry protocol on the left was performed without enhancement.  The one on the right did use enhancement.  There’s the same amount of protein in both sections.  But the signal is much darker on the right than on the left.  

We have to pause for a moment and contemplate what this means if we were to use image analysis to measure the intensity of signal.  Would our image analysis results have anything to do with the actual protein content in this tissue?

Clearly, enhancement is not the only factor that can cause misleading results with image analysis if we would compare the results from two different laboratories using different protocols. 

Unfortunately, there are also parameters that also interfere with image analysis, but this is beyond the scope of this presentation.

Let us conclude now that immunohistochemistry test is not a quantitative test, and that the sensitivity of immunohistochemistry does correlate well with intensity of signal since intensity is dependent on many factors, other than the amount of protein.

Another question for the audience: which chromogranin test shows higher analytical sensitivity?  This image is taken from the NordiQC website.  They have many useful and illustrative images.

This is pancreas.  And we see that islets of Langerhans stained stronger on the right than on the left.  Do not think that this is a trick question.  It is not. 

If you are not sure and you answer with anybody, please feel free to let your mind go.  In other words, which test would you prefer to have in your lab?

The answer to this question lies not in the islets of Langerhans, but elsewhere.  Instead of focusing on the Islets, we must look to what is happening in tissues that we know to have a low level of expression of chromogranin.

The cross section of the appendix depicted here in row two, shows accents, which are demonstrated as positive on the left but a negative on the right. 

Similarly, this fully differentiated neuroendocrine tumor with a low expression of chromogranin that is depicted in the bottom row, shows positivity on the left.  And it’s negative on the right.

Therefore, the immunohistochemistry protocol on the left is more sensitive, despite being lighter in signal intensity, and despite the fact that both protocols use the same primary antibody.

How is this possible?  It is possible because these two laboratories use differently designed immunohistochemistry protocols. 

This image helps to reinforce a statement about proportionality between the analyte and the results we achieved with an immunoassay. 

This is a comparison between immunofluorescence and immunohistochemistry.  It is theoretically much easier to develop an immunofluorescence immunoassay that is linear, than to develop an immunohistochemistry assay that is even partially linear.

We have mentioned that qualitative assays are proportional.  Proportional and linear relationships both create a straight line on a graph.  Proportional assays start at 0, while a linear assay may start at +1, +2, +5, or anything else on the y-axis.

Linearity means that if there is more antigen in the tissue, then there will be more intense signal.  This is not so for immunohistochemistry.  We see that immunohistochemistry is not linear.  And it’s certainly not proportional.

The concern is that we do not know what this curve looks like for any of our clinical immunohistochemistry assays.

In this example, the same lung cancer was stained by the 5A4 clone for ALK. 

These three laboratories that participated in the Canadian immunohistochemistry quality control proficiency statement program achieved a very different intensity of signal, respect of the fact that this is the same tumor, with the same tissue processing, and the same clone.

We wonder which of these tests have the highest diagnostic sensitivity.  In other words, is intensity of signal related to our ability to properly stratify patients for targeted therapy?  Can we say that the most intensely stained sample was tested with the most diagnostically sensitive assay? 

We cannot.  All three tests showed the same diagnostic sensitivity in this proficiency test in realm. All three protocols correctly identified the patients that were positive for ALK, and therefore, eligible for treatment with crizotinib.  This was also confirmed by FISH.

Pathologists typically prefer to see stronger signals that are easier to interpret.  But the reality is that we should not be too harsh, for as the protocols that still perform as they should for clinical practice, even if we may not like how they look.

Papers like this one do not do any justice to immunohistochemistry.  This is not the only paper that states that immunohistochemistry may not be so good over tests for precision medicine. 

Most such publications use arguments that immunohistochemistry does not measure precisely the amount of expressed protein and that this is a problem.  Let us examine this more and see just how big of a problem it really is.

We are looking at an immunohistochemistry slide of a patient with acute myeloid leukemia.  The immuno-histochemistry test is empty in one.  As a hemato-pathologist, I’m only interested in whether I can see signal.

Why?  Because normal non-mutated nucleophosmin is 100% in the nucleus, while mutated NPM1 is not so efficient between - - in the nucleus.  And it’s also present in the cytoplasm. 

We can see that in this case, the cytoplasm is staining.  And therefore, we can conclude that antiem-1 is mutated.  The test is validated to work like this.  If we performed DNA sequencing for the presence of this mutation, we would get the same results.

Do I want to know exactly how much of the mutated protein is there?  No, I do not.  Clinically, it makes no difference whether the amount of protein is many times more or less.  In this validated assay, it only matters that the presence of cytoplasmic staining correlates 100% with mutated NPM1.  No measurement.  No matter.

In the case of lung cancer with an ALK translocation and the AML patient with NPM1 mutation, the exact intensity of signal does not determine whether we are getting the correct result. 

In lung cancer, we would stratify the patients properly for crizotinib, in respect of the intensity of staining.

And in AML, the patients with an NPM1 mutation do better than those without.  We would still stratify the patients for appropriate therapeutic considerations based on the under-presence or absence of signal in the cytoplasm, without any measurement of the intensity of signal.

This paper from 2006 illustrates that a question of quantification in immunohistochemistry is a big one and cannot simply be dismissed. 

Please consider that every qualitative test ultimately has a quantitative component because of the cutoff point, especially at the point where the reaction switches from being negative to being positive. 

This point is essentially quantitative in nature.  However, this does not mean that the test needs to be proportional or linear or that we must measure how much protein is there. 

It only means that it is essential that a proper threshold be achieved between a positive and negative sample.

From the published literature, we know that clinical trials did not show that more intense staining for PD-L1 in lung cancer correlated better with survival of patients with lung cancer.

What the trial showed is that if more than 50% of cells of the tumor are positive, there is a good correlation with positive response to treatment with pembrolizumab.  Different cutoff points, such as 1% cutoff point or other may be applicable to other drugs. 

We come back now to Hodgkin lymphoma and the BCL6 immunohistochemistry test.  I only want to know that Hodgkin’s cells are negative, or at least at most, or many, are negative for BCL6.

Measuring the amount of BCL6 here is similar to measuring the amount of NPM1 in AML and does not make sense. Having lots of NPM1 in the nucleus is irrelevant if it’s not present in the cytoplasm, having lots in the cytoplasm, rather than less, is also relevant.

Therefore, each immunohistochemistry test is based on the biology of the underlying disease, as well as specific applications, which ultimately depend, especially in the area of targeted therapies, on how these markers were used in clinical trials.

I would not recommend that we try to make immunohistochemistry look better and then look in the clinical trials, even if not very good.  The best test for targeted therapy is the one that was used in the clinical trial, in respect of whether we were happy or not happy with these types of tests.

It is important to remember this distinction about what we consider to be quote, unquote “better”.  To develop a test that is better-looking than the one used in the - - regional clinical trials, we can use all sorts of interesting lab tweaks. 

However, to develop a test that is better at predicting which patients will respond to a particular therapy than the one used in the original clinical trial, this would require that we conduct a new clinical trial altogether.

What we consider quote, unquote “better” and easier to work with in pathology and better from a clinical and meaningful perspective, could be completely different.

This image from the NordiQC site illustrates that it is possible to develop an immunohistochemistry assay that represents what is happening at the genetic level.  The breast cancers are both labeled with ISH.

The one on the left shows no amplification of HER-2 gene, but the one on the right does show amplification.  We see how well the intensity of the complete membranous staining correlates in these two examples. 

More amplification of the gene, more protein expression is demonstrated by immunohistochemistry.  Therefore, some immunohistochemistry tests are more linear than others. 

And to keep it so, when that really matters, such as in this example, we need to follow international guidelines, such as ASCO-CAP guidelines, regarding pre-analytical, analytical, and post-analytical component.

However, linearity is not always useful.  Sometimes, it is very difficult or impossible to develop a more linear test without affecting the robustness of the test. 

By this, I mean that when immunohistochemistry tests are finally choose to be linear, they may lose analytical sensitivity for some samples, especially those that had suboptimal, pre-analytical conditions.

The results with HER2 shown on this slide would be very, very difficult to achieve with samples that were sub-optimally fixed.

We are emphasizing here that even when the test is considered to be quantitative, as in this example, they are still basically qualitative, because we need to observe that there is membranous staining, and that the whole cell circumference is positive.

Now it is time to go back to the future of immunohistochemistry. 

This future was introduced by the College of American Pathologists when they published this paper.  Before they published this paper, the term “validation of immunohistochemistry tests” was often used.  But we are not sure how many people had a good handle on what that really meant.

It just happens that by publishing this paper, the CAP did exactly this.  They could open any box on the shelf.  But the one they opened Pandora’s box. 

By this, I mean that by publishing this paper, they opened the floodgates to more questions about validation and about immunohistochemistry as a test that anybody could predict.

There was no way back.  These two questions needed to find their answers.

Some excellent papers on primary antibody validation were published at about the same time, as was this paper from David Rimm’s group.

This is a summary diagram of the primary antibody validation.  Clinical immunohistochemistry labs collectively ignored such papers because there is no way that any normal lab could do this.  There is also no good evidence that clinical laboratories should do this for immunohistochemistry validation.

So what is the difference between this paper and the CAP paper?  The answer may be intuitive to some.  But for the most part, it was not that simple of an answer.

Therefore, an international team of immunohistochemistry experts, especially experts from ETA programs that provide proficiency testing for immunohistochemistry, and have extensive experience in evaluating the results of many clinical laboratories, agreed that there needs to be further qualification of many issues related to validation.

Who knew international organizations emerged out of this need for more oversight regarding biomarkers used for targeted therapy, ISIMM, and IQN Path?  The details about this organization is out of scope of this presentation.  But you could visit their website and read more about them.

Here is a screenshot of the International Society for Immunohistochemistry and Molecular Morphology Web page.  I’d like you to join ISIMM.  There is not much now on the website, because it’s completely new.  But just join. 

It is a major academic forum for immunohistochemistry biomarkers.  It is important and necessary that you do join.  Good things will come from it, and you could be part of it.

Here is the International Quality Network for Pathology Web page.  This is an umbrella organization for EQA programs that develop proficiency testing for biomarkers. 

You cannot join this organization, because EQA programs are the members, not individuals.  There will be lots of good work coming from this organization, too.

These two organizations spearheaded further evolvement of our understanding of biomarker testing and validation principles.  The product of the moment was a series of full-wide papers on the “Evolution of Quality Assurance for Clinical Immunohistochemistry in the Era of Precision Medicine.”

It has been recognized that the ISO 9000 definition of validation also applies to immunohistochemistry testing, any immunohistochemistry test, not just predicting immunohistochemistry tests.

Therefore, we have to completely understand this definition.  And I’m going to read it to you.  Validation is “confirmation, through the provision of objective evidence, that requirements for a specific intended use or application have been fulfilled.”

We need to deconstruct this definition in three parts in order to really understand what it’s trying to tell us.  The first one is intended use, or intended application, being synonymous with purpose.

The second component is that there’s provision of objective evidence.  And that is fulfilled by collecting evidence that the relevant test performer’s characteristics have been identified and addressed. 

And finally, validation process provides a high degree of assurance that these needs will be met.  All of these three components are very important for validation.

So here is a list of the titles.  These papers are now in submission to Applied Immunohistochemistry and Molecular Morphology. 

And as I previously mentioned, this initiative was born out of pressing need to develop QA principals that will fit the purpose for new applications of immunohistochemistry and high expectations of its accuracy. 

Clearly, we cannot say much about these papers here, but please watch for them.  And when they are published, please read them and the similarity principals in your laboratory where appropriate.

Every laboratory test, including every immunohistochemistry test, needs to be developed and moderated for its specific purpose.

However, in all that, how hepatologists using immunohistochemistry, there’s not always a line with the purpose for which a test was developed.

This is frequently a source of frustration and a potential source of false negative or false positive tests.  Papers run from this evolution series emphasizes the importance of test validation, and the importance of aligning tests used to the purpose for which the test was developed.

The test’s purpose is also closely related to who the end user is of the immunohistochemistry test, and what ultimately the results will be used for.

Essentially, immunohistochemistry test results are used either by pathologists for diagnostic purpose, or by treating physician, often an oncologist, for making clinical decisions related to targeted therapy, for follow-up, or for other uses.

Some examples are shown here.  Pathologists often use TTF-1 or cytokeratin 7 for diagnostic classification of metastatic carcinoma of unknown primary region.

Clinicians may use immunohistochemistry tests for various applications as listed in this table.  The most typical uses are those for treatment of ER- PR-positive breast cancer or ALK-positive lung cancer.

When immunohistochemistry tests are used for other purposes for which they were not validated for, it may lead to confusion and/or patient harm. 

This paper shows that when a HER2 immunohistochemistry test that was developed for breast cancer is used for uterine papillary serious carcinoma, it significantly overestimates HER2 amplification.  Therefore, it is not fit for purpose.

Let’s move to even more territory, PD-L1.  There are currently different tests that are developed to detect the same molecule, but for different purposes.

Here is the list.  And here is the wisdom.  Do not use one PD-L1 test instead of another if you do not have proper and strong evidence that your test is performing the same as the companion diagnostic you develop during the clinical trial. 

Why?  Simply because it may not be suitable purpose as shown in the HER2 example.  We also know that we need different HER2 tests with different readout scoring for gastric cancer.  So where HER2 is going this way is not that different from PD-L1.

Different and untypical results are not unique to predictive or type 2 immunohistochemistry tests.  Here’s an example of class 1, or type 1, immunohistochemistry tests, where the CD34 immunohistochemistry test is completely different for different applications, in this case, soft tissue tumors versus bone marrow.

We need higher analytical sensitivity in the bone marrow.  And for the readout, we often count 500 cells in order to determine percent-positive cells, very different than what we do with CD34 in soft tissue tumors.

Now we will remember that validation requires evidence.  This evidence is gathered by demonstrating test performance characteristics.  You may not like the introduction of test performance characteristics in diagnostic immunohistochemistry, because it just complicates the matters. 

True, but TPCs are present whether we know it or not.  Tests will perform one way or the other, whether we pay attention to it or not.  So is it not better, then, if we know what they’re doing?

In my personal view, this paper intends to summarize that successful predictive biomarker qualification is only possible by proper validation.  For this proper validation, we need evidence. 

So this paper defines some of the TPCs that need to be demonstrated for immunohistochemistry testing.

The summary of the proposal is shown here.  Basically, it is reproducibility, sensitivity, and specificity that needs to be demonstrated for immunohistochemistry tests.

This paper also emphasized that immunohistochemistry is indeed a descriptive test.  However, it does not make immunohistochemistry a bad test.  That descriptive test provides nominal results, meaning positive versus negative, or 0 to 3+, or percent-positive cells.

What this means is that a bad lab test is the one that is not properly validated for a specific test.  Any test - -, if properly validated for a specific use, and especially if found to be suitable in clinical trials, is a good test for that use.

As mentioned before, clinical laboratories need to validate immunohistochemistry tests.  Which validation will be used? 

Clinical validation?  No, this is often done in clinical trials.  Diagnostic validation?  No, this is usually done in pathology publications demonstrating the validity of a specific marker or a specific diagnosis. 

Think:  DOG1 as a new marker of GIST, highly sensitive and highly specific marker.  Should we do that type of validation in our clinical immunohistochemistry labs?  Of course not.  We will do technical validation. 

And for this type of validation, we need to demonstrate several test performance characteristics.  Not all of them are applicable to all markers.  Please refer to the upcoming for white papers for details.

We mentioned so far that validation needs to demonstrate that a test is fit for purpose, that there should be evidence for this statement, and finally, that it needs to be demonstrated at a high level of assurance.

This ISO 9000 does not say how high a high level of assurance should be.  Our resources for this definition are professional organizations, such as ASCO and CAP.  We all use their guidelines on required concordance for breast cancer markers.

We could also state now that this level of assurance is likely usable and desirable for all predictive immunohistochemistry markers.

Furthermore, the term “validation” is meaningless if not properly framed.  This term is being tossed around in many situations. 

It is not inappropriate to do so, but for practical reasons, it would work better if we were to define validation characteristics when we use this term.  For example, there’s a huge difference between clinical, diagnostic, and technical validation. 

The part that’s responsible for each of these spheres of validation are completely different.  And also, the methods that we use are completely different.  Therefore, it is good to specify the sphere of validation that we mean when we talk about immunohistochemistry biomarker validation. 

It is also very important to say what is being validated.  Are we planning immunohistochemistry protocol validation?  Is it lot-to-lot validation of different reagents?  Is it primary antibody lot-to-lot validation?  Is it validation of the equipment, or maybe even something else?

Clearly, we want to limit immunohistochemistry validation to technical validation in clinical laboratories.  However, I the lab itself, we are most often concerned with validation of immunohistochemistry protocol, which is just one component of technical validation altogether. 

We should also be concerned with immunohistochemistry readout validation.  Let’s examine this further.

Once we have decided that we are to perform technical validation, we may or may not do totally completely technical validation.  Compete validations would include all possible test performance characteristics that can apply to immunohistochemistry. 

Since this may not be possible, practical, or even necessary, the expert group has identified the terminology for different tiers of technical validation, depending on the number of test performance characteristics that will be included in the validation process.

We see here in the tier 1, which is a basic evaluation, we demonstrate analytical, sensitivity, basic analytical specificity, and analytical reproducibility. 

If it includes two other test performance characteristics.  There, we have to also demonstrate extended analytical specificity, reportable range, and accuracy.

For complete, or tier 3 validation, we also include pre-analytical reproducibility or pre-analytical robust.

This slide illustrates what is usually covered by technical immunohistochemistry validation.  And that is mainly the analytical phase, and rarely the preanalytical phase, if at all.

This is a deficiency that we could potentially overcome if we manage to develop proper tissue tools that will help us perform proper validation for pre-analytical parameters.

The analytical phase of any test is completed once the results are generated.  The immunohistochemistry readout is a part of the analytical phase.  And it also needs to be validated. 

This is particularly emphasized by some of the new  - - for PD-L1 where the readout of the test is so complex, that it takes at least a whole day, and also, continuing practice to first become proficient, and then to keep proficiency for deciding whether 1% or 50% of tumor cells are positive or not.

Depending on the type of readout, the validation may include accuracy of the readout, or both precision and accuracy of the readout.

Accuracy should be sufficient for nominal results, like positive versus negative, but precision is required if percent-positive cells needs to be reported. 

Many times in the clinical laboratory, we face the question of whether we need to revalidate an immunohistochemistry test.  And if yes, then how, exactly, would we do that?

The scope of validation refers to, better yet, performing an initial validation or revalidation.  Once we decide that we are doing the revalidation, we need to consider why we are doing it.

For example, if there was a change in the test performance characteristic, but the labs did not initiate any changes to the immunohistochemistry protocol or readout, we would only need to do secondary revalidation.

Finally, the tools.  Several types of tools are described.  And the choice of which tool to use depends on the evidence that we want to demonstrate.  So there is a link between the tool and the test performance characteristics that need to be defined.

This is a very important and practical paper, because it is showing what tools are necessary for technical validation, and that they need to be prepared in advance of validation.  It shows that most tissue tools are designed as multi-tissue blocks with various types of benign tissues or various types of tumors. 

For details, please refer to the fourth paper when the series is published. 

Today, I only want to mention iCAPCs.  iCAPCs are immunohistochemistry critical assay performance controls.  iCAPCs are relatively easy to make and are very useful as on-site controls for daily testing.

If you use this type of tissue on-site control, you are demonstrating analytical sensitivity, basic analytical specificity, and analytical reproducibility for every immunohistochemistry slide that is run.  iCAPCs are very powerful tools.

We see here also that we can use the TMA design.  It is not critical that you actually have the equipment the TMAs.  Multi-tissue blocks would be sufficient.




     First one is:  Will it be appropriate to use IHC to stain positive section without the enhancement steps to determine the optimal working volumes of monoclonal antibodies for immunofluorescence?

DR. TORLAKOVIC:  That’s a different question, because the question is actually about immunofluorescence rather than immunohistochemistry.  In immunofluorescence, we do not use actual enhancements. 

But if we switch immunohistochemistry—perhaps this was a typo—then for immunohistochemistry, would it be better to use protocols without enhancements when we are developing the protocol in order to determine the optimal primary antibody concentration or conditions of the assay? 

And that is absolutely true.  The enhancement can be used more as a decorative step in immunohistochemistry.  We should be able to develop a readable and good test without enhancement.  And it is up to a local preference whether you want these signals to look darker or not.

They don’t have to necessarily look darker, but you should be able to develop proper signals, even without enhancement.  That’s my answer to that.

Next question:  There are several key challenges in a diagnosis of HER2 for positive breast cancer.  Is pre-analysis issues, which may affect the accuracy of HER2 testing in non-excision sample types, one of these challenges?

DR. TORLAKOVIC:  Absolutely.  Pre-analytical conditions are critical for the success of immunohistochemistry assays. 

If the tissue is not fixed properly or soon enough, if you have long  ischemic time, there is nothing in the world that will put them back and that you will be able to detect them properly. 

So I think that, when we think about pre-analytical conditions, the most important one is that the fixation has to start in a timely manner.  After you’ve started the fixation, also, the fixation has to be controlled regarding the temperature, as well as regarding the time. 

And if you follow the ASCO for guidelines, we will do well.

What happens when the guidelines are not followed and whether our results are then appropriate, are they accurate of the patient’s true status, is another question.

And ASCO guidelines, or any other guidelines, for that matter, will tell you that you can change the fixation conditions if you can actually validate that the result that you will produce by not following exact limitations in the guidelines, are still equivalent to that, that they have very high concordance between then.


How do HER2 testing of core needle biopsies compare with cervical specimens?  Actually, it’s a two-part question here.  And what about retesting HER2 status upon disease recurrence?

DR. TORLAKOVIC:  Well, this is a different question.  That is not a technical question.  And I think that not being the best expert, and not doing this daily and following up, I should not be probably your best person to ask about it.

     I know that in most places, at least in our institution, we do repeat testing.  But whether there is a strict requirement in every institution to do so, to repeat it, that, I’m not sure. 

     There are a number of papers addressing this issue, whether there is a good correlation in the results before an  - -.    And even if there is plenty of—let’s just talk about any marker, whether there will be a correlation between the primary and then biopsy and then follow-up biopsy or metastatic cure.

     There are a number of papers addressing this issue of how the immune-phenotype changes, and so on.  But even the published evidence is not sometimes sufficient to tell whether other oncologists are going to be happy with that, and whether they will still not request us to retest the samples. 

     Though, we do know that in breast cancer, you may get different results in samples in metastatic tumors or a biopsy afterwards.

     But as I said, I’m not your best pathology expert.  And correct me if I’m wrong in any of this.


Next question.  Some antibodies have a dual purpose, such as ER for treatment of breast cancer.  But it can also be used to determine if breast origin is a class I, not II.  And some are used for multi-purpose.  Do you suggest multiple validations for the antibodies for every different purpose?

DR. TORLAKOVIC:  Yes, this is a very, very difficult question.  But it’s a very proper question.  Let me just comment.  I don’t think we use ER so much today as a class 1, because I think we have many other markers than before that would suggest whether there is breast primary region or not.

     The principal of the question is wonderful.  So if I was going to use, for example, ALK1 test for lymphoma, can I use that ALK1 test also for lung cancer?  Do I have to revalidate it?

     And I don’t know if this should be an answer only for type 1, or that was put in the question because there’s a type 1 versus type 2, or if I should answer this generally speaking.

     I think ALK1 example is also good, because we started to use ALK1 as a marker off anaplastic large-cell lymphoma.  And then it switched for other ALK expressions in cancers, especially in lung cancer.

So what happened in the very beginning with this ALK marker, was that the assay that was used for ALK in lymphoma was applied to lung cancer.  And the sensitivity of the assay was about 60 something percent.  And then the immunohistochemistry for ALK was collectively accused of not being sensitive enough.  And FISH was favored.  It is a much better, much more sensitive test.

It turns out that the protocol was not properly validated to be fit for use for lung cancer.  And we know today that by changing this protocols and changing primary antibodies, we now have a different ALK1 test for lung cancer that is virtually 100% sensitive.

So it is important that every marker is validated for a specific use.  And that initial validation may or may not be good for a purpose or may not be good for another purpose.  But you will not know until you test that and demonstrate it that it is the case.

We had this question from two different folks.  Where can we get more information about how to use iCAPCs?

DR. TORLAKOVIC:  Well, the first paper where this concept was published—I believe it was maybe a year or two years ago now—in Applied Immunohistochemistry and Molecular Morphology.  And the title was the “Standardization of Positive Controls in Diagnostic Immunohistochemistry”.

     And you will find there, also another paper that is called “Standardization of Negative Controls”.  I recommend both of them to be read together. 

But this is where this principal of how you combine different tissues to give it a more information about the marker, has been introduced.  And we are now developing it further. So you can read a little bit there. 

But look for future works of ISIMM, because this International Society for Immunohistochemistry and Molecular Morphology will collaborate in the very near future with other professional organizations or international societies in order to develop further iCAPCs for all different types of subspecialty practices.


Next question is:  Do you routinely do validations that include decalcified specimens?

DR. TORLAKOVIC:  Yes.  A pre-analytical component is essential for immunohistochemistry results.  When any test is being validated, it has to be validated for a specific purpose. 

If we are using the test in decalcified samples, then we have to validate the marker to demonstrate that it’s going to work such samples.  So we have to do special validation for all of our bone marrow markers. 

And if you’re going to use any of the other markers in other decal procedures, such as, let’s say, femoral head, and you’re using formic acid for that, there’s probably a limited number of markers that you would be considering, maybe to show that there is metastatic cancer or melanoma. 

Maybe you’re not going to have to validate 150 or 300 markers that you have in your laboratory, but only those that you know that typically could be ordered in such circumstance.  But it has to be validated with the tissue processing that is being used for actual testing.


Is it acceptable to use tissue from outside your laboratory for validation, or should one only use tissue fixed and processed in their own laboratory?

DR. TORLAKOVIC:  That is also an excellent question, because if we say that tissue processing is essential, are we not limited then to our own tissue processing?

     Hopefully, our assays are actually validated for standard tissue processing, which means, again, that results obtained in one laboratory will be equivalent to results obtained in another laboratory.

We use “equivalent tissue processing” or “standard tissue processing” in proficiency testing. 

When we are supplying the tissues for proficiency testing, we are not trying, most of the time, to trick people, or laboratories, to see how they’re going to perform that was fixed one month, or the tissue that was fixed five minutes. 

We are sending the tissues that are standardly fixed.  And then we are judging laboratory success.  It would be very unfair if you would do that, and that had no relevance to your testing. 

So in other words, yes, you can use material from other laboratories if it is equivalent and if it is standard tissue processing, meaning that your formal in formulation is almost identical and that conditions of timing and conditions are identical.

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