CME Slides Forum - M. J. Mihatsch

WHAT CAN THE CLINICIAN LEARN FROM PROTOCOL BIOPSIES?

Michael J. Mihatsch, Basel, Switzerland

Chair: Josep M. Grinyo, Barcelona, Spain

Mehmet Sever, Istanbul, Turkey

Dr M.J. Mihatsch
Institute for Patholopgy
University Hospital Basel
Basel, Switzerland

So, Dear Chairman, Ladies and Gentlemen I can tell you what I hate, I hate to give a talk at 8 o’clock in the morning.

What we will do is we will discuss the significance of protocol biopsies and the first question is what would you like to see in a transplant biopsy at any time after transplantation? Well, I think you all agree that you would like to see no relevant pathology.

Well, I’ll show you such an example 10 years after transplantation and you see this biopsy here and this shows no relevant pathology in the medulla nor in the renal cortex.

Well, what is no relevant pathology? No relevant pathology are morphological findings in a protocol biopsy minus morphological findings in a zero hour biopsy.

This means that after transplantation no pathology developed. But what is normal in zero or implantation biopsies? Well, normal maybe defined as biopsies showing less than 10% of obsolescent glomeruli. Arteriolar hyalinosis which is slight. Tubular atrophy and interstitial fibrosis which is minimal. Intimal fibrosis in arteries which is also minimal but no segmental glomerular lesions, unspecific, no diffuse fibrosis, no interstitial inflammation.

So, this gives me the opportunity to introduce some of these morphologic lesions and one of the most important is arteriolar hyalinosis. Well, what you see here in unspecific arteriolar hyalinosis which is brought about by hypertension or diabetes, you see that the protein deposits are situated underneath the endothelium but the most important point is that the smooth muscle cells all around are well preserved.
On the upper left panel you see intimal fibrosis which maybe found even in implantation biopsies and adjacent to it you have inflammatory infiltrates which can easily be mistaken for borderline lesions in protocol biopsies and on the lower left panel you have interstitial fibrosis, focal interstitial fibrosis in blue and obsolescent glomeruli.

Well in summary, when you look at the large number of zero hour biopsies only 70-80% of these biopsies show really normal findings and 20-30% show more than normal findings.

Now, we will deal with protocol biopsies and first of all, I will show you the results of unselected cases compared with zero hour biopsies in the indication biopsies.

Well, we selected 560 zero hour biopsies and compared these with 550 protocol biopsies and 510 indication biopsies all performed about at the same time 147 days after transplantation. Well here we have excluded all specific pathology for example, in the protocol biopsies 20 cases of glomerulonephritis, 14 of polyoma virus nephropathy.

The good information is first of all, that arteriolar hyalinosis, unspecific arteriolar hyalinosis which maybe found after hypertension decreases in protocol biopsies.

We found this in 43% of all zero hour biopsies, however only in 23 % in protocol biopsies and in indication biopsies also in 26%. The bad information however, is that focal interstitial fibrosis which was already present in 24 % of zero hour biopsies dramatically increased to 50% in protocol biopsies and it was the same in indication biopsies.
Well, focal interstitial fibrosis of a more severe degree increased from 9.2% to 24.3% was very similar in indication biopsies. Diffuse fibrosis and subcapsular scars are of little significance.

Well, the bad information is that 12% of all patients or of all biopsies, protocol biopsies shows a calcineurin inhibitor arteriolopathy, the figure was 20.7% in indication biopsies. Glomerular lesions and tubular lesions are of less significance.

When we compare now protocol biopsies after 3 months and after 6 months, we see that there is still a further increase of focal interstitial fibrosis and this is even more dramatic when we look at the more severe forms of interstitial fibrosis which increased from 3 months with 11.7% to 32.9% after 6 months.

While with respect to the calcineurin inhibitor lesions there were no significant differences between 3-6 months after transplantation.

So, the most important lesion found in protocol biopsies is focal interstitial fibrosis and tubular atrophy which are the driving forces for these lesions. The driving forces are arteriolar hyalinosis and calcineurin inhibitor arteriolopathy which result in glomerular obsolescence and on the other hand, transplant vasculopathy in arteries.

Just to show you the difference between unspecific arteriolar hyalinosis and calcineurin inhibitor arteriolopathy. This is unspecific arteriolar hyalinosis I have shown already. Here you see for comparison calcineurin inhibitor arteriolopathy and the difference is that the protein deposits are situated in place of the smooth muscle cells. So the smooth muscle cells have undergone necrosis and this is the major difference between both. The lesions in arteries are shown here.

This is a classic lesion due to rejection in arteries where you have a thickening of the intima with more or less in intimal infiltrates and a fibrosis but without elastosis. This is quite different from what you can see in zero-hour biopsies as an unspecific intimal fibrosis which holds always elastosis.

So focal interstitial fibrosis and tubular atrophy are brought about by a reduction in blood flow. So the tubular interstitial space depends on the integrity of the vascular tree. As soon as there’s a narrowing of the arteries, as soon as there is a narrowing of the arterioles in case there’s obsolescence of glomeruli the blood flow in the tubulointerstitial space is reduced and the consequence is tubular atrophy and interstitial fibrosis.

Let me summarise what we found concerning the unspecific lesions. The most vulnerable phase is the first 3 months after transplantation with respect to unspecific lesions, calcineurin inhibitors related lesions significantly increase within the first 3 months. Interstitial fibrosis and tubular atrophy persistently increase up to 6 months and even later but all lesions are slight in the beginning. Severe arteriolar hyalinosis and calcineurin inhibitor arteriolopathy may call for therapeutic consequences.

Now, let me come to the rejection related lesions as seen in protocol biopsies. On the left hand side you see a classic picture of interstitial cellular rejection while you have interstitial oedema, you have massive infiltrates and here the hallmark of interstitial cellular rejection is tubulitis where you can find mononuclear infiltrates in the tubules.

But in other cases you have minor lesions with focal interstitial infiltrates with or without tubulitis. While in some cases and not in all, there maybe a marker of the activity of the rejection process to be seen and this is HLA-DR expression in the tubular epithelium.

When we compare protocol biopsies and indication biopsies, we see that all the lesions which are found in indication biopsies borderline lesions, acute rejection including tubulointerstitial infiltrates, vascular lesions, transplant glomerulitis they are also present in protocol biopsies, however with a lower prevalence especially the more severe forms of rejection. C4 d deposition was found in 5% in protocol biopsies and 9% in indication biopsies. The indicator of an active rejection process HLA-DR expression in the tubular epithelium was present in 16% in protocol biopsies and in 38.6% in indication biopsies.

Furthermore, the lesions related to chronic rejection, transplant glomerulopathy is not significant but already 11% of protocol biopsies showed signs of chronic lesions such as transplant vasculopathy and this was in the same order as in indication biopsies.

Well, you can see here the rejection score and just arbitrary score was slightly was much more severe in indication biopsies. Among all the protocol biopsies 44% showed no rejection at all, 30% in the indication group.

But now when we compare protocol biopsies after 3 months and after 6 months, you see that there’s no significant difference at all, so there’s no further progression of rejection related lesions between month 3 and month 6 and when you look at protocol biopsies after 1 year there is even a decrease to be seen.

Well, also with respect to the chronic lesions there is no difference between month 3 and month 6 although you see there’s an increases in transplant vasculopathy but this difference is not significant.

Well, when we summarised what we found concerning rejection we can say in protocol biopsies the same lesions are present as in indication biopsies but less severe, so there’s nothing special in protocol biopsies with respect to rejection. Borderline lesions and acute rejection develop in about 50% of the patients within the first 3 months and do not increase thereafter.

Well, in the second part we will look at patients with repeat biopsies, so patients who had two biopsies after 3 and 6 months.

These patients come mainly from Basle and we analysed here 333 biopsies coming from 144 patients.

When you look here at the acute rejection borderline lesions, you see again also in this sub sample there’s no significant difference between month 3 and month 6. However there’s a clear cut difference with respect to the indication biopsies which show quite more severe rejection.

Interestingly, with respect to the HLA-DR positivity no differences were found, this means that active signs of rejection were very common also in protocol biopsies.

However, there was a difference for C4 d positivity in peritubular capillaries was much more common in indication biopsies than in protocol biopsies.

However, there was a clear cut increase form 4% to 10% nearly for C4d between month 3 and month 6 in protocol biopsies.
When we compared these 2 time points, we found no rejection at all after 3 and 6 months in roughly 1/3 of all patients. No rejection was present in the biopsy after 3 months in roughly 50% and no rejection was present after 6 months in roughly 50%. There was an increase in rejection in also 1/3 of the patients and an improvement in 25% no change in 7%.

So, what we see here is that this rejection process in the kidney transplant is a dynamic process and when we perform a protocol biopsy we do not really know whether we are on the increasing side of this rejection process, whether we are on the top or whether we are on the decreasing side of this process.

So let me summarise. Borderline or acute rejections are present in about 50% of protocol biopsies.
About 30% of biopsies show increase in rejection and improvement of rejection respectively independent of therapy which I have not shown. About 30% of protocol biopsies show neither rejection after 3 or 6 months.

What is the functional impact of rejection processes or other lesions in protocol biopsies?

Well, here I show you the baseline data. Baseline was the lowest in S-creatinine within the first month after transplantation. There was no significant difference after 3 months, 6 months and 9 months and you see this here in the delta creatinine as well. I will show you only one slide here because no differences were found for all subgroups we analysed.

When we compared patients with clear cut Banff classification I or II, treated and not treated then no significant difference was found with respect to the s-creatinine.

From this point of view I can summarise morphological lesions in protocol biopsies have on the one hand no impact on renal function, this is a definition. Therapy of rejection has no impact on renal function within the following 3 months.

Well, the answer is no. Here we looked at three different groups of patients. Patients having no rejection at all after 3 and 6 months and only 5% of these patients had in later indication biopsies rejection. While here in this group in one of the 2 protocol biopsies signs of rejection were present. 25% of these patients developed later overt rejection in indication biopsies and had also clinically overt rejection. In this group here in both biopsies after 3 and 6 months signs of rejection were present and 35% of these patients developed later overt clinical rejection. So these patients showing rejection in 1 or in 2 biopsies are at a higher risk of developing overt clinical rejection. On the other hand, even in this group 65% of the patients did not.

So, what we do not know is why one patient has a decrease in rejection spontaneously and the other patient a increase so that overt clinical rejection is finally present.

Let me summarise this part. Patients with signs of rejection in protocol biopsies are at a higher risk to develop overt clinical rejection. Signs of rejection in 1 or 2 biopsies increase the risk for overt clinical rejection. 3.5 and 5 times respectively and the selection of patients for therapy however needs further studies.

In conclusion, we need more and better tools to identify patients at risk of developing overt clinical rejection since the goal is to avoid both over and under treatment. On the basis however and this is well documented in the literature, on the basis of all published literature both unspecific and rejection related lesions significantly influence the long-term graft survival.

Well, this review by Doctor Seron and Moreso in Kidney International is strongly recommended for this topic of protocol biopsies in renal transplantation. Thank you very much.

Chairman: Thank you very much Doctor Mihatsch for this very nice comprehensive talk. Now are there any questions or comments from the floor?

Question: Doctor Mihatsch your data concerning the lack of benefit of the treatment in cases of subclinical rejection is quite similar to that recently reported by --- but in his experience the incidence of subclinical rejection is much lower than in your material, I think it was around 5% in patients treated with tacrolimus and MMF.

Dr. Mihatsch: Well yes 95% of all patients had calcineurin inhibitors and well, some cyclosporine others tacrolimus. Well, I think it is clear and when we look back let’s say 10 years or even 20 years, especially when you belong to the dimer source of transplant pathology like me you know, we have developed over the last 20 years better and better immunosuppressive protocols and what we have experienced and which is documented in all studies is that we have less and less acute rejection episodes within the first year after transplantation. Well, this makes it even so difficult in clinical trials to find a good end point for the clinical trial because 20 years ago it was simple because you had a rejection episode, you could count rejection episodes however, when you compare to today clinical therapeutic regimen well the acute rejection episodes are rare in both arms, in the control arm as well as in the experimental arm. So it may be necessary to develop something better especially some better markers for the activity of a renal rejection process even in a protocol biopsy. In case we have better markers it would be possible also to better select patients for therapy.

Chairman: Ok. Would you please go to the microphone and identify yourself. I see one at the rear at the back.

Question: Mr Holmberg from Helsinki. I was just wondering when you showed us that there was no progression of the lesion between 3 and 6 months in your protocol biopsies. When you did protocol biopsies did it not influence the treatment of your patients or did you just do it for later studies?

Dr. Mihatsch: Yes, well in Basle the clinician decides himself whether he wants to treat or not. When we compared both protocol biopsies after 3 and 6 months, some were treated and some were not treated and also in patients in which there was no therapy at all the lesion vanished after 6 months.

Question: Karovitch from France. Thank you very much for your nice lecture. Just could you advise us how many protocol biopsies should we perform during the first year? 0, 1, 20 or 100?

Dr. Mihatsch: The best would be every other day you know. Well, look I say this here especially in Sweden because while very close in Finland – developed this – technique and I think we have to read again all these publications from this time. Well, it’s probably 20 years ago. What we learnt at that time is that in all kidney transplants there is an influx of cells you know and these cells come and these cells go and in some patients overt rejection and I thank you very much for your question because I’ve been thinking now for more than a year about how many protocol biopsies should really be done. What I would recommend today is after 1 and after 6 months would be a good idea. But there are as many different protocols as you want performed in different centres, 1 month, 3 months, 6 months, 12 months and so on. But 1 month would be a good time point and 6 months would be also a good time point in my mind.