PATHOLOGY/CORRELATIVE STUDIES SECTIONS - MARKERS: WHAT WE KNOW AND DON'T KNOW by Adi Gazdar, MD

SLIDES & TRANSCRIPTS
Tuesday, June 19

PATHOLOGY/CORRELATIVE STUDIES SECTIONS - MARKERS: WHAT WE KNOW AND DON'T KNOW

Adi Gazdar, MD
Presented by John Minna, MD

Slide 1: Introduction

You were expecting maybe Adi Gazdar, but unfortunately he has uveitis, which is severe, and I understand involves both eyes. So he has left me with something of a difficult job here. He sent me the slides, but he didn't give me any text. So we are going to do a little bit of winging it. In going over these slides, the original title of this talk, is "Markers, What We Know and What We Don't Know." As it turns out, Adi's part is going to be what we know. Then I have a slide at the end about what we don't know, which I am an expert on. And that actually will just represent a vast ocean of lack of information.
So the first set of slides that Adi wanted to talk about referred to the atypical adenomatous bronchoalveolar carcinoma sequence.

Slide 2: Atypical

I wanted to make the point that it's very important to visualize these images, as we have seen lots of examples of this high technology, and to correlate the imaging data with the pathology. This is a case where this has been done. This is a case of atypical adenomatous hyperplasia. I presume this is a ground glass lesion here.

Slide 3: Peripheral Lung Adenocarcinomas

And then when you look at this under the microscope, you see that it lies at the beginning of the carcinogenesis sequence. It's characterized by the atypia of the cells lining the alveolar spaces in the so-called lipidic pattern, where they do not elicit a stromal response in the alveolar septi. This presumably progresses, perhaps not in the same patient, but at least over large populations, to bronchoalveolar carcinoma, which also grows in a lipidic pattern, but has many cytological features that are more advanced than the atypical adenomatous hyperplasia.This further advances into an invasive carcinoma, which is characterized by the formation of stromal tissues. This is actually a key feature for prognosis, as Dr. Shimosato has already discussed.

Slide 4: Atypical Adenomatous Hyperplasia

We have seen examples where these are well-circumscribed lesions, again, rather minimal atypia, which is visible not only on the histologic section, but also on the cytology. I won't spend a lot of time on the morphology here.

Slide 5: AAH

But one can see from the discussions earlier on that this is not always a straightforward diagnostic distinction between these various entities, and there is always some hope that we may get some additional information out of molecular studies. So the molecular studies that have been done on atypical adenomatous hyperplasia are these. Some of these are immunohistochemical assays, a proliferative index, which is largely measured by Ki-67. This is increased, but at a relatively minimal level in the cases in which it's been examined. The Ki-67 proliferative index is about 3%, whereas in invasive tumors it's on average, about 15%. The cyclin D1 gene is overexpressed in these tumors. There is CEA expression in the atypical adenomatous change. There is a fairly frequent loss of 3P and 9P. There is often, as we already mentioned, a K-ras mutation, and this varies from 15-40%, depending on the center that is doing the evaluation. P53 overexpression is visible at a protein level by immunohistochemistry, but rarely is it the massive overexpression that one sees in association with mutations. In fact, mutations in atypical adenomatous hyperplasia are rare, as is loss of retinoblastoma gene P16, and activation of telomerase. So there are some molecular correlates. I think all these point out the relatively benign nature of this process in relationship to invasive carcinoma, where we have many more abnormalities of molecular markers.

Slide 6: AAH

Now the question then might arise, "is this really a kind of reactive change or not?" One of the ways that we have to address this is the Humara assay, which actually measures monoclonality. And from a series of 10 informative cases of atypical adenomatous hyperplasia, and 7 corresponding bronchoalveolar carcinomas cases from 5 patients, all were monoclonal, and the contiguous lesions had the same abnormalities. So we think that this is a progression of the same clonal lesion.

Slide 7: AAH and BAC

. Aneuploidy can occur in atypical adenomatous hyperplasia. Again, it is at a lower level than in bronchoalveolar carcinoma. It's less frequent, and the degree of aneuploidy is usually not as severe as you see in invasive cancer.

Slide 8: Tuberous Sclerosis

There are some genetic associations with this process, or at least a similar process. Tuberous sclerosis is associated with a lesion, and I have to refer to my notes, as a multi-focal micronodular pneumocyte hyperplasia, or MNPH. This is the part Adi needed to be here for.
But anyway, this is a lesion that is associated with mutations in two genes of the tuberous sclerosis complex, located on chromosome 9q and 16q. There is often loss of heterozygosity, or mutations in lymphoangioleiomyomatosis, another disease which occurs in association with tuberous sclerosis. And frequently there is loss at these loci in sporadic type bronchoalveolar carcinoma and atypical adenomatous hyperplasia. So we think that this may be in some way associated with the predisposition to develop these two lesions.

Slide 9: AAH

So all this molecular data tends to support the morphological findings, and to point to the idea that atypical adenomatous hyperplasia is indeed a true neoplastic process, and it may in fact be a precursor lesion for non-mucinous bronchoalveolar carcinoma.

Slide 10: Markers

So what markers do we have that might be relevant to the clinical management of these individuals? Well, here Adi diverges a bit, and he's talking about the markers that he has done primarily on individuals who are smokers. Many of them are cancer-free. So we have a list of surrogate markers that we think might be useful in following these patients, at least for risk, if not for diagnostic purposes. So there are several possible specimens that might be tested for these surrogate tissues that might be tested for molecular markers. One of these is oral pharyngeal brushings. From the central lung we can get sputum and bronchial brushings, and from the peripheral lung, BAL.

Slide 11: Study

In this population of smokers, and these were heavy smokers with at least 30 pack years, Adi evaluated various methylation markers, including the RAR-beta, the H-cadherin P16, and the ras SF1A.

Slide 12: Aberrant Methylation

And when you look at the combined data here, you see that there is frequent methylation at all these sites, with the most frequent being the retinoic acid receptor promoter. But there was relatively low levels of methylation in the smoking population for all of these methylation markers.

Slide 13: Promoter Methylation

It's of interest, and I think this represents the same patient, that these markers may not be the same for each different surrogate tissue. In this particular patient the oral pharynx was negative, sputum was positive for P16 and RAR-beta, and on down the line. So it seems that there are multiple clones that exist in the airways, that may not actually represent a single progenitor population, but rather a field cancerization effect.

TOP

Slide 14: Results

So the overall results were that there was a highest frequency again for RAR-beta. That at least one gene is methylated in 47% of this high risk smoking population. This is a lower incidence than you see in tumors, and these methylation changes were more frequent in patients in the central airway than in the peripheral airway specimens. Then finally, the results from sputum and from bronchial brushes were comparable. So sputum then becomes a potentially useful marker for further following this patient population for risk, and for possibly diagnosis later on down the line.

Slide 15: Specimens for Testing

So here is the list of specimens that Adi recommends that we collect in future studies for early detection of AAH and bronchoalveolar carcinoma. One would be the excision specimens and fine needle aspiration material. And this can be saved preferably fresh or frozen. These specimens will also as a matter of course, be available in paraffin blocks, and also in alcohol, where RNA is fairly readily extractable. BALs should be collected when this is feasible. And possibly these large trials should be collecting accessible sputum that do not require intervention, such as sputum, bronchial biopsies, which can be obtained from diagnostic procedures, and then bronchial brushes as well.

Slide 16: Molecular Tests

And the tests in favor at the moment would include morphometry and ploidy, loss of heterozygosity, the presence or absence of ras mutations, immunostains for these various markers, and evaluation of the methylation status of multiple genes.

Slide 17: Markers

Now this is a list of markers that are currently available, that we know something about. But there are lots that we don't know. I would like to refer Ruth Katz's discussion of molecular cytogenetics. Here it is possible to get information about atypical adenomatous hyperplasia. I think the risk for development of invasive cancer at an in situ level, and I think we can do this with the molecular probes that are available. We can look for a chromosome copy number, which is an indication of chromosomal instability.I think that these lesions are genetically simpler than invasive carcinomas. So my hope is that we will be able to look at these lesions for evidence of translocations. Now there is a recent paper by Adi himself on a translocation involving chromosome 19 that affects the notch gene. This patient is a person who had adenocarcinoma. I think this is an example of these relatively simple abnormalities that we are likely to find in these minimally aneuploid tumors that perhaps make techniques such as sky FISH for looking at detailed chromosomal changes more interesting than it is in these invasive tumors, where the karyotype is very highly abnormal. The other thing that I think will be of interest is to evaluate global patterns of gene expression. And I think this is going to be a challenge, because these lesions are small. It's difficult to harvest enough RNA, and this is especially complicated by the fact that it is now perceived that if you have a 5-millimeter area of fibrosis, then your chances of getting metastases are considerably increased. So pathologists are going to want to evaluate as much tissue as possible, and it's going to limit the amount of material that is available for RNA analysis. This is something that I think we need to talk about. I think another issue that needs to be brought up is, is atypical adenomatous hyperplasia really a consequence of field change? And is it possible that just by removing one of these lesions, we will have no effect on the long-term outcome of these individuals? As a corollary, are there molecular targets that could effectively target these AAH lesions so that these patients who possibly could progress even if a single lesion is removed, could they be offered some chemotherapeutic agent to negate the field effect? Then finally, a particularly timely issue, since this meeting actually conflicts with an animal model workshop that is going on in Boston tomorrow, what animal models would be relevant to the development of atypical adenomatous hyperplasia and bronchoalveolar carcinoma? My impression from looking at the few animal models that I have been able to evaluate is that mouse adenocarcinomas tend to be very morphologically similar to these peripheral adenocarcinomas, more so than the central, highly aneuploid and very aggressive tumors that we see in the human population. So I think that's where we are at. I guess we'll be talking about more possibilities for future studies in the discussion period. So I think we will end here, and turn the floor over the surgeons. Thank you very much.