Director of Pathology
Peter MacCallum Cancer Centre
This edition of Cancer Forum explores the “Current face of cancer pathology”. Pathology has the unfortunate reputation of being a rather traditional and static subject. The truth is that it is a vast, fascinating and constantly changing discipline.
In the late 1970s, when I was a pre-clinical medical student, university medical faculties and medical students alike understood that pathology was the basis of medicine and, as such, it occupied a major place in medical curricula. It was usual in those days for medical students to have over 240 hours of contact time with pathologists, to spend many hours studying microscope slides and bottled specimens and to witness numerous autopsies. The first edition of Robbins’ Pathological Basis of Pathology, a formidably thick text, described in great detail the clinical and pathological features of all the common and important medical conditions. There is no doubt that this exposure helped develop excellent observational, descriptive and correlative skills and that these contributed substantially to the clinical acumen of the graduates. An enquiring mind, however, was left to speculate about what caused the majority of the common diseases such as cancer. These secrets still lay hidden in the tissues.
By the early 1980s diagnostic anatomical pathology was beginning to change. The routine application of immunohistochemistry to diagnostic tissue sections began to focus the pathologist’s attention on the expression of individual proteins that served as markers of cell lineage. Initially, there were few antibodies available that were useful in a diagnostic setting and many only worked in frozen tissue sections.
Nevertheless, the impact of immunohistochemistry was profound and it quickly replaced electron microscopy, which had revolutionised tumour classification 20 years earlier. Anatomical pathology had made its first tentative steps away from its reliance on morphological interpretation and towards functional analysis. Twenty years later most pathology laboratories now routinely perform immunohistochemistry on paraffin-embedded tissues, using over 30 highly reliable and well-characterised commercially-available antibodies. These enable the vast major categories of diagnostically-challenging tumours to be identified.
In the first article of this Forum, Leong and Leong discuss the latest generation of immunological markers that are being used in cancer pathology. These antibodies cover a wide range of uses from tumour subclassification (eg CD5 and CD23 in low grade B cell lymphomas, E-cadherin in breast cancer, and CD117 in gastrointestinal stromal tumours), predicting the primary site of a metastatic tumour (eg CK7/20, GCDFP-15, TTF-1), identification of specific somatic chromosomal alterations (eg cyclin D1, ALK, FLI-1, WT-1), germline line mutations (eg MLH1, MSH2, and MSH6 in hereditary non-polyposis colon cancer) and the identification of therapeutic targets (eg HER-2 in breast cancer).
In the second article, Cummings discusses a relatively new use of immunohistochemistry – the detection of micrometastatic disease. Studies performed by her and others have exposed some of the limitations of routine surgical pathology practice. By carefully examining axillary lymph nodes from women with breast cancer using antibodies that distinguish tumour cells from normal lymph node cells, they found that approximately 25% of cases that were reported as lymph node negative (N0) actually contain small numbers of tumour cells that were missed by routine sectioning and staining of lymph nodes. The clinical significance of these micrometastases, however, is still unclear. Do they represent passive drainage of tumour cells via the lymphatics that would have be cleared anyway by tumour immunosurveillance mechanisms or do they represent clinically aggressive disease? Should these patients be offered adjuvant therapy? The uncertainty and concern regarding these findings has recently led to a re-defining of the TMN classification for the staging of breast cancer. Pathologists are now required to examine the axillary lymph nodes in more detail and measure all lymph node tumour deposits. It will however take several more years before we will know how best to treat women with these early stage nodal metastases. These findings will also, no doubt, apply to all other types of cancer.
In the third article Field discusses another application of immunohistochemistry – the detection of a therapeutic target (HER-2) in patients with breast cancer. Since the early 1990s pathologists have become accustomed to performing immunohistochemistry for oestrogen (ER) and progesterone receptors (PR) on breast cancers as both prognostic markers and as predictive markers for treatment with tamoxifen. Although this is now part of routine practice, it is disturbing that there are still significant concerns regarding quality assurance and inter-laboratory variability in ER and PR reporting. Similar problems emerged with HER-2 immunostaining, which is now routinely performed in the larger laboratories. It took approximately 10 years from the initial discovery, in 1987, that HER-2 was over-expressed in a subset of breast cancers to the requirement for pathologists to select those patients who are eligible for treatment with the HER-2 inhibitor, Herceptin. Although we have had almost 20 years of experience with immunohistochemistry developing a reproducible HER-2 test remained a significant challenge.
Immunohistochemistry is essentially a qualitative test in which antibodies are selected and the conditions are manipulated to ensure a clear positive or negative result. HER-2 immunostaining presented a new paradigm for immunohistochemistry because it required the test to be quantitative. Although a standardised HER-2 antibody kit (HercepTest) was commercially available it was prohibitively expensive for most diagnostic laboratories which understandably resorted to cheaper in-house tests. Most laboratories now reliably identify patients who are clearly eligible or non-eligible for treatment, however there remains a group where immunohistochemistry is less helpful. Field’s laboratory is able to separate this indeterminate group by using fluorescence in situ hybdridisation (FISH) to semi-quantitate the degree of HER-2 gene amplification. This represents the first application of FISH technology to diagnostic anatomical pathology practice.
In the fourth article I discuss, from a pathologist’s perspective, the recent introduction of another predictive tumour marker, c-kit (CD117). The c-kit burst onto the pathology scene about two years ago following the spectacular success of Glivec in treating patients with malignant gastrointestinal stromal cell tumours (GISTs). Within weeks our laboratory was inundated with requests to perform CD117 immunostaining on a wide range of tumour types. The requests were all initiated by oncologists who were, understandably, keen to enrol their patients onto a Glivec trial. The trial protocols required eligible patients to have tumours that showed strong staining for CD117, thus placing the onus for therapeutic decision upon the pathologists. It soon became clear that there were discrepant results between laboratories and it was a struggle to validate this test for this purpose while under intense pressure from clinicians who had patients calling them daily for the results. There are apparently several hundred similar drugs in the developmental “pipeline” and considerable effort will need to be expended to ensure that participating pathology laboratories have a reliable test in place before the clinical trials commence.
In the fifth article, Farshid describes the morphological appearance of breast cancers that occur more frequently in women who carry a germline BRCA1 mutation. Extensive phenotype-genotype correlative studies have identified a number of morphological features of particular tumours that appear to be good predictors of a germline mutation in a tumour-predisposing gene. Certain morphological features of hereditary non-polyposis colorectal cancer (HNPCC)-associated colorectal cancers (eg extensive mucinous and signet-ring areas) have already been incorporated into clinical criteria used to screen patients for the underlying mutations. It is likely that pathological criteria will soon be incorporated into the genetic screening protocols for patients with breast cancer, particularly those occurring in women under 40 years of age. These observations and protocols mean that pathologists now need to be aware of these features, and their significance, and that they have a duty of care to inform the referring clinician that the patient may require referral to a familial cancer clinic.
All of the previous major advances in pathology have been underpinned by the introduction of new technology. In the sixth article Venter et al describe the impact that microarray-related technologies are having on pathology. Whereas immunohistochemistry, FISH and genetic analysis are all based on a single gene or protein, these new technologies can analyse over 30,000 genes simultaneously. Already, there are several landmark studies that have demonstrated that genome-wide analysis has the potential to provide new insights into cancer classification and diagnosis and to generate a vast number of prognostic and predictive markers. The speed of progress at the moment is breathtaking and the amount of data that needs to be assessed and validated is already vast. Five years ago the prospect of being able to analyse the whole genome was the stuff of fantasy; now there are serious plans to begin to incorporate these technologies into diagnostic pathology practice. We are, right now, on the cusp of the genomic revolution. What will diagnostic pathology be like in five, 10 or 15 years from now? It is a daunting but exciting prospect.
Many of the advances that will be discussed in the forthcoming articles, were derived from research performed on tissue samples. Although fixed paraffin-embedded tissues are valuable for histological and DNA-based studies, unfixed-fresh tissues are usually required for major research initiatives. As the molecular revolution progressed these fresh tissues became increasingly valuable. It became common practice for researchers to obtain excess tissues, without the patient’s knowledge, from pathology laboratories for their research projects. Pathologists were the gatekeepers of this process as they were responsible for determining whether the tissue was in excess to that required for diagnosis. These research projects often involved single diseases or a single gene or protein and researchers built up their personal tissue collections to support their own research.
As the genomic era dawned about five years ago, it became apparent that there was a pressing need for comprehensive well-curated collections of fresh tissue samples that were readily accessible to researchers. Tissue banks began to emerge in almost every hospital and research institution. Coincident with these developments was the emergence of widespread community concern regarding the ethical use of human tissues, particularly those obtained from autopsies, for research. Institutions with tissue collections and tissue banks responded by paying more attention to the ethical issues that related to the ownership of tissue samples; the consent to collect, store, use and transfer tissues, and the need to protect patient confidentiality. These necessary measures, however, have somewhat restricted access to tissues for research and it is a painstaking process to obtain tissues from other institutions. Presently, there are moves to improve access and streamline the process by networking existing tissue banks with common collection protocols and ethics application processes. In the last article Zeps discusses the ethics and logistics of using tissue samples in research and describes the archival tissue bank network he established in Western Australia.
The rate of change is accelerating and it is a thrilling time to be a cancer pathologist. I hope that I am still around to read the 15th edition of Robbins’ Pathological Basis of Disease. What secrets will have been revealed?