Preparación de muestras para estudios microscópicos

Basic biomedical research

Cancer diagnosis and treatment have changed greatly in recent decades due to numerous technological advances and a deeper understanding of tumour biology. For many centuries, tumour diagnosis was based on physical examination and personal and family medical histories, and treatment was largely restricted to symptom-oriented relieving of suffering. Surgical procedures were used only in few cases – mostly external tumours involving the skin. When medicine became a natural science-oriented disci- pline, the bases and concepts of cancer diagnosis and treatment also changed.

Aims of patient-centred cancer medicine

The success or failure of cancer treatment is decisively dependent on the characteristics of the tumour and characteristics of the patient. In addition to physical conditions of the patient such as age, previous illness, and co-medication, social, psychological, and envi- ronmental factors also influence treatment to varying

conscious and unconscious degrees. But it is the char- acterization of the biological characteristics of the tumour that creates the necessary prerequisite for personalized (also called stratified or individualized) cancer medicine.

To do this, tumour cells are examined for certain bio- logical characteristics – called biomarkers – at the level of the genes (DNA), primary gene products (RNA), and proteins. Depending on the biomarker, their identification may provide:

– prognostic information: assessment of the probable course of an illness, for example the risk of relapses and metastases

– predictive information: assessment of the findings allows the most promising treatment concept to be chosen

– and pharmacodynamics information on the effects and side-effects of drugs.

Prof. Holger Moch, MD

Director of the Institute of Surgical Pathology, Zurich University Hospital Matthias Rössle, MD

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Diagnostic examinations that take into consideration as far as possible all of these molecular changes of tumour cells form the basis of optimized treatment. New-generation substances that have the highest and most targeted effectiveness and at the same time minimized undesired side-effects play an important role here.

Foundations of morphological cancer diagnosis Cellular pathology, which was decisively shaped by Robert Remak and Rudolf Virchow, assumed that the development of all diseases was at the cellular level (“omnis cellula e cellula”); this led to a radical change in cancer diagnosis. Since the second half of the nine- teenth century, practically all potentially cancerous findings are examined morphologically at the level of tissue and/or cell. There is a certain irony in the fact that Virchow, as late as in 1860, wrote in an editorial that microscopes were not necessary in tumour diag- nosis.1 _{He was of the opinion that skin carcinomas} do not develop from the surface epithelium but in- stead in connective tissue. (In fact carcinomas start in epithelial cells, the outermost cell layers of skin and mucosal tissue.)

Advances in microscope and staining techniques, especially the development of various histochemical and immunohistochemical staining methods, make more and more detailed pictures of tumours and their specific cellular and subcellular characteristics possible. In the past 20 to 30 years, new techniques have also been developed, including DNA cytometry, fluorescence in situ hybridization, and comparative genomic hybridization, which can detect changes in the chromosomes that have a role in cancer.

Development of modern cancer diagnosis

The decoding of the human genome more than 10 years ago and the development of new techniques make possible today ever faster and more extensive

analysis of all genes (genome), gene products (tran- scriptome), proteins (proteome), and metabolic prod- ucts and pathways (metabolome) of cells. The first of these tests to be used were PCR-based multigene expression assays (for example, EndoPredict and Oncotype DX) to assess risk in breast cancer. To determine the most appropriate treatment, previ- ously mostly single gene mutations or chromosomal changes were examined. Examples are the presence of the KRAS gene in its normal variant as a prerequi- site for antibody treatment with cetuximab of meta- static colorectal cancer, and overexpression of the HER2 gene for treatment of breast cancer with the monoclonal antibody trastuzumab.

Today – especially in tumour research and increas- ingly also in tumour diagnosis – new sequencing techniques (“next-generation sequencing”) make it possible to decode the complete genome more and more quickly and less expensively. Frequently, only those gene segments that contain the information on the production of the proteins are sequenced. In these segments most of the known disease-causing mutations, which make up only about one per cent of the entire genetic information of an organism are found. Pilot studies have demonstrated the impor- tance of implementing such techniques in everyday oncological practice.2

A great advantage of these techniques is that in a single examination the existing tumour material can be examined for numerous genetic changes. For ex- ample, material obtained from an adenocarcinoma in the bronchi of the lung can at the same time be analysed for mutations and translocations (rear- rangements of whole segments of chromosomes). This not only allows us to find out what cancer-spe- cific gene changes there are but also to find out how

61 heterogeneously they appear within the diverse cells

of the tumour sample. Based on these analyses the decision can be made as to what surgery, radiation therapy, and chemotherapy treatment types can be used and in what sequence and combination they could be used optimally.

Opportunities and challenges

One of the greatest challenges when using modern molecular techniques like next-generation sequenc- ing is that at present we have little experience in how medicine should deal with the information generated. Especially in cases where genetic errors are found in very few tumour cells (in the extreme case, in one tu- mour cell), the relevance of the diagnostic finding for progression and treatment of the tumour is not clear. Here there is a great need for research, mainly for prospective clinical studies.

In this connection, diverse questions need clarifica- tion: What conditions must a tumour sample fulfil to be considered representative? Are a few cells suffi- cient – for example cells that are taken from a tumour by endoscopic fine-needle aspiration – for determin- ing prognostic, predictive, and pharmacodynamic biomarkers? Does examination of the heterogeneity of the primary tumour allow conclusions to be drawn as to the characteristics of newly discovered meta- stases, or do they have to be analysed again? Although in principle cell and tissue material that has been routinely extracted and fixed can be used for these modern examinations, it has been found that depending on the amount of material and its han- dling, the results vary in quality.3 _{The problem is that} up to now there are no standardized and certified protocols for these procedures. At present no cross- institutional quality assurance such as interlaboratory tests for the latest sequencing methods has been es- tablished.

But the great potential of modern cancer diagnosis is evident. Thanks to sequencing of the entire tumour genome or all protein-encoding DNA segments, it will be possible in the future to identify in individual tumours not only the on-average two to eight driver mutations that are responsible for the malignant alterations of the cells but also to find other gene changes that are less relevant for the development of the cancer.4 _{Up to now, these so-called passenger} mutations have not been searched for, despite the fact that they can have considerable treatment con- sequences.

The increasing importance of pathology

For pathologists, these developments mean, for one, that in the future they must include clinicians more in pre-analysis aspects such as sample selection and preservation. For another, based on their morpho- logical expertise, they must decide what amounts of sample tissue have sufficient tumour cell material for the further analyses. It is also centrally important that pathologists not only present the findings to the treating physicians but also – for example in interdis- ciplinary tumour boards – critically comment on and classify them. For pathology as a special discipline, this means that it must develop more into a clinically and treatment-relevant branch of medicine and take on a more active role than before in oncological research and care.

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Prof. Holger Moch, MD

Holger Moch studied medicine at Humboldt-Universität zu Berlin. From 1988 to 1990 he worked as a resident in pathology at the Char- ité University Hospital – Univer- sitätsmedizin Berlin. The following three years Moch was a pathology resident at the University of Basel. In 1994 he was a research fellow at the University of California San Francisco and a visiting fellow at Harvard Medical School in Boston. In 2001 he became director of the Surgical Pathology Unit at Basel University Hospital and three years later chairman of the Institute of Surgical Pathology at Zurich University Hospi- tal. In 2004 he was named full professor of pathology at the University of Zurich and has been director of the Institute of Surgical Pathology at Zurich University Hospital since then. He is a member of the Scientific Committee of the Swiss Cancer League and the Swiss Cancer Research foundation, as well as the Cancer Commission of the canton of Zurich.

Tel. +41 44 255 25 00 [email protected]

www.klinische-pathologie.usz.ch

Matthias Rössle, MD

Matthias Rössle studied medicine at the University of Regensburg and Ludwig-Maximilian-University in Mu- nich, Germany. He earned a Doctor of Medicine at Ludwig-Maximilian- University in 2001 and was a resident at the Institute of Pathology there from 2001 to 2003. In 2003 he came to Switzerland to work at the Institute of Pathology at Cantonal Hospital Lucerne and then at Cantonal Hospital St. Gallen. Rössle is board certified as a pathologist (Facharzt) and board certified in cytopa thology

(FMH-Schwerpunkt); he earned the Proficiency Certificate in Head & Neck Sonography in 2012. Since 2010 he has been a senior physician at the Institute of Surgical Pathology at Zurich University Hospital, on Holger Moch’s team. Tel. +41 44 255 96 86

[email protected] www.klinische-pathologie.usz.ch References

1. Virchow R. Die Kritiker der Cellularpathologie.

Archiv für pathologische Anatomie und Physiologie und für klinische Medicin. 1860; 18(1– 2): 1–14. 2. Roychowdhury S, Iyer MK, et al. Personalized

oncology through integrative high-throughput sequencing: a pilot study. Sci Transl Med. 2011; 3(111): 111ra121.

3. Teichman A, Storz M, et al. Whole genome and transcriptome amplification: practicable tools for sustainable tissue biobanking? Virchows Archiv. 2012; 461(5): 571– 80.

4. Vogelstein B, Papadopoulos N, et al. Cancer Genome Landscapes. Science. 2013; 339(6127): 1546 – 58.

63 Basic biomedical research