Catálogo de publicaciones - libros

The standard method for the detection of disseminated epithelial tumour cells is still immunocytochemistry despite some concerns such as relative low sensitivity and subjective evaluation. Several approaches have been made to develop sensitive and specific polymerase-chain reaction assays comparable to those in use for detection of minimal residual disease in haematological malignancies. The major problem is the absence of specific genetic aberrations in solid cancer. Thus, researchers focused on amplification of so-called tissue-specific expressed genes such as epithelial structure proteins or messenger RNA of tumour markers or tumour-associated proteins. Most assays were described as highly specific valuable tools by the developers, and subsequently as non-specific by investigators. This chapter describes the mechanisms leading to so-called ‘false-positive’ and ‘false-negative’ results, and discusses the strength and weakness of RT-PCR for detection of solid cancer cells. Furthermore, strategies are discussed for development of reverse-transcriptase polymerase-chain reaction systems and for using and increasing their specificity.

The prognosis of head and neck cancer is largely determined by the radicality of treatment: residual tumour cells will grow out and develop in manifest local recurrences, regional recurrences and distant metastases. Classical diagnostic methods such as radiology and histopathology have limited sensitivities, and only by molecular techniques minimal residual cancer or disseminated tumour cells can be detected. In tissue samples containing the normal tissue counterpart of a tumour only (pre)cancer cell-specific markers can be exploited, whereas in other samples tissue-specific markers can be used. Currently, there are two main methodologies in use, one based on antigen-antibody interaction, and the other based on amplified nucleic acids. The most commonly used nucleic acid markers are mutations or alterations in tumour DNA (tumour-specific markers) or differentially expressed mRNA (tissue-specific markers). The limits of detection of these molecular assays can reach levels of a single tumour cell in a background of 2 107 normal cells. The assays are, however, often complex, demand a large experience and are usually laborious. Nevertheless, the data collected with these assays enable the elucidation of unexplained clinical phenomena. Further technical developments might allow implementation in clinical practice once the relevance has been assessed in large prognostic trials with long-term follow-up. In this chapter a number of the molecular assays used for (pre)cancer cell detection in head and neck cancer patients will be presented.

The early and clinically occult spread of viable tumour cells to the organism is becoming acknowledged as a hallmark in cancer progression, since abundant clinical and experimental data suggest that these cells are precursors of subsequent distant relapse. Prospective clinical studies have shown that the presence of such immunostained cells in bone marrow is prognostically relevant with regard to relapse-free and overall survival of breast cancer patients. As current treatment strategies have not resulted in a substantial improvement of breast cancer mortality rates so far, it is noteworthy to consider the intriguing options of immunocytochemical screening of bone marrow aspirates for occult metastatic cells. Besides improved tumour staging, such screening offers opportunities for guiding patient stratification for adjuvant therapy trials, monitoring response to adjuvant therapies, which, at present, can only be assessed retrospectively after an extended period of clinical follow-up, or for specifically targeting tumour-biological therapies against disseminated tumour cells.

The most important factor affecting the outcome of patients with invasive cancers is whether the tumor has spread, either regionally (to regional lymph nodes) or systemically (to the bone marrow). However, a proportion of patients with no evidence of systemic dissemination will develop recurrent disease after primary therapy. Clearly, these patients had occult systemic spread of disease that was undetectable by methods routinely employed (careful pathological, clinical, biochemical and radiological evaluation). This early dissemination of tumor cells is known as occult metastases (or micrometastases). In addition, the success of adjuvant therapy is assumed to stem from its ability to eradicate occult metastases before they become clinically evident. Therefore, methods for the detection of occult metastases in patients with the earliest stage of cancer, i.e., prior to detection of metastases by any other clinical or pathological analysis, have received a great deal of attention. This chapter focuses on the detection and significance of occult metastatic cells in the peripheral blood bone marrow and lymph nodes of patients with breast cancer.

About 20% to 40% of men who undergo a radical prostatectomy for localized prostate cancer will relapse with progressive disease that frequently results in bone metastases. In addition to numerous studies of the primary tumour, there has been increased attention paid to disseminated cells shed by the tumour and detected in the peripheral blood and bone marrow. It would appear logical that the prostate cancer cells in the blood and the bone marrow that remain following a radical prostatectomy may be more informative in revealing prognostic information than those of the primary tumour which is removed at surgery. The evidence for existence of these disseminated cells in the peripheral blood and bone marrow of prostate cancer patients was first described in the early 1990s using a prostate specific antigen reverse transcriptase polymerase chain reaction (PSA RT-PCR). In general, these studies revealed the presence of disseminated PSA+ epithelial cells (presumed to be prostate cancer cells) early in the disease course and more prevalent in advanced disease than in early disease. Most showed that the bone marrow was more frequently positive than the peripheral blood. As a staging tool, these studies generally showed that PSA RT-PCR was not highly informative since even patients with low stage and early disease frequently showed evidence of disseminated PSA+ cells. Over the past few years, there has been a rapid development of technologies for isolating these disseminated cells for further study. The first step was the enrichment of circulating tumour cells, followed by attempts to isolate and characterize individual cells. The enriched cells have been analysed by a variety of methods, including flow cytometry, immunohistochemistry, and fluorescent in situ hybridisation. Biological characterisation of the cells have included the assessment of telomerase activity, androgen receptor status, and genomic alterations such as loss of heterozygosity and microsatellite instability. Attempts are under way to extend this analysis by utilizing gene expression micro-arrays and array Comparative Genomic Hybridisation (array-CGH). Although these efforts encompass challenges much will be learned about the character of these disseminated cells, including their process of trafficking to distant sites, their potential to seed and grow at these sites and their tendencies for cell dormancy.

Metastasis to lymph nodes or distant organs is a well-known feature of poor prognosis in potentially resectable non-small cell lung cancer (NSCLC). However, a significant number of lymph node negative patients die early of metastatic disease. Therefore, it has to be assumed that in some patients an early tumour cell dissemination has occurred which is clearly underestimated by current staging procedures. Recently, it has been shown, that an early dissemination of individual carcinoma cells to regional lymph nodes or bone marrow can be detected by using sensitive immunocytochemical techniques with monoclonal antibodies against epithelium-specific proteins. The incidence of immunohistochemically positive patients varies between 30% and 70% depending on the type of primary tumour, the immunohistochemical staining procedure used and especially on the primary monoclonal antibody. The detection of disseminated tumour cells in lymph nodes or bone marrow by immunocytochemistry is associated with a poorer prognosis in lung cancer. In conclusion, the immunohistochemical detection of early disseminated tumour cells in lymph nodes or bone marrow can help to obtain a more exact identification of patients with an unfavorable prognosis. Whether the identified patients will gain from an adjuvant therapy needs to be evaluated in further studies.

A substantial proportion of patients (40% to 50%) with supposedly localized esophageal cancer who had undergone curative surgical treatment with complete tumour removal suffer from a metastatic tumour relapse within 24 months after surgery. A reason for such an early tumour relapse in these patients might be a minimal tumour cell dissemination (minimal residual disease, MRD) present at the time of operation, which cannot be detected by clinical and routine histopathological tumour staging procedures. Over the past 10 years, more sensitive immunohisto-/-cytochemical and nucleic acid based assays have been developed that are based on the detection of epithelial cell-or tumour-associated marker proteins and are able to detect single tumour cells or small tumour cell clusters present in lymph nodes classified as tumour-free by conventional histopathologic analysis, bone marrow or blood. Here we present an overview of recent studies concerning the prevalence and prognostic value of occult tumour cells in lymph nodes and bone marrow of patients with esophageal cancer identified by antibody or nucleic acid based assays.

Metastatic spread is a major factor in the prognosis of cancer patients. Early detection and eradication of circulating tumor cells prior to the development of metastases could help to improve the outcome of patients after tumor resection. Disseminated tumor cells have been detected in different compartments of the body using cytological and immunostaining methods and, more recently, using different molecular biological techniques. However, the specificity and the sensitivity of the methods and their prognostic impact are still being debated. This chapter gives an overview over the published studies regarding the prognostic relevance of the detection of disseminated tumor cells in lymph nodes, bone marrow, blood and peritoneal cavity in colorectal, gastric and pancreatic carcinoma patients.

A number of specific genes encoding for melanosomal proteins are selectively expressed in melanocytes and melanomas. For detection of circulating melanoma cells, the expression of the tyrosinase gene is most widely used. Several cohorts of melanoma patients from single institutions have been analyzed by various research groups for the presence of circulating melanoma cells in all stages of disease. The percentage of patients with evidence for occult tumor dissemination has been correlated with the stage of disease in several, but not all, reports. Two prospective analyses suggest that the PCR result is of prognostic value in melanoma. Several laboratories have found PCR evidence for circulating melanoma cells in the great majority of untreated patients with Stage IV disease, other groups have reported much lower frequencies. Taken together, there is a wide range of results. Methodological differences are likely to account for this discrepancy. With the availability of true quantitative real-time reverse transcriptase (RT)-PCR systems, accurate quantification of tyrosinase transcripts over a range of 1 to 10,000 tumor cells per milliliter of blood is possible. Quantitative real-time RT-PCR systems also dramatically improve quality control, since exact quantitation of housekeeping gene mRNA facilitates determination of sample quality. Two large clinical trials are currently under way within the EORTC and in the US to adequately determine the clinical usefulness of PCR detection of minimal residual disease in melanoma.