Detection of occult metastasis in patients with breast cancer.

The most important factor affecting the outcome of patients with invasive cancer is whether the tumor has spread, either regionally (to regional lymph nodes) or systemically. However, a proportion of patients with no evidence of systemic dissemination will develop recurrent disease after primary "curative" therapy. Clearly, these patients had occult systemic spread of disease that was undetectable by routinely employed methods (careful pathological, clinical, biochemical, and radiological evaluation). 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.

Occult metastases in patients with breast cancer.

The presence of occult metastases in the lymph nodes, bone marrow, or both of these compartments may not only define patients who are at higher risk for recurrence and death but also may identify biologically distinct mechanisms of tumor spread (e.g., lymphatic vs. vascular dissemination). Use of techniques to detect occult metastases may also allow the identification of a biologically important population of cells, i.e., those cells constituting the earliest metastatic population of tumor cells. Thus, techniques that identify occult metastases may be valuable in furthering our understanding of the events regulating tumor dissemination.

Her-2/neu expression in prostate cancer: high level of expression associated with exposure to hormone therapy and androgen independent disease.

PURPOSE: HER-2/neu is a proto-oncogene that encodes a transmembrane receptor belonging to the family of epidermal growth factor receptors. Increasing evidences indicates that HER-2/neu may contribute to hormone resistance in prostate cancer. We investigated HER-2/neu expression in primary, androgen dependent and advanced androgen independent prostate cancer, and its potential value as a marker of disease progression. MATERIALS AND METHODS: Immunohistochemical testing was performed to investigate HER-2/neu expression in 81 patients with prostate cancer, including 31 with pathological stage C disease treated with radical prostatectomy without preoperative androgen ablation therapy (untreated group), 30 with pathological stage C disease treated before surgery with androgen ablation therapy (treated group) and 20 with advanced androgen independent prostate cancer (androgen independent group). Tumors were classified based on the percent of tumor cells showing HER-2/neu membrane immunoreactivity as low (50% or less) and high (50% or greater) expression. RESULTS: Of the 31 prostate tumors in the untreated group 9 (29%) showed high HER-2/neu expression versus 15 of 30 (50%) in the treated and 17 of 20 (85%) in the androgen independent groups. The difference in HER-2/neu expression was significant in the untreated and androgen independent (p <0.001) and in the treated and androgen independent (p = 0.016) groups. There was a significant association of Gleason score with HER-2/neu expression in the untreated group (p = 0.038) but not in the treated group. No association was found of tumor substage with HER-2/neu expression. In the untreated group patients with tumors showing high HER-2/neu expression had a decreased survival rate (p = 0.044). CONCLUSIONS: High HER-2/neu expression is highly associated with exposure to hormone therapy and androgen independence. It may contribute to androgen independence in prostate cancer and identify patients with prostate cancer more likely to have disease progression, particularly those not exposed to previous hormone therapy.

Bioassay of prostate-specific antigen (PSA) using microcantilevers.

Diagnosis and monitoring of complex diseases such as cancer require quantitative detection of multiple proteins. Recent work has shown that when specific biomolecular binding occurs on one surface of a microcantilever beam, intermolecular nanomechanics bend the cantilever, which can be optically detected. Although this label-free technique readily lends itself to formation of microcantilever arrays, what has remained unclear is the technologically critical issue of whether it is sufficiently specific and sensitive to detect disease-related proteins at clinically relevant conditions and concentrations. As an example, we report here that microcantilevers of different geometries have been used to detect two forms of prostate-specific antigen (PSA) over a wide range of concentrations from 0.2 ng/ml to 60 microg/ml in a background of human serum albumin (HSA) and human plasminogen (HP) at 1 mg/ml, making this a clinically relevant diagnostic technique for prostate cancer. Because cantilever motion originates from the free-energy change induced by specific biomolecular binding, this technique may offer a common platform for high-throughput label-free analysis of protein-protein binding, DNA hybridization, and DNA-protein interactions, as well as drug discovery.

Antigen retrieval techniques: current perspectives.

Development of the antigen retrieval (AR) technique, a simple method of boiling archival paraffin-embedded tissue sections in water to enhance the signal of immunohistochemistry (IHC), was the fruit of pioneering efforts guided by the philosophy of rendering IHC applicable to routine formalin-fixed, paraffin-embedded tissues for wide application of IHC in research and clinical pathology. On the basis of thousands of articles and many reviews, a book has recently been published that summarizes basic principles for practice and further development of the AR technique. Major topics with respect to several critical issues, such as the definition, application, technical principles, and further studies of the AR technique, are highlighted in this article. In particular, a further application of the heat-induced retrieval approach for sufficient extraction of nucleic acids in addition to proteins, and standardization of routine IHC based on the AR technique in terms of a test battery approach, are also addressed. Furthermore, understanding the mechanism of the AR technique may shed light on facilitating the development of molecular morphology.

Antigen retrieval immunohistochemistry and molecular morphology in the year 2001.

The impact of the antigen retrieval (AR) technique upon diagnostic immunohistochemistry (IHC) and upon research has been demonstrated by numerous of articles and more than a dozen major reviews. The specific aim of this survey of the field is to examine potential new approaches to retrieval of nucleic acid and protein from archival paraffin-embedded tissue for molecular biology-based diagnostic procedures that form the basis of the emerging field of molecular morphology. Any new approach must incorporate the principles of standardization and improved reproducibility. The ultimate goal will be to understand the mechanisms of fixation (by formalin) and "unfixation" (by AR). In the interim, the diligent application of practical procedures that have been shown to be tried and true is the least that we must demand from ourselves and our laboratories.

Occult metastasis.

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. However, a proportion of patients with no evidence of systemic dissemination will develop recurrent disease after primary 'curative' therapy. Clearly, these patients had occult systemic spread of disease that was undetectable by methods routinely employed (careful pathological, clinical, biochemical and radiological evaluation). In addition, the success of adjuvant therapy is assumed to stem from its ability to eradicate occult metastases before they become clinically evident [1]. 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.

Aseptic technique for cell culture.

This unit describes some of the ways that a laboratory can deal with the constant threat of microbial contamination in cell cultures. A protocol on aseptic technique is described first. This catch-all term universally appears in any set of instructions pertaining to procedures in which noncontaminating conditions must be maintained. In reality, aseptic technique encompasses all aspects of environmental control, personal hygiene, equipment and media sterilization, and associated quality control procedures needed to ensure that a procedure is, indeed, performed with aseptic, noncontaminating technique. Although cell culture can theoretically be carried out on an open bench in a low-traffic area, most cell culture work is carried out using a horizontal laminar-flow clean bench or a vertical laminar-flow biosafety cabinet. Both are described here.

Sterilization and filtration.

This unit on sterility in the tissue culture environment describes methods for sterilization of liquid and dry goods used for tissue culture and filtration of liquids to prevent contamination of cultures.

Suprasellar germinoma in three lake whitefish (Coregonus clupeaformis).

Suprasellar germinomas were identified in three wild-caught lake whitefish (Coregonus clupeaformis) from the St. Lawrence River, Quebec, Canada. Histologically, the three tumors expanded the subarachnoid space of the ventral surface of the brain immediately adjacent to the pituitary gland and, in one case, infiltrated the adjacent neuropil. These tumors were characterized by nests and sheets of round cells with a high mitotic rate, separated by a scant amount of loose fibrovascular stroma. The stroma was infiltrated by a moderate number of small mononuclear cells, including rare CD3-immunoreactive lymphocytes. This is the first report of intracranial germinoma in a fish species.

LEA.135 expression: its comparison with other prognostic biomarkers for patients with primary breast carcinoma.

The purpose of this retrospective study was to examine the prognostic value of expression of luminal epithelial antigen (LEA.135) for recurrence and overall survival of patients with primary invasive breast carcinoma by both univariate and multivariate analyses. The possible prognostic value of LEA.135 was also compared with some widely utilized prognostic biomarkers such as c-erbB 2, topoisomerase II.alpha (TPII.alpha), MIB 1, estrogen receptor (ER) and progesterone receptor (PR), as well as age of the patients and clinicopathologic parameters. The study was carried out by immunohistochemical methods on formalin-fixed/paraffin-embedded tissue sections in a series of 225 patients with median follow-up of 8.5 years. Prognostic significance of the biomarkers was determined by two-sided p value. In this series of patients, among the age and clinicopathologic parameters, only age, was significantly associated with a decreased overall survival (logrank p = 0.027). Among the prognostic biomarkers, TPII a expression at high (> 50% positive cells) or moderate (6-50% positive cells) level was associated with an increased rate of recurrence (logrank p < 0.001). However, the association of TPII.alpha expression with a decreased overall survival failed to reach a statistically significance. Expression of c-erbB 2 showed a trend of being associated with an increased probability of recurrence, but the association did not reach statistical significance. The remaining biomarkers were not associated with either the probability of recurrence or overall survival. LEA.135 expression was observed in 163 (72.4%) of the 225 patients. The patients with high (> 50% positive cells) or moderate (6-50% positive cells) level of LEA.135-positive cancer cells showed a significantly decreased probability of recurrence (logrank p < 0.001) and an increased overall survival (logrank p < 0.001) compared with those with LEA.135-negative cancer cells. The association remained significant by multivariate analysis for recurrence (likelihood ratio test p < 0.001) and overall survival (likelihood ratio test p < 0.001) when assessed with other prognostic parameters. Furthermore, the combination of LEA.135 with other prognostic biomarkers stratified four subgroups of patients with distinct clinical outcome. The subgroup of patients who were LEA.135+/TPII.alpha- showed the lowest probability of recurrence and the longest overall survival compared with those who were LEA.135-/TPII.alpha+ (logrank p < 0.001). Interestingly, the patients whose cancer cells were LEA.135+/TPII.alpha+, LEA.135+ MIB.1+ or LEA.135+/c-erbB 2+ experienced a decreased probability of recurrence and an increased overall survival compared with those with LEA.135-/TPII.alpha+, LEA.135- MIB.1+ or LEA.135-/c-erbB 2+ (logrank p < 0.001). The results demonstrated that LEA.135 is an independent and favorable prognostic biomarker for patients with primary invasive breast carcinoma, that the loss of LEA.135 expression is associated with aggressive phenotype of cancer cells during the breast cancer progression, and that its continued expression seems to override the adverse effects of expression of an oncogene or cell proliferation-associated molecules.

LEA.135 expression: an independent and favorable prognostic biomarker for patients with primary invasive breast cancer.

The prognostic significance of LEA.135 expression, detected by immunohistochemistry in formalin-fixed and paraffin-embedded tissue sections, was evaluated and compared with the widely utilized clinicopathological parameters for patients with primary invasive breast carcinomas. Pathological parameters such as tumor size, histological tumor type, histological grade, nuclear grade, lymph node (LN) status, bone marrow (BM) status, as well as age of patient at initial diagnosis together with follow-up in years were available for this group of patients (n = 178). Among these parameters, tumor size, histological tumor type, histological grade, LN status, and BM status were individually and significantly associated with increased probability of recurrence by univariate analysis. By multivariate analysis, however, only tumor size, LN status, and BM status remained statistically significant. LEA.135-positive patients showed a statistically significant probability of not recurring (77 +/- 5% at 5 years after surgery) compared with patients who were LEA. 135-negative (49 +/- 6% at 5 years after surgery) (log-rank p < 0. 001). Furthermore, the association remained statistically significant by multivariate analysis (log-rank p = 0.019), demonstrating that LEA.135 expression independently and significantly identified breast cancer patients with favorable clinical outcome. In addition, there was a statistically significant association between loss of LEA.135 expression and poor prognosis when patients were stratified by pathological parameters. Furthermore, a subgroup of patients who were LEA. 135-positive/LN-negative experienced a decreased rate of recurrence compared with those who were LEA.135-negative/LN-negative (16% vs. 27%, respectively). A similar result was also obtained when BM-negative patients were stratified on the basis of LEA. 135-positive or LEA.135-negative subgroups for recurrence (18% vs. 43%, respectively). Most interestingly, the patients whose cancer cells were LEA.135-positive/LN-positive experienced a much lower rate of recurrence than those whose cells were LEA. 135-negative/LN-positive (29% vs. 57%, respectively). The results clearly demonstrate that LEA.135 expression was a significantly independent and favorable prognostic marker for patients with primary invasive breast carcinoma by both univariate and multivariate analyses.

Bone marrow and lymph node assessment for minimal residual disease in patients with breast cancer.

The immunocytological detection of disseminated epithelial cells in bone marrow in patients with breast cancer has been performed at many hospitals and institutes since the early 1980s. Despite numerous publications in this field, it has not been possible to standardize the method and establish the 'ideal' antibody, either nationally or internationally. Molecular biological methods using PCR technology could extend the diagnostic spectrum. However, one of the major problems in breast cancer is the lack of a disease-specific marker gene. As a result, immunocytology is still the standard procedure for tumour cell detection. The detection of disseminated single cells in bone marrow in primary breast cancer (also known as minimal residual disease) is a new prognostic factor for disease-free and overall survival. This has been demonstrated in two large (N > 300) groups and several small to medium groups (N = 50-300). As a marker of dissemination in a target organ for metastasis this prognostic factor corresponds much more closely to the tendency of breast cancer to early haematogenic spread. Tumour cell detection may predict the course of the disease better than the axillary lymph node status. Bone marrow aspiration and detection of disseminated cells might replace lymph node dissection, at least in those patients with small tumours and no clinical signs of lymph node involvement. This strategy will soon be investigated in appropriate studies. Another possible clinical use might be in deciding on whether or not to give adjuvant systemic therapy to node-negative patients. Patients with positive tumour cell detection are at a higher risk of subsequent metastasis, even if the axillary nodes are histologically normal. The immunohistological or molecular biological detection of tumour cells in axillary lymph nodes might also be very useful, now that it has been shown that a considerable subset of patients determined to be node-negative by means of conventional methods, are positive according to these new techniques. These methods could be a useful supplement to sentinel node biopsy. A further potential use of this method is in monitoring therapy with new treatment modalities such as gene therapy and immunotherapy. Repeated bone marrow aspiration can provide information on the success of therapy in minimal residual disease (cytoreduction). Immunocytochemical investigation of individual cells may be useful in studying the pathogenesis of metastasis, in particular in the skeleton. Phenotyping of cells might allow statements to be made on the metastatic potential of cells and the question of cell dormancy. It remains to be hoped that this aspect of minimal residual disease will be granted more attention in future.

Genetic and molecular markers of urothelial premalignancy and malignancy.

The molecular genetic changes reported in bladder tumors can be classified as primary and secondary aberrations. Primary molecular alterations may be defined as those directly related to the genesis of cancer. These are frequently found as the sole abnormality and are often associated with particular tumors. There are characteristic primary abnormalities involved in th production of low-grade/well-differentiated neoplasms, which destabilize cellular proliferation but have little effect on cellula "social" interactions or differentiation, as well as the rate of cell death or apoptosis. Other molecular events lead to high-grad neoplasms which disrupt growth control, including the cell cycle and apoptosis, and which have a major impact on biological behavior. A primary target leading to low-grade papillary superficial bladder tumors resides on chromosome 9, while p53 gene alterations are commonly seen in flat carcinoma in situ. Other molecular alterations must be elucidated, as many non-invasive neoplasms have neither chromosome 9 nor p53 alterations. Novel approaches utilizing tissue microdissection techniques an molecular genetic assays are needed to shed further light on this subject. Secondary genetic or epigenetic abnormalities may be fortuitous, or may determine the biological behavior of the tumor. Multiple molecular abnormalities are identified in most human cancers studied, including bladder neoplasms. The accumulation, rather than the order, of these genetic alterations may be the critical factor that grants synergistic activity. In this regard, it is noteworthy that many of the genes that are altered act upon the two recognized critical growth and senescenc pathways, TP53 and RB. These particular molecular aberrations may be especially important to evaluate for their use in the management of bladder cancer because of their commonality in progressive forms of the disease. Thus, clinical trials are underway to explore their use in specific situations, particularly in the surgical management of locally advanced disease, and to determine whether adjuvant chemotherapy in such patients may be of benefit. The use of molecular alterations in the management of non-invasive bladder neoplasms remains to be firmly established. Our knowledge of molecular alterations important in bladder cancer progression is far from complete, and further study is necessary to further elucidate cruci pathways involved in progression and therapeutic response. As per preneoplastic conditions, difficulties in identifying and interpreting the significance of phenotypic changes have imposed certain limitations, as has an evolving nomenclature and issues of reproducibility in interpreting morphologica criteria. Nevertheless, molecular alterations involving chromosome 9q and the INK4A locus in papillary superficial tumors vs changes in chromosomes 14q and 8q, p53 and RB in flat carcinoma in situ lesions may indicate a molecular basis for early events that lead to varying pathways in urothelial tumorigenesis. Studies aimed at revealing the clinical relevance of genet instability, as well as molecular or epigenetic alterations, in urothelium and preneoplastic lesions of otherwise morphologicall normal appearance are needed to further advance knowledge in the field. Clinical advances in bladder cancer will be facilitated by novel animal models paralleling the human disease. Molecular diagnostics, particularly specific antigen expression, fluorescence in situ hybridization and microsatellite analyses, have show great promise as screening and follow-up methodologies, and may supplement urine cytology in the diagnosis and characterization of new and recurrent disease. In addition, the use of high-throughput genomic/proteomic assays, linked to comprehensive databases, and coupled with robust bioinformatics will be key elements in elucidating the components of regulatory and signaling pathways involved in bladder tumorigenesis and cancer progression.

Role of immunohistochemical detection of lymph-node metastases in management of breast cancer. International Breast Cancer Study Group.

BACKGROUND: This study was designed to ascertain whether immunohistochemical methods could improve the detection of metastases in primary breast-cancer patients whose axillary lymph nodes were classified, by conventional methods, as disease free. METHODS: Ipsilateral lymph nodes (negative for metastases by routine histology) from 736 patients (participants in Trial V of the International [Ludwig] Breast Cancer Study) were examined by serial sectioning and staining with haematoxylin and eosin (two sections from each of six levels) and by immunohistochemistry of a single section (with two anticytokeratins AE-1 and CAM 5.2). After median follow-up of 12 years, disease-free and overall survival were estimated by Kaplan-Meier methods. FINDINGS: Occult nodal metastases were detected by serial sectioning and haematoxylin and eosin in 52 (7%) of 736 patients and by immunohistochemistry in 148 (20%). Only two (3%) of 64 invasive lobular or mixed invasive lobular and ductal cancers had node micrometastases, detected by haematoxylin and eosin, compared with 25 (39%) by immunohistochemistry. Occult metastases, detected by either method, were associated with significantly poor disease-free and overall survival in postmenopausal but not in premenopausal patients. Immunohistochemically detected occult lymph-node metastases remained an independent and highly significant predictor of recurrence even after control for tumour grade, tumour size, oestrogen-receptor status, vascular invasion, and treatment assignment (hazard ratio 1.79 [95% CI 1.17-2.74], p=0.007). INTERPRETATION: The immunohistochemical examination of ipsilateral axillary lymph nodes is a reliable, prognostically valuable, and simple method for the detection of occult nodal metastases. Immunohistochemistry is recommended as a standard method of node examination in postmenopausal patients.

Detection and clinical importance of micrometastatic disease.

Metastatic relapse in patients with solid tumors is caused by systemic preoperative or perioperative dissemination of tumor cells. The presence of individual tumor cells in bone marrow and in peripheral blood can be detected by immunologic or molecular methods and is being regarded increasingly as a clinically relevant prognostic factor. Because the goal of adjuvant therapy is the eradication of occult micrometastatic tumor cells before metastatic disease becomes clinically evident, the early detection of micrometastases could identify the patients who are most (and least) likely to benefit from adjuvant therapy. In addition, more sensitive methods for detecting such cells should increase knowledge about the biologic mechanisms of metastasis and improve the diagnosis and treatment of micrometastatic disease. In contrast to solid metastatic tumors, micrometastatic tumor cells are appropriate targets for intravenously applied agents because macromolecules and immunocompetent effector cells should have access to the tumor cells. Because the majority of micrometastatic tumor cells may be nonproliferative (G0 phase), standard cytotoxic chemotherapies aimed at proliferating cells may be less effective, which might explain, in part, the failure of chemotherapy. Thus, adjuvant therapies that are aimed at dividing and quiescent cells, such as antibody-based therapies, are of considerable interest. From a literature search that used the databases MEDLINE(R), CANCERLIT(R), Biosis(R), Embase(R), and SciSearch(R), we discuss the current state of research on minimal residual cancer in patients with epithelial tumors and the diagnostic and clinical implications of these findings.