Small Number Of Cancer Cells Research Articles

Mouse Induced Glioma-Initiating Cell Models and Therapeutic Targets

Both stem cells and cancer cells are thought to be capable of unlimited self-renewal. Moreover, a small number of cancer cells express stem cell markers, including CD133 and ATP-binding cassette transporters through which the cells can pump out anti-cancer drugs or specific fluorescence dyes such as Hoechst33342, suggesting that either cancer cells resemble stem cells or that cancers contain stem cell-like cancer cells, called cancer-initiating cells (CICs) or cancer stem cells. Using the common characteristics of tissue-specific stem cells, malignant tumors and cancer cell lines were shown to contain CICs, which self-renew and are tumorigenic. CICs are also resistant to both irradiation and chemotherapy. These findings suggest that CICs are critical targets for successful therapy. However, CICs have not been well characterized, due to a lack of specific markers. We recently established mouse glioma-initiating cell (GIC) lines by overexpressing oncogenic HRas(L)⁶¹ in p53-deficient neural cells. These cells form transplantable glioblastoma multiforme (GBM) with features of human GBM when as few as 10 cells are transplanted in vivo, suggesting that these GIC-like cells are enriched in CICs. Characterization of these GICs showed that they expressed little or no Sox11. The overexpression of exogenous Sox11 in GICs blocked their tumorigenesis by inducing their neuronal differentiation, which was accompanied by decreased levels of a novel oncogene, plagl1. These findings suggest that Sox11 and Plagl1 work as a tumor suppressor and oncogene, respectively, in GBM and are potential therapeutic targets.

ChemInform Abstract: Utilization of Polymerase Chain Reaction Technology in the Detection of Solid Tumors

BACKGROUND Most cancer detection tests currently performed are based on either antibody assays to a marker protein with altered expression in cancer patients or on imaging studies to identify characteristic lesions. Generally, for a positive result, these detection assays require that a tumor have a significant volume of cancer cells. Advances in diagnostic techniques and technology may allow for cancer detection at earlier stages, when the tumor burden is smaller and potentially more curable. The molecular techniques of polymerase chain reaction (PCR) and reverse transcriptase PCR (RT-PCR) are highly sensitive methods for detecting a small number of cancer cells. Over the past few years, numerous clinical studies have used PCR techniques to detect physical alterations of genes, such as mutations, deletions, translocations and amplification, the presence of oncogenic viruses, and the expression of genes specific to tissue, cancer, and metastasis. The current status of PCR as a method for detecting marker genes in the management of solid tumors is reviewed. METHODS A review of the literature on the clinical utility of PCR and RT-PCR in the detection of solid tumor micrometastasis was conducted. RESULTS Amplification by PCR is a highly sensitive method to determine gene expression. A single cell expressing a tumor marker among 10-100 million lymphocytes can be detected by the PCR assay. This approach has been used to detect tumor cells in approximately 18 different solid tumor types, with melanoma and carcinoma of the breast and prostate the most widely investigated to date. PCR-based assays have been used to detect cancer cells in biopsies of solid tissue, lymph nodes, bone marrow, peripheral blood, and other body fluids. Several studies have reported a high specificity and sensitivity of tumor marker detection and a high correlation between PCR results and the presence of metastatic disease. However, in a few studies, PCR assays have not consistently demonstrated a higher sensitivity and specificity of detection than traditional modalities for many types of cancer. There has been a wide range in sensitivity and specificity among the studies, which may be partly attributed to the lack of uniformity among the PCR protocols used in different studies. CONCLUSIONS PCR can detect tumor marker-expressing cells that are otherwise undetectable by other means in patients with localized or metastatic cancer. Reports from various study groups have lacked uniformity in their protocols, and this has prevented adequate comparison. The clinical utility of this assay as a tool for the prognosis and management of cancer patients remains and area of active investigation. PCR is a powerful tool in the study of the biology of cancer metastasis and will likely serve as a useful adjunct to clinical decision-making in the future. Cancer 1998;82:1419-42. © 1998 American Cancer Society.

Fertility preservation in cancer patients using stored ovarian tissue: clinical aspects

This review covers the medical approach and laboratory guidelines needed for fertility preservation using stored ovarian tissue in cancer patients. Indications, careful patient selection and timing are essential. Techniques for tissue harvesting and storage are evaluated. Up-to-date information on publications reporting on transplantation, recovery of endocrine function, pregnancy and delivery of healthy babies is reviewed as well as relevant data on safety measures to detect cancer cells in stored ovarian tissue. Recent literature review indicates 12 pregnancies, five deliveries of healthy babies and two ongoing pregnancies after transplantation of ovarian tissue using different methods. To increase the safety of ovarian tissue cryopreservation-reimplantation procedures, algorithm and methods to identify tumor involvement in the ovaries and detection of small numbers of cancer cells in ovarian tissue were recently reported. Cryopreservation of ovarian tissue has been practiced for over a decade in an attempt to preserve fertility before the commencement of potentially sterilizing chemotherapy. With more than a few recent reports on pregnancies and deliveries after transplantation of ovarian tissue, there will be more patients requesting the storage of ovarian tissue in order to preserve fertility prior to cancer treatments.

ID genes mediate tumor reinitiation during breast cancer lung metastasis.

The establishment of distant metastases depends on the capacity of small numbers of cancer cells to regenerate a tumor after entering a target tissue. The mechanisms that confer this capacity remain to be defined. Here we identify a role for the transcriptional inhibitors of differentiation Id1 and Id3 as selective mediators of lung metastatic colonization in the triple negative [TN, i.e., lacking expression of estrogen receptor and progesterone receptor, and lacking Her2 (human epidermal growth factor receptor 2) amplification] subgroup of human breast cancer. Although broad expression of Id1 has recently been documented in tumors of the rare metaplastic subtype, here we report that rare Id1-expressing cells are also present in the more common TN subset of human breast tumors but not in other subtypes. We also provide evidence that Id1 expression is enriched in clinically obtained hormone receptor negative lung metastases. Functional studies demonstrate that Id1 and its closely related family member Id3 are required for tumor initiating functions, both in the context of primary tumor formation and during metastatic colonization of the lung microenvironment. In vivo characterization of lung metastatic progression reveals that Id1 and Id3 facilitate sustained proliferation during the early stages of metastatic colonization, subsequent to extravasation into the lung parenchyma. These results shed light on the proliferative mechanisms that initiate metastatic colonization, and they implicate Id1 and Id3 as mediators of this malignant function in the TN subgroup of breast cancers.

Stem cell-like cancer cells in cancer cell lines

Both stem cells and cancer cells are thought to be capable of unlimited proliferation. Moreover, a small number of cancer cells express stem cell markers, including CD133 and ATP-binding cassette transporters, by which the cells can pump out specific fluorescence dyes, such as Hoechst33342, as well as anti-cancer drugs, suggesting that either cancer cells resemble stem cells or cancers contain stem cell-like cancer cells, called "cancer stem cells (CSCs)". Using the common characteristics of tissue-specific stem cells, it was demonstrated that many types of tumors and cancer cell lines contain CSCs, which self-renew, express stem cell markers, and are tumorigenic. It was also shown that CSCs are resistant to anti-cancer drugs and irradiation. Thus CSCs might be a crucial target for the therapy. Because tumors contain CSCs and recruited normal stem cells, both of which contribute to tumorigenesis, it is difficult to separate CSCs from tumors. By contrast, cancer cell lines do not have any contaminating normal stem cells that quickly loose mulitpotentiality and differentiate in normal culture condition, suggesting that cancer cell lines could be an attractive alternative source of cells for CSC research. In this review I summarize the recent progress in CSC research using cancer cell lines.

Detection of cancer cells and gene expression of cytokines in the peritoneal cavity in patients with gastric cancer

The gene expression of the cytokines interleukin-2 (IL-2) and IL-10 in peritoneal washings was examined in relation to the presence of cancer cells in the peritoneal cavity in patients with gastric cancer. Total RNA was extracted from 50-ml peritoneal wash samples from 124 patients (gastric cancer, n = 110; controls, n = 14). Carcinoembrionic antigen (CEA) messenger RNA (mRNA) was used to identify the number of cancer cells in peritoneal wash samples by a real-time reverse transcription-polymerase chain reaction (RT-PCR) method, which method was also used to assay the IL-2 and IL-10 gene expression levels. In the 14 control samples, CEA mRNA was not detected, while CEA mRNA was detected in 2 of the 51 stage I gastric cancer patients. Thus, the specificity of this method for the detection of cancer cells in peritoneal wash samples was 97% (63/65). The CEA-based real-time RT-PCR method demonstrated greater prognostic impact than the traditional cytological method. IL-2 gene expression in peritoneal wash samples that were CEA mRNA-positive was suppressed compared with that in peritoneal wash samples that were CEA mRNA-negative, while IL-10 gene expression did not differ according to the CEA mRNA findings. The detection of small numbers of cancer cells in peritoneal wash samples from patients with advanced gastric cancer is a good marker for peritoneal metastatic recurrence. In the peritoneal cavity, cancer cells may escape from immune surveillance by controlling the expression of cytokines.

Novel heteroduplex method using small cytology specimens with a remarkably high success rate for analysing EGFR gene mutations with a significant correlation to gefitinib efficacy in non-small-cell lung cancer

We conducted a feasibility study to examine whether small numbers of cancer cells could be utilised for analysis of the EGFR gene status using the loop-hybrid mobility shift assay, which is a modified heteroduplex technique. Cytology specimens obtained by transbronchial abrasion were successfully used for analysis of the EGFR gene status in 50 of 52 (96.2%) patients diagnosed with class V non-small-cell carcinoma. Furthermore, the relationship between the EGFR gene status and clinical outcome was analysed in 25 patients treated with gefitinib. Overall, 10 of 11 patients with EGFR mutations in exon 19 or 21 showed tumour regression with gefitinib treatment, compared to only two of 14 patients with wild-type EGFR. The response rate was significantly higher in the EGFR mutation group than in the wild-type EGFR group (90.9 vs 14.3%, P=0.00014). Logistic regression analysis revealed that EGFR mutations in cytology specimens represented an independent predictor of the gefitinib response. The overall and progression-free survivals were significantly longer in the EGFR mutation group than in the wild-type EGFR group (P<0.05). In conclusion, cytology specimens could be useful for analysing the EGFR status in the majority of patients with non-small-cell lung cancer to determine whether they are likely to benefit from gefitinib treatment.

Combined therapy of an established, highly aggressive breast cancer in mice with paclitaxel and a unique DNA‐based cell vaccine

Here, we describe the enhanced benefits of treating a highly aggressive breast cancer in mice with a combination of paclitaxel and immunization with a unique DNA-based cell vaccine. An adenocarcinoma was isolated from a spontaneous neoplasm that arose in the mammary gland of a C3H/He mouse (H-2(k)) (SB5b cells). The vaccine was prepared by transfer of genomic DNA-fragments (25 kb) from the breast cancer cells into a mouse fibroblast cell line (LM), modified to enhance its immunogenic properties. As the transferred DNA is integrated, and replicated as the recipient cells divide, the vaccine could be prepared from relatively small numbers of cancer cells (10(7) = 4 mm tumor). SB5b cells were injected into the mammary fat pad of naïve C3H/He mice, which are highly susceptible to the growth of the cancer cells. When the tumors reached 3 mm, the mice were injected s.c. with a noncurative dose of paclitaxel. Six days later, when immune competence returned, the mice received the first of 3 weekly s.c. injections of the vaccine. The combined therapy induced robust cellular immunity to the breast cancer, mediated by CD8+ and NK/LAK cells, which resulted in prolonged survival. The immunity was specific, as immunization with a vaccine prepared by transfer of DNA from B16 melanoma cells into the fibroblasts failed to induce immunity to the breast cancer. This type of vaccine raises the possibility that an analogous strategy could be used in the treatment of breast cancer patients at an early stage of the disease.

New molecular diagnostic methods in head and neck cancer

Initial cancer evaluation includes assessment of histologic appearance, tumor grading, assessment of lymph node status, and presence of metastasis. However, traditional diagnostic methods such as histopathology and radiology are not sensitive enough to detect small numbers of cancer cells and are limited in their ability to predict response to treatment. Recently, there has been considerable progress in molecular diagnostics in these areas. Using molecular-based technologies, it is now possible to detect cancer early in asymptomatic individuals, identify minimal residual disease at histopathologic normal surgical margins, more precisely assess tumor burden in cancer patients, and more accurately assess the prognosis of the patients. Examples of these applications in the evaluation of head and neck cancer are reviewed here. However, despite the great promise of these new molecular approaches for cancer detection, much of the current technology limits their implementation into routine clinical use.

Irrigation Volume Determines the Efficacy of “Rectal Washout”

Rectal stump washout has been recommended to prevent implantation of exfoliated malignant cells in the anastomosis after anterior resection for rectal cancer. The aim of this study was to investigate its efficacy, particularly the extent to which the volume of irrigation fluid might influence the efficacy of tumor cell elimination and whether tumor characteristics might influence the result. The study comprised 30 consecutive patients operated on by anterior resection for rectal cancer. After cross-clamping the rectum below the tumor, a washout sample was collected for examination after every incremental 500 ml of saline irrigation up to 2 liters. The presence of shed cancer cells was correlated with the washout volume and tumor characteristics. Cancer cells were found in 29 of 30 patients (97 percent) in the first sample of irrigation fluid and decreased gradually in frequency and number with increasing irrigation volumes. No cancer cells were demonstrated after 1.5 liters of irrigation in patients with tumor below the peritoneal reflection, whereas cancer cells were still present in one-fourth of the patients with tumor located above the peritoneal reflection. Finally, only a small number of cancer cells was confirmed in one patient after 2 liters of irrigation. The irrigation volume determined the efficacy of rectal washout. With our method, 1 1/2 liters of saline irrigation appears to clear contents from cancer cells in patients with tumors below the peritoneal reflection whereas at least 2 liters is recommended for patients with tumor above the peritoneal reflection.

Prognostic impact of Fas ligand on hepatocellular carcinoma after hepatectomy.

The expression of Fas ligand on tumor cells may counterattack the host's immunity and worsen the prognosis. Knowledge of the prognostic impact of Fas ligand on patients with hepatocellular carcinoma (HCC) after hepatectomy is still limited. Fas ligand expression in HCCs was examined in 59 patients who underwent hepatectomy for HCC. The prognosis was analyzed and correlated to the expression of Fas ligand. Expression of Fas ligand was detected by immunohistochemical staining in 27 of the 59 HCCs (45.8%). The Fas ligand was expressed in only a small number of cancer cells. However, even though only a few cancer cells expressed it, the prognosis for patients whose HCCs showed Fas ligand expression was worse than that for patients with an HCC without Fas ligand expression. The mean disease-free survival was only 10.83 +/- 1.90 months when HCCs expressed Fas ligand compared with 43.51 +/- 7.02 months for those without Fas ligand expression ( p = 0.0007). The overall patient survival was 28.34 +/- 4.08 months when the HCC expressed Fas ligand compared with 55.31 +/- 5.37 months for HCC without Fas ligand expression ( p = 0.0003). The expression of Fas ligand did not correlate with the presentation of other prognostic factors. Fas ligand expression is thus an independent prognostic factor for HCC. Thus the HCC expressing Fas ligand has a worse prognosis than the HCC without Fas ligand expression.

Pulmonary adenocarcinoma with massive lymphocyte infiltration: report of three cases

We report on three cases of pulmonary adenocarcinoma with lymphoid stroma. In these cases the lesions were small pulmonary adenocarcinomas, less than 2 cm in diameter, and histologically diagnosed as poorly or moderately differentiated adenocarcinomas. Histologically, the tumors were characterized by a small number of cancer cells and numerous infiltrating lymphoid cells, especially CD8 positive T-cell lymphocytes. No Epstein–Barr virus (EBV) genomes were demonstrated by in situ hybridization in the tumor cells of any of the tumors. Lobectomy and regional lymph node dissection were performed and intrapulmonary and mediastinal lymph node metastases were demonstrated in one of these cases (pT4N0M0, pT1N2M0 and pT1N0M0). Two patients are alive and the other died of pneumonia 26 months after the operation. None of the patients experienced recurrence of carcinoma. This unique type of adenocarcinoma characterized by massive lymphocyte infiltration could be referred to as “pulmonary adenocarcinoma with lymphoid stroma”.

Differential Uptake of 18F-fluorodeoxyglucose by Experimental Tumors Xenografted into Immunocompetent and Immunodeficient Mice and the Effect of Immunomodification

PURPOSE: To study the contribution of immunologic background to the uptake of fluorine-18-fluorodeoxyglucose (18F-FDG) by the tumor tissues. METHODS: The uptakes of 18F-FDG to the same experimental tumor model (SCCVII) xenografted into immunocompetent and immunodeficient (athymic) mice were compared. In addition, the immunomodifying effect of steroid on the uptake of 18F-FDG by these tumors was investigated. RESULTS: The uptake of 18F-FDG by the tumors in immunocompetent mice was significantly higher than that in immunodeficient (athymic) mice. Although steroid pretreatment had no effect on the tumor uptake in immunodeficient mice, it significantly decreased the tumor uptake in immunocompetent mice. CONCLUSION: The higher tumor uptake of 18F-FDG observed in immunocompetent mice, modulated by steroid pretreatment, was contributed by the host immune reaction, probably cellular immunity employed by T-lymphocytes. These findings can clinically conclude that the intense accumulation of 18F-FDG in the metastatic lymph nodes, which contain only a small number of cancer cells, was caused by the enhanced uptake of 18F-FDG by activated T-lymphocytes due to host immunity against cancer cells present in metastatic lymph nodes.

Detection of minimal residual disease

Minimal residual disease (MRD) is defined as the presence of a small number of cancer cells in hematological malignancies usually below 10 10 cells (that amount approximately corresponds to 1g of the total cancer cell burden in a human body). The ability to distinguish residual malignant cells among a normal population of cells is inherently dependent on the presence of identifiable cellular or subcellular features specific to the malignant clone. Unique combinations of surface, cytoplasmic, or nuclear molecules that are expressed by the malignant clone and are not present in normal cell population allow multiparameter flow-cytometric detection of MRD. This chapter presents an overview of the most current technology used for defining the advancement in MRD. It also focuses on MRD in acute and chronic leukemias and some lymphoproliferative disorders. In both acute and chronic leukemias and also in multiple myeloma, bone marrow (BM) is the primary and most significant compartment for MRD analysis.

Cytological recognition of invasive squamous cancer of the uterine cervix: Comparison of conventional light-microscopical screening and neural network-based screening

Cytologic recognition of invasive or microinvasive cancer of the uterine cervix may present substantial difficulties. In this study, we compared conventional light-microscopical screening of 109,104 cervical smears and neural network-based screening (NNS) of 245,527 smears, all obtained by the spatula-Cytobrush method. Two populations of Dutch women were included in the study: those receiving smears within the framework of the Dutch population screening program ("routine smears") and those receiving smears for other reasons, discussed in the text ("interval smears"). There were 71 smears, from an equal number of biopsy-confirmed invasive squamous carcinomas, 28 of which were microinvasive. The "interval smears" yielded a statistical valid higher prevalence of invasive cancer than "routine smears." Except for 5 smears that contained no evidence of abnormality ("sampling errors"), no false-negative errors occurred in the 52 NNS cases, whereas 4 such errors occurred in the 19 conventionally screened cases. By measuring the amount of cancerous material present in each smear (mapping), it could be documented that NNS was effective even in smears with a small number of cancer cells, whereas the 4 conventional false-negative screening errors occurred in smears of this type. The study showed that cells derived from invasive cancer of the cervix may have large bland nuclei that do not fit the images commonly associated with squamous cancer cells. Neural network-based screening of cervical smears was more effective than conventional screening in the diagnosis of invasive squamous cancer of the uterine cervix.

Detection of telomerase activity in biopsy samples of colorectal cancer

Telomerase is a ribonucleoprotein that synthesizes telomeric DNA onto chromosomal ends. The expression of telomerase is thought to be required for cellular immortality and oncogenesis. To investigate the role of telomerase in the pathogenesis of colorectal cancer, we analysed telomerase activity in biopsy samples of colorectal cancer and colonic adenomas. Using a polymerase chain reaction-based assay, we examined telomerase activity in 52 samples of colorectal cancer, 12 colonic adenomas and 30 normal colonic mucosa samples obtained by endoscopic biopsy. Telomerase activity was detectable in 88.5% (46/52) of colorectal carcinomas, in 50% (6/12) of colonic adenomas but not in normal colorectal mucosa. There was no correlation between telomerase activity and tumour location, type, size and differentiation (P > 0.05). It was concluded that telomerase activation plays a role in the evolution of colorectal cancer, and that measurement of telomerase activity in biopsied colorectal mucosa samples may provide information both as a diagnostic marker to detect small numbers of cancer cells, and as a screening method for patients at high risk for colorectal carcinoma.

Utilization of polymerase chain reaction technology in the detection of solid tumors.

Most cancer detection tests currently performed are based on either antibody assays to a marker protein with altered expression in cancer patients or on imaging studies to identify characteristic lesions. Generally, for a positive result, these detection assays require that a tumor have a significant volume of cancer cells. Advances in diagnostic techniques and technology may allow for cancer detection at earlier stages, when the tumor burden is smaller and potentially more curable. The molecular techniques of polymerase chain reaction (PCR) and reverse transcriptase PCR (RT-PCR) are highly sensitive methods for detecting a small number of cancer cells. Over the past few years, numerous clinical studies have used PCR techniques to detect physical alterations of genes, such as mutations, deletions, translocations and amplification, the presence of oncogenic viruses, and the expression of genes specific to tissue, cancer, and metastasis. The current status of PCR as a method for detecting marker genes in the management of solid tumors is reviewed. A review of the literature on the clinical utility of PCR and RT-PCR in the detection of solid tumor micrometastasis was conducted. Amplification by PCR is a highly sensitive method to determine gene expression. A single cell expressing a tumor marker among 10-100 million lymphocytes can be detected by the PCR assay. This approach has been used to detect tumor cells in approximately 18 different solid tumor types, with melanoma and carcinoma of the breast and prostate the most widely investigated to date. PCR-based assays have been used to detect cancer cells in biopsies of solid tissue, lymph nodes, bone marrow, peripheral blood, and other body fluids. Several studies have reported a high specificity and sensitivity of tumor marker detection and a high correlation between PCR results and the presence of metastatic disease. However, in a few studies, PCR assays have not consistently demonstrated a higher sensitivity and specificity of detection than traditional modalities for many types of cancer. There has been a wide range in sensitivity and specificity among the studies, which may be partly attributed to the lack of uniformity among the PCR protocols used in different studies. PCR can detect tumor marker-expressing cells that are otherwise undetectable by other means in patients with localized or metastatic cancer. Reports from various study groups have lacked uniformity in their protocols, and this has prevented adequate comparison. The clinical utility of this assay as a tool for the prognosis and management of cancer patients remains and area of active investigation. PCR is a powerful tool in the study of the biology of cancer metastasis and will likely serve as a useful adjunct to clinical decision-making in the future.

Sensitive detection of p53 gene mutations by a 'mutant enriched' PCR-SSCP technique.

For the rapid and sensitive detection of p53 'hot spot' mutations, we combined polymerase chain reaction based single-strand conformational polymorphism (PCR-SSCP) analysis with sequence specific-clamping by peptide nucleic acids (PNAs) in a one-step reaction tube protocol. For this purpose, we designed two PNA molecules comprising aa 246-250 of exon 7 and aa 270-275 of exon 8, respectively, to suppress the amplification of wild-type p53 allelic variants during PCR amplification. Using this method in a survey of 20 brush cytology samples from lung cancer patients, we were able to detect five p53 point mutations occurring in codons 248, 249 and 273 which could not be retrieved by conventional PCR-SSCP. Thus, allelic suppression by PNA molecules opens a way to largely improve the sensitivity of existing PCR-SSCP protocols (approximately 10-50-fold) and could be useful in the detection of 'hot spot' oncogene lesions in histological samples containing only a small number of cancer cells.

Prediction of Relapse of Pediatric Acute Myeloid Leukemia by Use of Multidimensional Flow Cytometry

Most patients receiving therapy for acute myeloid leukemia (AML) enter an interval in which leukemic blast cells cannot be detected by light microscopy (i.e., morphologic remission). However, many of these patients experience a subsequent relapse. Multidimensional flow cytometry, which allows the discrimination of antigens expressed on normal and malignant cells, can detect small numbers of cancer cells in bone marrow or peripheral blood specimens. This technique enables the detection of one leukemic blast cell among 10(3) to 10(2) normal regenerating hematopoietic cells. We determined whether the presence of residual leukemic blast cells, identified in the bone marrow of pediatric patients with AML by use of multidimensional flow cytometry, would be predictive of subsequent leukemic relapse. Multidimensional flow cytometry was performed on 205 marrow specimens collected throughout the course of treatment from 39 patients who had achieved morphologic remission. The analyses employed monoclonal antibodies directed against CD45 in combination with mixed pairs of monoclonal antibodies directed against 10 other antigens. A time-varying Cox regression analysis that controlled for sample time intervals, age, sex, morphologic classification of disease, and white blood cell count at diagnosis was used to relate the multidimensional flow cytometric results to the risk of relapse after achieving remission. Reported P values are two-sided. Thirty-five of the 39 patients had bone marrow specimens available from the time that first morphologic remission was achieved. Leukemic blast cells were detected in the specimens from 19 (54%) of these 35 patients. Twenty-five of the 35 patients did not receive an allogeneic (i.e., from a different genetic background) bone marrow transplant during first morphologic remission, and 13 of 14 with residual leukemic cells experienced a relapse at a median time of 153 days after diagnosis (range, 48-863 days). Nine of the 11 patients who did not receive an allogeneic bone marrow transplant and lacked evidence of leukemic blast cells at first morphologic remission relapsed at a median time of 413 days after diagnosis (range, 321-794 days). Among the 10 individuals who received an allogeneic bone marrow transplant during first morphologic remission, five were positive for leukemic blast cells and five were negative; one of these patients (positive for leukemic blast cells) experienced a relapse 265 days after diagnosis, and three others died of transplant-related complications. The estimated risk of relapse during intervals of multidimensional flow cytometric positivity (i.e., intervals of remission for which the immediately preceding cytometry measurement was positive) was 2.8 times greater than that during negative intervals (95% confidence interval = 1.1-7.0; P = .02). Multidimensional flow cytometry identifies residual leukemia in more than half of the patients with AML who are in morphologic remission. The detection of leukemic blast cells in these patients by multidimensional flow cytometry is predictive of a more rapid relapse.

Reverse transcriptase- polymerase chain reaction (RT-PCR) to detect prostate cancer micrometastasis in the blood.

Since the high mortality rate of prostate cancer is directly associated with the incidence of cancer metastasis, early detection of metastasizing prostate cancer cells in the blood has very important clinical implications. Conventional screening and staging modalities often fail to identify patients who may have prostate cancer that is already metastatic [1], and it has been estimated that 60% of new patients may have asymptomatic micrometastasis. It is therefore of great importance to be able to define which patients are at risk for the development of further metastatic disease [2,3]. It has been suggested that the detection of circulating prostate cancer cells in the peripheral blood may allow assessment of disease state [4], although the exact clinical significance of circulating cancer cells remains unclear. Animal experiments using solid tumors have shown that only two cells per milliliter of blood may be enough for tumor metastasis from hematogenous dissemination [5–7]. Based on these studies, a technique to detect circulating cells must be highly sensitive, detecting ten tumor cells in a five milliliter blood sample. Although immunohistochemical and other immunological techniques that use antibodies against tumor-associated antigens or tissue-specific antigens are helpful in detecting the primary site of metastatic disease and the presence of small foci of metastatic disease, these techniques are not sensitive enough to detect such a small number of cancer cells in a large quantity of nonneoplastic cells. It is through the development of Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and new tumor markers that the desired sensitivity for this approach becomes possible. The high sensitivity and specificity obtained from this technique as well as the small quantity of test material required makes RT-PCR a definite front runner in the race for early detection of micrometastasis in prostate cancer.