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For decades it has been assumed that T cells reject tumors essentially by direct killing. However, solid tumors are composed of malignant cells and a variety of different nonmalignant cells, referred to as tumor stroma. Stromal cells, such as endothelial cells, fibroblasts and inflammatory cells, often support tumor growth. Here, we discuss new findings showing that the tumor stroma is an important target during T-cell-mediated tumor rejection. Cytotoxic molecules and cytokines produced by T cells inhibit or destroy the stromal 'infrastructure', thereby withdrawing essential resources and leading to tumor infarction and subsequent T-cell-mediated elimination of residual tumor cells. These findings are important for the development of more effective and specific immunotherapies for cancer.
Trends Mol Med 2005 May
PMID:Immunotherapy: target the stroma to hit the tumor. 1588 10

Multiparametric immunophenotyping of multiple myeloma (MM) and other plasma cell (PC) dyscrasias represents an attractive approach not only for research purposes but also in clinical practice. Based on well-established antigenic patterns, discrimination between myelomatous and normal PCs can be easily achieved in various types of samples, and this can be particularly valuable for the differential diagnosis between MGUS and MM and for monitoring residual disease in the latter. In addition, immunophenotyping may be an alternative and more reproducible method than morphology for evaluating PC infiltration, as well as for specifically analyzing DNA content and the cell-cycle distribution of different subsets of PCs. Despite the widespread use, standardization of methods and protocols still remains a challenge. In this chapter, we describe in detail the protocols and precise instructions for specimen collection, sample preparation, together with the methods for staining PCs and flow cytometry, data acquisition, and data analysis, including the more recent developments in the field. We highlight the most frequent limitations, and provide troubleshooting and practical recommendations that could help to solve them. The goal of this chapter is to emphasize the relevance of methodological issues in order to obtain reproducible and high-quality results regarding the phenotypic analysis of PCs.
Methods Mol Med 2005
PMID:Immunophenotyping of plasma cells in multiple myeloma. 1596 91

We present the case of a 6-year-old male who received an allogeneic bone marrow transplant as part of treatment for acute lymphoblastic leukemia. The patient relapsed 5 months after transplantation and received additional chemotherapy. He acquired an angioinvasive fungal infection that required transfusion of granulocytes. Approximately 5 weeks after relapsing (181 days after transplant), a bone marrow specimen was taken for molecular engraftment analysis and flow cytometry to assess graft loss as well as residual disease. The engraftment results generated by the multiple short tandem repeat loci tested were inconsistent, and alleles were present at several loci that were of neither patient nor donor origin. An error in specimen identification was initially considered. Further investigation into the circumstances surrounding procurement of the patient's bone marrow aspirate revealed that the patient had received a granulocyte transfusion approximately 10 hours before the bone marrow specimen was taken. In addition, morphological and flow cytometric analyses of the same bone marrow aspirate demonstrated a significant degree of peripheral blood contamination. We determined that the unknown alleles in the bone marrow engraftment specimen were derived from the donor of the transfused granulocytes. This case illustrates that white cell transfusion can lead to erroneous bone marrow engraftment results, particularly if only one microsatellite locus is used to monitor engraftment.
J Mol Diagn 2005 Aug
PMID:Bone marrow engraftment analysis after granulocyte transfusion. 1604 15

Caveolin-3 (Cav-3) is a principal structural protein of caveolae membrane domains. Animal studies have revealed that Cav-3 is expressed in skeletal and cardiac myocytes but absent in other types of cells. Recent studies have shown that abnormalities in the Cav-3 gene are associated with some forms of muscular dystrophy, while skeletal muscle abnormalities have been observed in Cav-3 transgenic and knockout mice. In this study the authors evaluated the distribution of Cav-3 in normal human tissues and compared the expression of Cav-3 with that of myogenin and myoD1 in rhabdomyosarcoma (RMS), malignant mixed mullerian tumor (MMMT), and an array of neoplasms that mimic RMS to assess the utility of Cav-3 as a diagnostic marker for tumors with skeletal muscle differentiation. In nonneoplastic human tissues, crisp membrane staining for Cav-3 was present in cardiac and skeletal myocytes and occasionally in arterial smooth muscle cells and prostatic stromal cells, while other cell types were negative for Cav-3. Eighty-eight percent (21/24) of RMS studied were positive for Cav-3. Positive staining was generally observed in the more maturely differentiated tumor cells but not the primitive tumor cells. Eight of nine cases of MMMT stained strongly with Cav-3 in their rhabdomyosarcomatous component but not in other components. Fifty-four other neoplasms (13 leiomyosarcomas, 8 neuroblastomas, 5 lymphomas, 6 Wilms tumors without skeletal muscle differentiation, 5 Ewing sarcomas, 4 malignant fibrous histiocytomas, 4 angiosarcomas, 6 malignant melanomas, and 3 synovial sarcomas) were negative for Cav-3 expression. Nearly all (96% [23/24]) cases of RMS were positive for myogenin, while 88% (21/24) were positive for myoD1. Primitive tumor cells showed significantly increased expression of myoD1 and myogenin; conversely, more differentiated tumor cells were negative or weakly stained. The rhabdomyosarcomatous component of MMMT stained focally with myogenin and myoD1, in contrast to the strong Cav-3 labeling in these cells. These results demonstrate that Cav-3 is specifically expressed in human cardiac and skeletal myocytes. Furthermore, its high specificity and relatively high sensitivity (88%) for tumors with skeletal muscle differentiation suggest that Cav-3 is a valuable marker for these tumors and may be used to assess the degree of differentiation of RMS and to identify residual tumor cells in post-chemotherapy specimens.
Appl Immunohistochem Mol Morphol 2005 Sep
PMID:Caveolin-3 is a sensitive and specific marker for rhabdomyosarcoma. 1608 47

With the development of fluorescence in situ hybridization (FISH), it was possible to detect the BCR-ABL fusion signal in both metaphase spreads and interphase cells of patients with chronic myeloid leukemia (CML). However, the use of FISH to detect residual disease in patients with CML post therapy was limited by the false positive rate using the early single fusion probes. Therefore, dual fusion probes that created a fusion signal on the derivative chromosome 9 in addition to the fusion sifnal on the Philadelphia chromosome or derivative chromosome 22 were developed. Using these second-generation probes, it was discovered that a significant proportion of CML cases has a sub-microscopic deletion at the site of the ABL-BCR fusion. This chapter outlines a testing strategy to identify deleltions of the derivative chromosome 9 and to use combinations of probes to identify residual disease in these cases.
Methods Mol Med 2006
PMID:Deletion of the derivative chromosome 9 in chronic myeloid leukemia. 1650 80

Assessing the level of residual disease in leukemia is vital for evaluating patients' response to treatment and for identifying those at high risk of relapse. This should enable early preemptive intervention to prevent the onset of hematological relapse in those patients. One of the most common translocations in acute myeloid leukemia (AML) is the t(8;21). t(8;21) AML is characterized by a relatively good prognosis. This chapter discusses both qualitative and quantitative (real-time quantitative reverse-transcription polymerase chain reaction [RQ-PCR]) protocols for the diagnosis and minimal residual disease (MRD) monitoring in t(8;21) AML. It also discusses the importance of choosing appropriate controls for each assay. The chapter provides a simple equation for assessing the sensitivity/reliability of RQ-PCR assays, which enables scientists to assess the accuracy and reliability of their data.
Methods Mol Med 2006
PMID:Diagnosis and monitoring of AML1-MTG8 (ETO)-positive acute myeloid leukemia by qualitative and real-time quantitative RT-PCR. 1650 83

EGFR and erbB-2 are targets for specific cancer therapy. The purpose of this study was to examine the frequency and clinicopathological correlations of gene amplification, protein expression, and mutations of EGFR and ERBB2 in serous carcinoma, the most common and aggressive type of ovarian cancer. Tissue microarray constructed of 398 carcinomas was examined by chromogenic in situ hybridization (CISH) and by immunohistochemistry. Cases with amplification of EGFR by CISH were further analyzed by fluorescence in situ hybridization. One hundred ninety-eight samples were analyzed for mutations in exons 18, 19, or 21 of EGFR and in exon 20 of ERBB2 using denaturating high-performance liquid chromatography and direct sequencing. Amplification of EGFR was present in 12% (41/333), low-level gain in 43% (144/333), and protein overexpression in 17% (66/379) of the tumors. Both increased copy number and overexpression of EGFR were associated with high tumor grade, greater patient age, large residual tumor size, high proliferation index, aberrant p53, and poor patient outcome. Furthermore, increased copy number of EGFR was associated with increased copy number of ERBB2. No mutations were identified in EGFR, whereas one tumor had an insertion mutation in exon 20 of ERBB2. Both amplification and protein overexpression of EGFR occur in serous ovarian carcinoma, but EGFR copy number has a stronger prognostic value. This makes EGFR amplification a potentially useful criterion for selecting patients in clinical trials testing the effect of EGFR inhibitors in serous ovarian carcinoma.
J Mol Med (Berl) 2006 Aug
PMID:Gene amplification, mutation, and protein expression of EGFR and mutations of ERBB2 in serous ovarian carcinoma. 1660 61

The evaluation of residual disease, which has prognostic value in the treatment of hematological malignancies, is currently assessed by scoring a limited number of cells by karyotyping and molecular cytogenetics. Real-time polymerase chain reaction (PCR) is an easier and more sensitive technique, enables analysis of a larger number of cells, and decreases sampling error. However, real-time PCR has been applied only to target transcripts of fusion genes. Here, we considered two real-time PCR strategies to quantify a number of cells carrying a partial deletion of chromosome 7 mixed with normal disomic cells. The first strategy was based on the amplification of two sequences, one on chromosome 7 and the other on chromosome 14. In the second strategy residual disease was assessed by the ratio between the two alleles of a bi-allelic marker, mapped on chromosome 7, measured with allele-specific assays. Precision and accuracy of the two approaches were tested by reference samples with nominal values of residual disease ranging from 2 to 95%. As expected the second strategy resulted in more precise and accurate monitoring within the range from 5 to 95%. Furthermore, this method may be applied to assess the number of dysplastic or neoplastic clones carrying any unbalanced chromosome changes.
J Mol Diagn 2006 May
PMID:Establishment and study of different real-time polymerase chain reaction assays for the quantification of cells with deletions of chromosome 7. 1664 8

The selective heat shock protein 90 (HSP90) inhibitor 17-allyamino-17-demethoxygeldanamycin (17-AAG) is currently in phase I/II clinical studies at numerous institutions. Heretofore, the biomarkers to detect 17-AAG bioactivity (Hsp70, Raf-1, and cyclin-dependent kinase 4) had to be analyzed by Western blot of cellular samples, either from tumor biopsies or peripheral blood leukocytes, a method that is both laborious and invasive. We have identified two new biomarkers [insulin-like growth factor binding protein-2 (IGFBP2) and HER-2 extracellular domain] that can be readily detected in patient sera by ELISA. Both secreted proteins are derived from or regulated by Hsp90 client proteins, raising hopes that they might be sensitive serum markers of HSP90 inhibitor activity. Several structurally unrelated HSP90 inhibitors dose-dependently decreased secretion of both IGFBP-2 and HER-2 extracellular domain into culture medium, and both proteins were more sensitive to HSP90 inhibitors than previously identified biomarkers. In sera from BT474 tumor-bearing mice, both IGFBP-2 and HER-2 extracellular domain were down-regulated by 17-AAG in a time-dependent and dose-dependent manner, coincident with the degradation of HER-2 and attenuation of AKT activity in the tumors. Furthermore, IGFBP-2 levels at the end of treatment correlated with residual tumor load, suggesting that IGFBP-2 might serve as an early indicator of therapeutic response. In addition, we also found that both IGFBP-2 and HER-2 extracellular domain levels are elevated in patient sera from several cancer types, suggesting that these novel secreted biomarkers could be valuable pharmacodynamic tools in clinical trials of HSP90 inhibitors.
Mol Cancer Ther 2006 May
PMID:Identification of new biomarkers for clinical trials of Hsp90 inhibitors. 1673 58

Monitoring multiple myeloma patients for relapse requires sensitive methods to measure minimal residual disease and to establish a more precise prognosis. The present study aimed to standardize a real-time quantitative polymerase chain reaction (PCR) test for the IgH gene with a JH consensus self-quenched fluorescence reverse primer and a VDJH or DJH allele-specific sense primer (self-quenched PCR). This method was compared with allele-specific real-time quantitative PCR test for the IgH gene using a TaqMan probe and a JH consensus primer (TaqMan PCR). We studied nine multiple myeloma patients from the Spanish group treated with the MM2000 therapeutic protocol. Self-quenched PCR demonstrated sensitivity of >or=10(-4) or 16 genomes in most cases, efficiency was 1.71 to 2.14, and intra-assay and interassay reproducibilities were 1.18 and 0.75%, respectively. Sensitivity, efficiency, and residual disease detection were similar with both PCR methods. TaqMan PCR failed in one case because of a mutation in the JH primer binding site, and self-quenched PCR worked well in this case. In conclusion, self-quenched PCR is a sensitive and reproducible method for quantifying residual disease in multiple myeloma patients; it yields similar results to TaqMan PCR and may be more effective than the latter when somatic mutations are present in the JH intronic primer binding site.
J Mol Diagn 2006 Jul
PMID:Application of self-quenched JH consensus primers for real-time quantitative PCR of IGH gene to minimal residual disease evaluation in multiple myeloma. 1682 10


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