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Stephen J Hamilton-Dutoit

Publications and source records attributed to Stephen J Hamilton-Dutoit.

9 recordsLinked to original sources
PubMed2005

Pathologic assessment of melanoma sentinel nodes: a role for molecular analysis using quantitative real-time reverse transcription-PCR for MART-1 and tyrosinase messenger RNA.

PURPOSE: Molecular analysis of melanoma sentinel nodes (SN) is sensitive, but poorly specific because metastases cannot be distinguished from benign nevus inclusions (BNI). We investigated whether quantitative reverse transcription-PCR (RT-PCR) detection of MART-1 and tyrosinase mRNAs could improve this specificity and contribute to SN assessment. EXPERIMENTAL DESIGN: Two hundred twenty SNs from 95 melanoma patients analyzed by extensive immunohistopathology and real-time quantitative RT-PCR. RESULTS: Using histopathology, SNs and patients were allotted to three diagnostic groups: (a) metastasis positive, (b) BNI positive, and (c) melanocyte-free. Median MART-1 and tyrosinase mRNA levels in SNs were significantly different in patients with metastasis compared with patients with BNIs (P < 0.05) and patients without melanocytic lesions (P < 0.001). However, a "gray-zone" was observed where distinction, based on mRNA levels, could not be made between the three groups. For both genes, the highest mRNA level recorded in each RT-PCR-positive patient was positively correlated with Breslow's tumor thickness. For SNs with metastases, tumor burden was significantly correlated to the mRNA level. Using the presence of a MART-1 RT-PCR signal to detect patients with metastases, a sensitivity of 100% and a negative predictive value of 100% were achieved when extensive immunohistology was used as reference. CONCLUSIONS: Quantitative RT-PCR MART-1 and tyrosinase mRNA analysis cannot be used alone for SN diagnosis because of its poor specificity for melanoma metastasis. However, in approximately one third of cases without RT-PCR evidence of MART-1 expression, extensive histopathologic SN investigation is not necessary, thus substantially reducing the cost of SN analysis. The level of melanocyte-associated mRNA is associated with both tumor thickness and tumor burden as measured histopathologically, suggesting that this may be of prognostic value.

Adult↗
PubMed2005

Improved diagnosis of mycobacterial infections in formalin-fixed and paraffin-embedded sections with nested polymerase chain reaction.

Traditional histological diagnosis of mycobacterial infection in formalin-fixed and paraffin-embedded (FFPE) tissues is insensitive and poorly specific. To improve this, we developed nested polymerase chain reaction (PCR) protocols for detecting a Mycobacterium genus-specific 65-kDa heat shock protein (HSP65) sequence and the M. tuberculosis complex-specific insertion sequence IS6110 in FFPE sections. Protocols were optimized on tissues from 20 patients with a final clinical diagnosis of mycobacterial infection. Amplicons were controlled by sequencing and restriction endonuclease digestion. PCR could detect as few as three mycobacterial genomes per reaction. Assays showed 100% sensitivity and specificity for both M. tuberculosis complex and M. avium complex infection. Paraffin blocks from a second group of 26 patients with histological evidence of necrotizing granulomas of unknown etiology were then analyzed as a surrogate group to test the assay under conditions similar to those applying during routine diagnosis. Twenty-three of these blocks contained amplifiable DNA; nine were positive for M. tuberculosis complex DNA and four for other types of mycobacterial DNA. Furthermore, digestion of HSP65 amplicons with NarI could distinguish M. tuberculosis from M. avium complex. In conclusion, our nested PCR assays can be used as reliable tools for the detection of mycobacterial infections in FFPE tissues. The assays are simple and rapid to perform and show improved sensitivity and specificity compared to previously reported protocols.

DNA, Bacterial↗
PubMed2004

Sentinel lymph nodes in malignant melanoma: extended histopathologic evaluation improves diagnostic precision.

BACKGROUND: The optimal technique for sentinel lymph node (SN) assessment in patients with melanoma is controversial. Molecular analysis (reverse transcriptase-polymerase chain reaction) detects significantly greater numbers of SNs with suspected micrometastases (up to 71%) than does routine histopathology (approximately 20%). The authors sought to identify possible reasons for this discrepancy and to determine whether using an extended histopathologic protocol could improve diagnostic precision. METHODS: Two hundred thirty-one SNs from 100 consecutive patients with cutaneous melanomas that measured 1-4 mm in thickness were bisected, and half of the lymph node was examined according to an extensive histopathologic protocol involving serial sectioning and immunohistochemical analysis of 3 melanocyte-associated markers (S-100, HMB-45, and Melan-A). RESULTS: Lymph node melanocytic lesions were frequent, with micrometastases and benign nevus inclusions (BNI) found in SNs in 28% and 28% of patients, respectively (4 SNs contained both). Melan-A was the most sensitive immunohistochemical marker and was positive in all BNI-positive SNs and 97% of micrometastasis-positive SNs. Although HMB-45 showed differential labeling in micrometastases compared with BNI (82% vs. 16%), immunohistochemistry could not distinguish between those lesions. Micrometastases were already identified on the first central level in 49% of positive SNs, whereas only 23% of SNs with BNI were diagnosed on the first level. CONCLUSIONS: Extensive serial sectioning with immunohistochemical analysis substantially increased the histopathologic detection of micrometastases and BNI in melanoma SNs to a level approaching the level reported for molecular techniques. The large number of BNIs represents an important potential source of imprecision (false positivity) in SN assays based on nonmorphologic methods.

Adult↗
PubMed2004

Quantification of melanoma mRNA markers in sentinel nodes: pre-clinical evaluation of a single-step real-time reverse transcriptase-polymerase chain reaction assay.

Although reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of melanocyte-associated mRNA can detect sentinel node melanoma metastases, most published assays are semi-quantitative methods of unknown sensitivity and precision, unsuitable for clinical use. We describe a single-step real-time quantitative RT-PCR assay for MART-1 and tyrosinase mRNAs, suitable for sentinel node analysis in a clinical setting. Using serial dilutions of melanoma cell line SK-MEL-28 RNA in water as a calibrator, we obtained linear calibration curves covering the range 0.5 to 10,000 arbitrary units (SK-MEL-28 melanoma cell equivalents). The sensitivity limit was 0.32 (MART-1) and 5 (tyrosinase) arbitrary units. Analytical imprecision was between 11% and 34%. MART-1 PCR efficiency was unaffected when samples were diluted with negative lymph node RNA rather than water, whereas tyrosinase PCR efficiency was halved. To evaluate the clinical suitability of our assay, we quantified melanocyte mRNAs in sentinel nodes with histologically verified micrometastases (n = 10) and benign nevus inclusions (n = 10), and in sentinel nodes without evidence of intranodal melanocytes (n = 10). We found significant differences in median melanocyte-derived mRNA levels comparing the three types of lymph nodes, suggesting that this quantitative molecular protocol may increase assay precision and be useful for the clinical evaluation of sentinel nodes.

Antigens, Neoplasm↗
PubMed2004

The influence of immunohistochemistry on mRNA recovery from microdissected frozen and formalin-fixed, paraffin-embedded sections.

Laser-assisted microdissection (LAM) is now widely used to obtain specific cell populations from heterogeneous tissues. A major disadvantage of LAM is poor tissue morphology during microscopy, in part because coverslips are not used. Immunohistochemical labeling can improve identification of target cells but may affect the subsequent analysis of the microdissected tissue. We studied the effect of immunohistochemistry (IHC) on mRNA recovery from labeled cells after microdissection from both frozen and formalin-fixed, paraffin-embedded (FFPE) sections, using Melan-A and Ki-67 staining in lymph nodes with metastatic melanoma as a model. We developed rapid protocols for immunostaining in an attempt to limit loss of mRNA during procedures. A sensitive real-time quantitative reverse transcription-PCR was used to measure mRNA. We found a marked decrease in the mRNA yield from 500 microdissected cells from frozen and paraffin sections after immunostaining for both markers. Recovery of mRNA decreased by up to 89%, comparing the immunostained with the routinely stained sections. Interestingly, the ratio between mRNA for the two markers was similar in all stains, indicating that immunostained sections may be used for mRNA analysis. We also investigated the effect of storing membrane-mounted sections for microdissection under different conditions. Slides mounted with paraffin sections could be stored at room temperature for up to 90 days with no significant decrease in mRNA recovery.

Actins↗
PubMed2003

XIAP-mediated caspase inhibition in Hodgkin's lymphoma-derived B cells.

The malignant Hodgkin and Reed-Sternberg cells of Hodgkin's lymphoma (HL) and HL-derived B cell lines were previously shown to be resistant to different apoptotic stimuli. We show here that cytochrome c fails to stimulate caspases-9 and -3 activation in cytosolic extracts of HL-derived B cells, which is due to high level expression of X-linked inhibitor of apoptosis (XIAP). Coimmunoprecipitation studies revealed that XIAP, apoptosis protease-activating factor-1, and caspase-3 are complexed in HL-derived B cell lysates. Even after stimulation with exogenous cytochrome c and dATP, XIAP impairs the proteolytic processing and activation of caspase-3. In cytosolic extracts, inhibition of XIAP by the second mitochondria-derived activator of caspases (Smac)/DIABLO, or immunodepletion of XIAP restores cytochrome c-triggered processing and activation of caspase-3. Smac or a Smac-derived agonistic peptide also sensitized intact HL-derived B cells for the apoptotic action of staurosporine. Finally, Hodgkin and Reed-Sternberg cells of primary tumor HL tissues also constitutively and abundantly express XIAP. The results of this paper suggest that high level XIAP expression is a hallmark of HL, which may play a crucial role in resistance to apoptosis.

Apoptosis↗
PubMed2003

Towards quantitative mRNA analysis in paraffin-embedded tissues using real-time reverse transcriptase-polymerase chain reaction: a methodological study on lymph nodes from melanoma patients.

Improved extraction techniques combined with sensitive real-time reverse transcriptase-polymerase chain reaction may allow detection of mRNA in formalin-fixed, paraffin-embedded (FFPE) materials, but the factors affecting mRNA quantification in clinical material using these methods have not been systematically analyzed. We designed analyses using real-time reverse transcriptase-polymerase chain reaction for quantification of MART-1, beta-actin, and beta(2)-microglobulin mRNAs. The analytical intra- and interassay imprecision (coefficient of variation) was in the range 10 to 20% for all three genes studied. Using these protocols, we studied the influence of tissue autolysis and length of formalin-fixation on mRNA detection in metastatic melanoma. Delay in freezing reduced detectable mRNA, although this was less than predicted and mostly occurred early in autolysis. MART-1, beta-actin, and beta(2)-microglobulin mRNAs were consistently detected in FFPE metastatic melanoma even after fixation for up to 3 weeks, although the total mRNA detected was markedly reduced in fixed compared with fresh tissues (up to 99%). Quantification of MART-1 was, however, possible if this was expressed relative to a housekeeping gene. The polymerase chain reaction product from FFPE tissues could be increased up to 100-fold amplifying short (<136 bp) compared with long amplicons. Variations in time before tissue processing and in fixation length seem to be less important sources of imprecision than previously assumed. Our findings suggest that quantitative analysis of mRNA in archive and routine diagnostic tissues may be possible.

Actins↗
PubMed2002

Loss of B cell identity correlates with loss of B cell-specific transcription factors in Hodgkin/Reed-Sternberg cells of classical Hodgkin lymphoma.

In classical Hodgkin lymphoma the malignant Hodgkin/Reed-Sternberg (HRS) cells characteristically constitute only a small minority of the tumour load. Their origin has been debated for decades, but on the basis of rearrangement and somatic hypermutations of their immunoglubulin (Ig) genes, HRS cells are now ascribed to the B-cell lineage. Nevertheless, phenotypically HRS cells have lost their B cell identity: they usually lack common B cell-specific surface markers such as CD19 and CD79a as well as Ig gene transcripts. Here we demonstrate that Ig promoters as well as both intronic and 3' enhancer sequences are transcriptionally inactive in HRS cell lines. This inactivity correlates with either reduced levels or even a complete lack of several B cell-specific transcription factors required for their expression: Oct-2, OBF-1, PU.1, E47/E12, PAX-5 and EBF. Moreover, we demonstrate that PU.1 and PAX-5 are significantly down-regulated in HRS cells in pathological specimens from primary tumour tissues. However, forced expression of these transcription factors can activate regulatory sequences of silenced B cell marker genes, and in one instance also transcription from a silenced endogenous locus. Thus, HRS cells are dedifferentiated B cells with global down-regulation of B cell-specific genes.

B-Lymphocytes↗