Reino Heikkilä

Quantitative RT-PCR detection of tumor cells in sentinel lymph nodes isolated from colon cancer patients with an ex vivo approach.

Objective:To investigate quantitative RT-PCR–based detection of tumor cells in lymph nodes (LNs) isolated from colon cancer patients by ex vivo sentinel lymph node (SLN) mapping. Summary Background Data:Although lymph node status is among the strongest prognostic factors in colon cancer patients, 20% to 30% of node negative patients experience disease recurrence. These patients may have LN metastases that are not detected by routine examination. Methods:Ex vivo SLN mapping was applied to 131 prospectively recruited patients undergoing curative surgery for primary colon cancer. The SLNs were analyzed for the presence of tumor cells by routine histology and real-time RT-PCR quantitation of cytokeratin 20 (CK20) and mucin 2 (MUC2) mRNA. Results:SLNs were identified in 125 (95%) of the 131 patients included. Routine histologic analysis of SLNs and other regional lymph nodes revealed LN metastases in 42 patients (N+), of which 29 (69%) had metastases detected in 1 or more SLNs (sensitivity, 69%; false negative rate, 31%). When analyzing the SLNs by quantitative RT-PCR, the sensitivity, compared with routine LN examination, was 37/42 (88%) for both the CK20 and the MUC2 mRNA markers. In addition, 46% and 27% of the patients’ node-negative by routine LN examination (N0) were positive for the CK20 and MUC2 mRNA markers, respectively, possibly reflecting the presence of occult tumor cells in their SLNs. Conclusions:Quantitative RT-PCR analysis of SLNs identified N+ patients with high sensitivity and revealed a subgroup of N0 patients with potential occult LN disease.

High-Fidelity DNA Polymerase Enhances the Sensitivity of a Peptide Nucleic Acid Clamp PCR Assay for K-ras Mutations

Sensitive detection of tumor-specific point mutations is of interest in both the early detection of cancer and the monitoring of treatment at a molecular level. Recently, peptide nucleic acid (PNA) clamp real-time PCR has provided a time-sparing and sensitive method for the detection of mutations in the presence of a large excess of wild-type DNA. We present the first report that the sensitivity of PNA clamp PCR is limited by the low fidelity of TaqDNA polymerase. Replication errors introduced by Taq polymerase in the PNA-binding site were amplified during PCR due to the resulting mismatches between PNA and DNA. To reduce the frequency of polymerase-induced errors, we developed a PNA clamp PCR assay for the detection of mutations in codons 12 and 13 of the K-ras gene based on a high-fidelity DNA polymerase. The sensitivity of our assay increased approximately 10-fold, significantly detecting mutant DNA diluted 20,000-fold in wild-type DNA (P = 0.025), compared with its detection at 2000-fold dilution (P = 0.039) when Taq polymerase was used. Our data suggest that the replication errors caused by Taq polymerase must be taken into consideration for PNA clamp PCR and for other methods based on selective PCR amplification, and that these assays can be enhanced by high-fidelity DNA polymerases.

Fluorescence-based method for measuring and determining the mechanisms of recombination in quantitative PCR.

We quantitatively measured the amount of recombinant molecules formed during PCR when the break point cluster region (BCR) cDNA was coamplified with a homologous internal standard using Taq polymerase. The products were analysed under denaturing conditions using capillary electrophoresis followed by detection of the fluorescently labelled products and the recombinant molecules were differentiated by their size. Early termination of chain synthesis and reannealing of incomplete fragments, to each other as well as to BCR and internal standard, is one mechanism for generating recombinants during PCR since prolonging extension time reduced, but did not totally suppress recombinant molecule formation. Template switching by the extending chain is another mechanism since recombinant molecules could be detected even after only one round of primer extension. The latter mechanism is probably facilitated by increasing number of templates. Thus, the large increase of recombinant molecules formed in plateau phase is mediated by direct amplification of the recombinants and de novo synthesis by template switching. The effect of additives on recombination could be quantitatively measured and both betaine and DMSO were effective in suppressing recombination. Thus, prolonging extension time, reducing the number of amplification cycles and incorporating additives in the PCR reaction, reduced recombinant molecule formation.

Heterogeneous distribution of K-ras mutations in primary colon carcinomas: implications for EGFR-directed therapy

PurposeK-ras mutations predict resistance against epidermal growth factor receptor (EGFR)-directed therapy of metastatic colorectal cancer (CRC). The purpose of this study was to analyze the distribution of K-ras mutations in primary tumors and corresponding sentinel lymph nodes (SLNs) from colon cancer patients.MethodsTumor biopsies and SLNs from 158 patients with non-metastatic colon cancer were analyzed for K-ras mutations in codons 12 and 13 by a sensitive and quantitative peptide nucleic acid clamp PCR assay.ResultsAnalyses of single fresh-frozen tumor biopsies revealed K-ras mutations in 67 (42%) of the patients. Apparently low levels of K-ras mutations in 13 of the mutated primary tumors and the presence of K-ras mutations in SLNs from seven patients with a wild-type primary tumor biopsy suggested possible intratumoral heterogeneity for 20 of the patients. To confirm this hypothesis, we analyzed tissue sections from all available formalin-fixed, paraffin-embedded (FFPE) tumor blocks from these 20 patients. Ten of the patients had a mixture of tissue sections positive and tissue sections negative for K-ras mutations, two patients had K-ras mutations in all sections, and eight patients had no detectable K-ras mutations in tumor FFPE tissue blocks. Among these eight patients, five had K-ras mutations detected in SLNs. Thus, evidence supporting a heterogeneous distribution of K-ras mutations was obtained for 15 patients.ConclusionsHeterogeneous distribution of K-ras codon 12 and 13 mutations within primary tumor, or between primary tumor and lymph node metastases, was demonstrated for 15 (20%) of 74 colon cancer patients having K-ras mutations. This may have implications for tissue sampling routines with regard to EGFR-directed therapy of CRC, both in adjuvant and metastatic settings.

Persistent tumor cells in bone marrow of non-metastatic breast cancer patients after primary surgery are associated with inferior outcome

BackgroundTo investigate the prognostic significance of disseminated tumor cells (DTCs) in bone marrow (BM) from non-metastatic breast cancer patients before and after surgery.MethodsPatients with non-metastatic breast cancer were consecutively recruited to this project during the years 1998–2000. Real-time RT-PCR quantification of a DTC multimarker panel consisting of cytokeratin 19, mammaglobin A and TWIST1 mRNA was performed in BM samples obtained from 154 patients three weeks (BM2) and/or six months after surgery (BM3). The results were compared to previously published data from pre-operative BM analyses for the same patients.ResultsDTCs were identified in post-operative BM samples (BM2 and/or BM3) from 23 (15%) of the 154 patients investigated. During a median follow-up of 98 months, 10 (44%) of these patients experienced systemic relapse as compared to 16 (12%) of 131 DTC-negative patients. Kaplan-Meier estimates of systemic recurrence-free- and breast-cancer specific survival demonstrated significantly shorter survival for patients with persistent DTCs in BM after surgery (p≤0.001). By multivariate Cox regression analyses, persistent DTCs after surgery was an independent predictor of both systemic recurrence-free- (HR = 5.4, p < 0.001) and breast-cancer specific survival (HR = 5.3, p < 0.001). Furthermore, the prognostic value of DTCs in BM was similar for pre- and post surgery samples. However, patients with DTCs both before and after surgery (BM1 and BM2/3) had a particularly poor prognosis (systemic recurrence-free survival: HR = 7.2, p < 0.0001 and breast-cancer specific survival: HR = 8.0, p < 0.0001).ConclusionsDetection of persistent DTCs in BM samples obtained after surgery identified non-metastatic breast cancer patients at high risk for systemic relapse, and with reduced breast-cancer specific survival. Furthermore, patients with positive DTC status both before and after surgery had a particularly poor prognosis.

Sensitive and quantitative one-step polymerase chain reaction using capillary electrophoresis and fluorescence detection for measuring cytokeratin 19 expression

An improved quantitative assay to measure cytokeratin 19 (CK19) expression has been developed. The assay utilizes reverse transcription and a one-step polymerase chain reaction (PCR), with capillary electrophoresis and fluorescent labelling, to separate and detect the PCR products. Calibration curves were constructed from a serial dilution of CK19 cDNA coamplified with a fixed amount of CK19 internal standard, which was found to be linear between 10 and 500 molecules. Quantitative measurement of CK19 in samples was carried out by coamplifying the cDNA with a fixed amount of internal standard. The values were calculated from the calibration curve. The integrity of RNA and cDNA synthesis was checked by quantitative measurement of the breakpoint cluster region (BCR) gene expression. The assay is sensitive, detecting < 10 CK19 transcripts, and reproducible with a coefficient of variation of approximately 10%. CK19 expression showed overlapping values when measured in samples from peripheral blood and bone marrow in operable breast cancer patients, in healthy volunteers or patients without epithelial cancer and in blood samples from patients with metastatic breast cancer. As the assay is easier to perform than traditional quantitative competitive PCR assays, it might be useful for quantitative measurement of other specific transcripts.

Thrombotic Microangiopathy in a Patient with Chronic Myelogenous Leukemia on Hydroxyurea

We describe the case of a 57-year-old woman with chronic myelogenous leukemia who was on hydroxyurea and developed a fatal thrombotic microangiopathy with renal, retinal and central nervous system involvement. There was no evidence of medullary or extramedullary leukemia transformation. Repeated examinations of the peripheral blood film revealed only minimal morphological changes of microangiopathic hemolysis. The diagnosis was made by postmortem examination of the kidneys, brain, meninges and retina. The underlying etiology may have been a paraneoplastic phenomenon of the chronic phase of CML or may have indicated the beginning of transformation to an accelerated phase. A late side effect of hydroxyurea therapy cannot be excluded.

Disseminated tumor cells in bone marrow assessed by TWIST1, cytokeratin 19, and mammaglobin A mRNA predict clinical outcome in operable breast cancer patients.

PURPOSE To investigate the prognostic relevance of disseminated tumor cells (DTCs) in bone marrow (BM) assessed by a multimarker mRNA panel consisting of TWIST1, cytokeratin 19 (CK19) and human mammaglobin A (hMAM) mRNA, in patients with early breast cancer. PATIENTS AND METHODS TWIST1 (gene name: TWIST1), CK19 (gene name: KRT19), and hMAM (gene name: SCGB2A2) mRNA was quantitated in BM samples from 191 operable breast cancer patients by real-time reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS Using the highest relative mRNA concentration of TWIST1 in the control population as a cut-off, 5 of the 191 breast cancer patients showed elevated TWIST1 mRNA levels in their BM by real-time RT-PCR. Two of these patients experienced a systemic relapse during a median follow-up of 98 months. Combining these results with previous hMAM and CK19 mRNA quantifications in the same BM samples, 12 (40%) of the 30 patients with BM positive for at least 1 marker (multimarker positive) experienced a systemic relapse as compared with 18 (11%) of the 161 patients with multimarker-negative BMs. The patients with multimarker-positive BM had significantly shorter systemic recurrence-free survival (P < .001, log-rank test), breast cancer-specific survival (P < .001), and overall survival (P = .03). The prognostic relevance of BM multimarker detection appeared to be independent of adjuvant treatment, although the difference was not statistically significant in the subgroup of patients who received adjuvant chemotherapy. Multivariate analysis demonstrated the BM multimarker panel status to be a strong independent predictor of clinical outcome. CONCLUSION Our results demonstrated the prognostic relevance of BM DTCs assessed by a multimarker mRNA panel consisting of TWIST1, CK19, and hMAM in operable breast cancer patients.

Quantitative analysis of cytokeratin 20 gene expression using RT-PCR and capillary electrophoresis with fluorescent DNA detection.

OBJECTIVE We developed a quantitative reverse-transcription polymerase chain reaction (RT-PCR) to determine CK20 expression in colorectal tumor and hematopoietic tissue. DESIGN AND METHODS Our method incorporates a calibrated PCR with an internal competitor and an external standard. RESULTS The RT-PCR assay is sensitive detecting 10 target molecules of CK20 in solution with one round of 38 amplification cycles. Genomic DNA contamination was eliminated by Dnase I digestion of total RNA. The inclusion of a calibrator in the quantitative RT-PCR analysis allowed for a high throughput of unknown samples within the same assay improving comparative analysis between the samples tested. Analysis of peripheral blood and bone marrow from 20 healthy volunteers revealed a low level of CK20 expression in all samples. CONCLUSION To study the clinical significance of CK20 expression as a marker of systemic metastatic disease it is essential to measure CK20 mRNA levels in hematopoietic tissue with sensitive quantitative RT-PCR. A sensitive and reproducible method, which is easily performed, is described.

Detection of occult metastases in sentinel lymph nodes from colon cancer patients by K-ras mutation peptide nucleic acid clamp PCR.

Objective:To identify colon cancer patients with occult lymph node metastases. Summary of Background Data:The prognostic value of regional lymph node (LN) metastases in colorectal cancer patients is well established. The disease recurrences nevertheless experienced by 20% to 30% of the LN negative patients suggest a potential for improvement in current LN diagnostics. We suspect that a subgroup of the patients that are LN negative by routine examination has occult LN metastases that are prognostically relevant. Methods:To identify these patients we applied ex vivo sentinel lymph node (SLN) mapping to colon cancer patients and analyzed the SLNs by a sensitive peptide nucleic acid clamp PCR (polymerase chain reaction) assay for K-ras mutations, using these mutations as a surrogate marker for tumor cells. Results:SLNs were identified in 158 (96%) of 164 prospectively recruited patients with localized colon cancer. Of the 158 patients with successful SLN mapping, 67 (42%) had K-ras mutations detected in their primary tumors. We analyzed the SLNs from these patients by peptide nucleic acid clamp PCR for K-ras mutations and found mutations in SLNs from 35 (52%) patients. At least one SLN from 14 (70%) of 20 patients with histologically proven regional LN metastases was positive for the K-ras mutation test. Interestingly, 21 (45%) of the 47 patients without known LN metastases had K-ras mutations detected in their SLNs. Conclusions:Sensitive detection of K-ras mutations in SLNs from colon cancer patients indicates the presence of occult metastases with potential prognostic implications.