Expression profiling and intracellular localization studies of the novel Proline-, Histidine-, and Glycine-rich protein 1 suggest an essential role in gastro-intestinal epithelium and a potential clinical application in colorectal cancer diagnostics.

BACKGROUND: The primary function of the intestines is the absorption of water and nutrients. Although our knowledge about these processes on the cellular level is extensive, a number of important intracellular elements remain unknown. Here, we characterize the novel proline-, histidine-, glycine-rich 1 (PHGR1) mRNA and protein on the molecular level and propose a functional role of the PHGR1 protein in the intestinal and gastric epithelium. METHODS: PHGR1 mRNA and protein expression in human tissues and cell lines were characterized by quantitative RT-PCR, in situ hybridization, Northern blotting, Western blotting, and immunohistochemistry. Glycosylation was assessed by a chemical deglycosylation assay, whereas intracellular localization was studied by immunofluorescent staining of cell line cells. PHGR1 mRNA levels in HT29 cells was reduced by RNA interference and the resulting global changes in gene expression assessed by microarray hybridization. RESULTS: PHGR1 mRNA and protein were found to be expressed specifically in epithelial cells of intestinal mucosa, with the highest expression in the most mature and differentiated cells. PHGR1 protein was found to be glycosylated and to localize to both the cytoplasm and nucleus. Transcript profiling and gene ontology analysis of HT29 cells subjected to PHGR1 knockdown suggested a functional relationship with transport and metabolic processes. Examination of PHGR1 mRNA and protein levels in lymph nodes with known colorectal cancer metastases indicated that they may serve as biomarkers for detection of such metastases. CONCLUSIONS: Functional analyses of the novel PHGR1 mRNA and protein suggest an essential role in gastrointestinal epithelium and a clinical application in detection of colorectal cancer lymph node metastases.

Hospital surgical volume and colorectal cancer survival in Norway: A nationwide cohort study.

BACKGROUND: Studies of hospital surgical volume and colorectal cancer survival are inconclusive. We investigated whether surgical volume was associated with survival of patients operated for colorectal cancer in Norway. METHODS: Using Cancer Registry of Norway data, we compared excess mortality from colorectal cancer by hospital surgical volume among 26,989 colon and 9779 rectal cancer patients diagnosed 2009-2020 and followed-up to 31.12.2021. Hospitals were divided into terciles according to their three-year average annual surgical volume; colon: low (< 22), middle (22-73), high (> 73); rectal: low (< 17), middle (17-38), high (> 38). We estimated excess hazard ratios (EHR) with flexible parametric models adjusted for age, year, stage, surgical urgency and surgery location (within/outside patient's residential health trust). RESULTS: Low-volume hospitals had the highest proportion of late-stage or acutely operated colon cancer patients. Colon cancer patients operated at low- versus high-volume hospitals had significantly increased crude excess mortality (EHR = 1.30; 95 % CI = 1.14-1.48) but no difference after adjustment for age, year, and stage (EHR = 0.97; 0.85-1.11). High-volume hospitals had the highest proportion of late-stage rectal cancer patients and patients operated outside their residential area. Rectal cancer patients operated at low- versus high-volume hospitals did not have significantly different excess mortality before (EHR = 0.84; 0.64-1.10) or after (EHR = 1.03; 0.79-1.35) adjustment for age, year, stage, surgical urgency and surgery location. After accounting for case-mix, hospital surgical volume was not associated with excess mortality from colon (P = 0.40) or rectal cancer (P = 0.22). CONCLUSION: Low hospital surgical volume was not associated with poorer colorectal cancer survival.

Prognostic relevance of occult metastases detected by cytokeratin 20 and mucin 2 mRNA levels in sentinel lymph nodes from colon cancer patients.

PURPOSE: To investigate the prognostic value of occult metastases detected by quantitative measurements of candidate biomarkers in sentinel lymph nodes (SLNs) from patients curatively resected for colon cancer. METHODS: Resection specimens from consecutive patients undergoing surgery for localized colon cancer were subjected to ex vivo SLN mapping. SLNs were examined for the presence of metastases by routine hematoxylin-erythrosin-safranin staining and by cytokeratin 20 (CK20) and mucin 2 (MUC2) mRNA quantification. The patients were stratified according to KRAS and BRAF mutation status and microsatellite instability status in their primary tumors. Survival end points were analyzed by Kaplan-Meier survival estimates and log-rank tests. RESULTS: A total of 817 SLNs were identified in 206 (97 %) of the 213 included patients. Routine histological examination of SLNs and other regional lymph nodes identified 63 patients with positive nodes (pN+), of which 42 (67 %) were positive in one or more SLNs (sensitivity 67 %, false-negative rate 33 %). On the basis of the CK20 and MUC2 mRNA levels in SLNs, occult metastases were suggested in 86 (60 %) and 52 (36 %) of the 143 otherwise LN-negative (pN0) patients, respectively. Survival analysis with a median 3.6-year follow-up revealed that MUC2 mRNA quantification had significant prognostic value in SLNs from all patients; however, occult SLN metastasis detection did not. CONCLUSIONS: Occult SLN metastases detected by CK20 and MUC2 mRNA quantification had limited prognostic value.

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

BACKGROUND: To investigate the prognostic significance of disseminated tumor cells (DTCs) in bone marrow (BM) from non-metastatic breast cancer patients before and after surgery. METHODS: Patients 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. RESULTS: DTCs 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). CONCLUSIONS: Detection 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.

Oligorecurrent nodal prostate cancer: Radiotherapy quality assurance of the randomized PEACE V-STORM phase II trial.

PURPOSE: Aim of this study is to report the results of the radiotherapy quality assurance program of the PEACE V-STORM randomized phase II trial for pelvic nodal oligorecurrent prostate cancer (PCa). MATERIAL AND METHODS: A benchmark case (BC) consisting of a postoperative case with 2 nodal recurrences was used for both stereotactic body radiotherapy (SBRT, 30 Gy/3 fx) and whole pelvic radiotherapy (WPRT, 45 Gy/25 fx + SIB boost to 65 Gy). RESULTS: BC of 24 centers were analyzed. The overall grading for delineation variation of the 1st BC was rated as 'UV' (Unacceptable Variation) or 'AV' (Acceptable Variation) for 1 and 7 centers for SBRT (33%), and 3 and 8 centers for WPRT (46%), respectively. An inadequate upper limit of the WPRT CTV (n = 2), a missing delineation of the prostate bed (n = 1), and a missing nodal target volume (n = 1 for SBRT and WPRT) constituted the observed 'UV'. With the 2nd BC (n = 11), the overall delineation review showed 2 and 8 'AV' for SBRT and WPRT, respectively, with no 'UV'. For the plan review of the 2nd BC, all treatment plans were per protocol for WPRT. SBRT plans showed variability in dose normalization (Median D90% = 30.1 Gy, range 22.9-33.2 Gy and 30.6 Gy, range 26.8-34.2 Gy for nodes 1 and 2 respectively). CONCLUSIONS: Up to 46% of protocol deviations were observed in delineation of WPRT for nodal oligorecurrent PCa, while dosimetric results of SBRT showed the greatest disparities between centers. Repeated BC resulted in an improved adherence to the protocol, translating in an overall acceptable contouring and planning compliance rate among participating centers.

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

PURPOSE: K-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. METHODS: Tumor 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. RESULTS: Analyses 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. CONCLUSIONS: Heterogeneous 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.

Regional variation in cancer survival in Norway.

BACKGROUND: Cancer services in Norway are intended to provide high quality services and equal access for all citizens. Still, regional variation in cancer survival has been reported. Currently, the public hospitals are organized in Health Trusts (HTs), respectively within one of four regional trusts (RHTs). We aimed to evaluate the extent and rank pattern of regional and intraregional variation in cancer survival systematically over the last three decades. We postulated that organizational reforms during this period might have modulated the variation. METHODS: Excess hazard ratios (EHR) of death from cancer were estimated for all individuals identified in The Cancer Registry of Norway as diagnosed with cancer from 1984 to 2018. The model covariates included continuous age at diagnosis, sex, cancer site, stage, 5-year time period of diagnosis and place of residence. In addition to analyses for all cancers combined, selected cohorts with predominantly centralized vs. not centralized primary surgery were evaluated. RESULTS: For all cancer sites combined and for the centralized surgery cohort, the range of variation in EHR among the four regions was in the order of 0.10. The ranks among the regions were fairly consistent over time. For the not centralized surgery cohort, the range of inter-regional EHR-variation was in the order of 0.10 - 0.15, with no consistent ranks. Intra-regionally, the ranges of EHR-variation were similar, but with more complex rank patterns. CONCLUSIONS: The range of inter- and intra-regional variation in cancer survival was minor, as compared to the general improvement in cancer survival in the period, with no evidence of effect from organizational reforms on regional variation.

The Multicenter, Randomized, Phase 2 PEACE V-STORM Trial: Defining the Best Salvage Treatment for Oligorecurrent Nodal Prostate Cancer Metastases.

Optimal local treatment for nodal oligorecurrent prostate cancer is unknown. The randomized phase 2 PEACE V-STORM trial will explore the best treatment approach in this setting. Early results on the acute toxicity profile are projected to be published in quarter 3, 2021.

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.

[Recurrence and survival after breast-conserving treatment of breast cancer]. / Residiv og overlevelse etter brystbevarende behandling av brystkreft.

BACKGROUND: The objective of this retrospective study was to determine, within one institution, local relapse rate and survival for women with early-stage breast cancer treated with breast-conserving surgery followed by radiotherapy. MATERIAL AND METHODS: All women with infiltrating early-stage breast cancer who underwent post-operative whole breast irradiation at our institution in the period 14.06.99-8.03.2002 were included in the study. A CT-based 3D dose calculation was performed in all patients. RESULTS: 222 women received 50 Gy whole-breast irradiation after breast-conserving surgery in the study period. 51 patients received adjuvant systemic therapy according to national guidelines. Median age at diagnosis was 59 years (34-82 years). Median tumour size was 12 mm (1-30 mm) and 86.5 % of the patients were N0. During a follow-up (median) of 96 months (28-111 months), local recurrence was observed in three of 222 patients (1.4 %; 95 % CI [0.5-4.4 %]) in the ipsilateral breast. The estimated 8-year breast-cancer specific survival was 95 % and total survival was 90 %. INTERPRETATION: Our data demonstrate excellent local disease control in women with low-risk early-stage breast cancer (about 25 % were on adjuvant systemic therapy) who undergo breast-conserving surgery and 50 Gy whole-breast irradiation.

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.

A small subgroup of operable breast cancer patients with poor prognosis identified by quantitative real-time RT-PCR detection of mammaglobin A and trefoil factor 1 mRNA expression in bone marrow.

PURPOSE: The utility of three different epithelial mRNA markers to detect clinically significant, disseminated tumour cells in bone marrow (BM) was explored. METHODS: Mammaglobin A (hMAM), trefoil factor 1 (TFF-1) and prostate derived Ets factor (PDEF) mRNA were quantitated by real-time RT-PCR in BM samples from 192 breast cancer patients undergoing surgery (control group: 26 healthy women). RESULTS: During a median follow-up of 72 months, four of the five hMAM BM-positive and three of the seven TFF-1 BM-positive patients experienced a systemic relapse. Kaplan-Meier survival analyses demonstrated significantly shorter recurrence-free-, breast-cancer-specific- and overall survival for both hMAM and TFF-1 BM-positive patients. In contrast, PDEF mRNA quantitation did not reveal any significant differences in the survival analyses. Multivariate Cox regression demonstrated hMAM mRNA BM expression to be an independent predictor of both overall- (hazard ratio = 5.896), breast-cancer-specific- (hazard ratio = 10.208) and systemic-recurrence-free survival (hazard ratio = 14.304). TFF-1 status was related to hMAM status (P < 0.001). CONCLUSION: Breast cancer patients with pre-operative elevated BM levels of hMAM and/or TFF-1 mRNA seem to constitute a small group of patients with a very poor prognosis.

The potential of cytokeratin 20 and mucin 2 mRNA as metastasis markers in regional lymph nodes of colon cancer patients investigated by quantitative RT-PCR.

PURPOSE: The presence of regional lymph node metastases is one of the most important prognostic factors in colon cancer. Nevertheless, up to 30% of the lymph node negative patients experience disease recurrence. Possibly, this patient group may be identified by more sensitive techniques than routine histopathological examination of the lymph nodes. METHODS: In the present study, we have evaluated the detection of colon cancer lymph node metastases by real-time RT-PCR quantitation of the epithelial-specific cytokeratin 20 (CK20) and mucin 2 (MUC2) mRNAs. RESULTS: Both assays were able to detect dilutions of tumor cells down to one tumor cell in 10(6) normal lymphocytes. CK20 and MUC2 mRNA were quantitated in 52 normal lymph nodes from 12 patients undergoing surgery for benign bowel diseases and in 144 primary colon tumors. The median tumor level of both markers were more than 10(4)-fold higher than the highest level in normal lymph nodes, indicating that the markers had a potential for metastasis detection in a clinical context. We applied the assays to 61 lymph nodes with known metastases detected by routine staining. Elevated CK20 or MUC2 mRNA levels were detected in 57 (95%) of the 61 LNs. CONCLUSIONS: Thus, CK20 and MUC2 quantitation by real-time RT-PCR seems to be a promising, sensitive tool to detect metastases in regional lymph nodes from colon cancer patients.

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.

The Prognostic Relevance of Sentinel Lymph Node Metastases Assessed by PHGR1 mRNA Quantification in Stage I to III Colon Cancer.

BACKGROUND: Regional lymph node (LN) metastasis is a strong and well-established prognostic factor in colon cancer, and recent data suggest a prognostic value of detecting micrometastases and isolated tumor cells in regional LNs. The aim of the study was to investigate the clinical relevance of detecting sentinel lymph node (SLN) metastases in colon cancer patients by measuring the novel metastasis marker PHGR1 mRNA. METHODS: Using quantitative reverse-transcription polymerase chain reaction, we measured PHGR1 mRNA levels in SLNs and primary tumors from 206 patients surgically treated for stage I to III colon cancer and 52 normal LNs from patients undergoing surgery for benign colon diseases. The prognostic impact of these findings was evaluated by Kaplan-Meier analysis and Cox proportional-hazards regression. RESULTS: Compared to normal LNs, elevated PHGR1 mRNA levels were detected in SLNs from 56 (89%) of the 63 patients with pN+ disease. Furthermore, 68 (48%) of the 143 node-negative (pN0) patients had elevated PHGR1 mRNA levels in SLNs, suggesting occult metastases. With a median follow-up of 7.2 years, a significantly shorter recurrence-free (P=.005) and disease-specific (P=.02) survival was observed in patients with elevated PHGR1 mRNA levels in SLNs. Multivariable modeling showed that the SLN PHGR1 mRNA level was an independent prognostic factor. However, when the survival analyses were restricted to pN0 patients, no significant prognostic information was found. CONCLUSION: Measuring PHGR1 mRNA in SLNs provided independent prognostic information on operable colon cancer patients but not in the pN0 subgroup.

[Sentinel lymph nodes in colorectal cancer]. / Vaktpostlymfeknuter ved kolorektal kreft.

BACKGROUND: Metastasis in regional lymph nodes is one of the most important prognostic factors in colorectal cancer. Nevertheless, recurrence may be expected in about 20% of lymph-node-negative patients. Detection of the lymph node(s) most likely to contain potential metastases, denoted the sentinel node(s), would be expected to contribute to the identification of patients with poor prognosis even if they have no lymph node metastases detected by routine analysis. MATERIAL AND METHODS: The literature databases PubMed and EMBASE were systematically searched with combinations of the words "sentinel, lymph node, node, colon, colorectal, rectum, carcinoma and cancer". Relevant reviews and original articles among primary hits, and cross-references therein, were extracted for further study. RESULTS AND INTERPRETATION: Mapping of sentinel lymph node(s) in patients with colorectal cancer is feasible. However, a standard histopathological analysis restricted to the sentinel lymph nodes is associated with too high false negative rates to be a realistic alternative to the present routine analysis of regional lymph nodes. New prospective studies are required to clarify whether immunohistochemical or molecular biological analysis of sentinel nodes can identify patients with a poor prognosis even though they have no lymph node metastases detected by routine analysis.

[Radiotherapy after breast-conserving surgery in the treatment of breast cancer: early results from a non-university hospital in Norway]. / Strålebehandling etter brystbevarende kirurgi.

BACKGROUND: In 1998 the first radiotherapy unit located outside a university hospital in Norway was established at Rogaland Central Hospital. MATERIAL AND METHODS: Results from 222 consecutive patients treated between June 1999 and March 2002 are presented. Median time to follow up was 25 months (range 8-41). All patients underwent a lumpectomy combined with a complete axillary dissection or a sentinel node biopsy. The entire breast was irradiated using 6MV photon energy to a total dose of 50 Gy. RESULTS: As of October 2002, there has not been registered any local breast failures. Three patients developed distant metastases and subsequently died from their disease. Contralateral breast cancer has occurred in one patient. The relative number of patients treated with breast conservation therapy, as compared to the total number of patients operated, has not changed after a unit of radiotherapy was established locally. INTERPRETATION: Our findings show that radiotherapy after breast-conserving surgery can be performed safely in a non-university hospital such as Rogaland Central Hospital.

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.

Bone marrow cytokeratin 19 mRNA level is an independent predictor of relapse-free survival in operable breast cancer patients.

PURPOSE: To study the prognostic significance of elevated cytokeratin 19 (CK19) mRNA levels in the bone marrow (BM) of operable breast cancer patients. PATIENTS AND METHODS: From 1998 to 2000, BM was collected from 195 consecutive breast cancer patients immediately prior to surgery and from 34 healthy volunteers. The patients received surgical and adjuvant treatment according to national guidelines at the time. We analyzed the level of CK19 mRNA in the BM samples from patients and normal controls using a real-time RT-PCR assay. The associations with known prognostic factors and the impact of pathological CK19 mRNA levels on patients' prognosis were investigated. RESULTS: Using the 99 percentile of the normal control group as a cut-off, 24 (12%) of the 195 patients and 1 (3%) of the 34 volunteers were diagnosed as CK19 mRNA positive. There was no correlation between CK19 BM status and the clinicopathological factors tested. During a median follow-up of 72 months, 7 (29%) of the 24 CK19 mRNA BM positive patients experienced systemic relapse compared to 20 (12%) of the 171 in the CK19 mRNA negative group. The patients with CK19 mRNA-positive BM had significantly shorter systemic recurrence-free survival (P=0.01) and overall recurrence-free survival (P=0.005). Multivariate Cox regression showed CK19 mRNA BM status to be an independent predictor of relapse. CONCLUSION: Detection of CK19 mRNA in the BM of breast cancer patients by real-time RT-PCR is an independent predictor of relapse-free survival in operable breast cancer patients.

Error propagation in relative real-time reverse transcription polymerase chain reaction quantification models: the balance between accuracy and precision.

Real-time reverse transcription polymerase chain reaction (RT-PCR) has gained wide popularity as a sensitive and reliable technique for mRNA quantification. The development of new mathematical models for such quantifications has generally paid little attention to the aspect of error propagation. In this study we evaluate, both theoretically and experimentally, several recent models for relative real-time RT-PCR quantification of mRNA with respect to random error accumulation. We present error propagation expressions for the most common quantification models and discuss the influence of the various components on the total random error. Normalization against a calibrator sample to improve comparability between different runs is shown to increase the overall random error in our system. On the other hand, normalization against multiple reference genes, introduced to improve accuracy, does not increase error propagation compared to normalization against a single reference gene. Finally, we present evidence that sample-specific amplification efficiencies determined from individual amplification curves primarily increase the random error of real-time RT-PCR quantifications and should be avoided. Our data emphasize that the gain of accuracy associated with new quantification models should be validated against the corresponding loss of precision.