FAIRLANE, a double-blind placebo-controlled randomized phase II trial of neoadjuvant ipatasertib plus paclitaxel for early triple-negative breast cancer.

BACKGROUND: This hypothesis-generating trial evaluated neoadjuvant ipatasertib-paclitaxel for early triple-negative breast cancer (TNBC). PATIENTS AND METHODS: In this randomized phase II trial, patients with early TNBC (T ≥ 1.5 cm, N0-2) were randomized 1 : 1 to receive weekly paclitaxel 80 mg/m2 with ipatasertib 400 mg or placebo (days 1-21 every 28 days) for 12 weeks before surgery. Co-primary end points were pathologic complete response (pCR) rate (ypT0/TisN0) in the intention-to-treat (ITT) and immunohistochemistry phosphatase and tensin homolog (PTEN)-low populations. Secondary end points included pCR rate in patients with PIK3CA/AKT1/PTEN-altered tumors and pre-surgery response rates by magnetic resonance imaging (MRI). RESULTS: pCR rates with ipatasertib versus placebo were 17% versus 13%, respectively, in the ITT population (N = 151), 16% versus 13% in the immunohistochemistry PTEN-low population (N = 35), and 18% versus 12% in the PIK3CA/AKT1/PTEN-altered subgroup (N = 62). Rates of overall and complete response (CR) by MRI favored ipatasertib in all three populations (CR rate 39% versus 9% in the PIK3CA/AKT1/PTEN-altered subgroup). Ipatasertib was associated with more grade ≥3 adverse events (32% versus 16% with placebo), especially diarrhea (17% versus 1%). Higher cycle 1 day 8 (C1D8) immune score was significantly associated with better response only in placebo-treated patients. All ipatasertib-treated patients with low immune scores and a CR had PIK3CA/AKT1/PTEN-altered tumors. CONCLUSIONS: Adding ipatasertib to 12 weeks of paclitaxel for early TNBC did not clinically or statistically significantly increase pCR rate, although overall response rate by MRI was numerically higher with ipatasertib. The antitumor effect of ipatasertib was most pronounced in biomarker-selected patients. Safety was consistent with prior experience of ipatasertib-paclitaxel. A T-cell-rich environment at C1D8 had a stronger association with improved outcomes in paclitaxel-treated patients than seen for baseline tumor-infiltrating lymphocytes. This dependency may be overcome with the addition of AKT inhibition, especially in patients with PIK3CA/AKT1/PTEN-altered tumors. CLINICALTRIALS.GOV: NCT02301988.

The cancer registry as an ally in monitoring treatment effectiveness.

OBJECTIVE: To evaluate if the cancer registry database can be used to monitor treatment effectiveness using nivolumab treatment of non-small cell lung cancer (NSCLC) as an example. METHOD: An observational inception cohort was used, where all registered cases of NSCLC with authorisation to initiate treatment with nivolumab were monitored retrospectively to evaluate disease characteristics and response to prior treatments. Current exposure to nivolumab was prospectively characterised and treatment outcomes classified based on the clinical information registered in the patient medical record. The main outcome measure used to assess treatment effectiveness was overall survival (OS). Secondary outcomes considered were progression free survival (PFS) as a measure of effectiveness and occurrence of Adverse Drug Reaction (ADRs) as a measure of safety. Data were analysed using SPSS, version 24. RESULTS: A total of 115 patients received treatment with nivolumab for NSCLC, between November 1st 2015 and July 31st 2016, and were registered in the database. The majority were non-squamous type (n=107). The median OS was 11.4 months {CI95%: 11.1-11.7}, with a 1-year survival of 44%, in line with clinical trial data. Median PFS was 5.4 months {CI95%: 2.8-7.9}. Treatment was discontinued in 82 cases, most frequently due to disease progression. There were 38 cases of ADRs documented in the patient medical chart, 21 of which led to treatment discontinuation. CONCLUSION: The analysed data suggest that the cancer registry is a powerful tool to monitor treatment effectiveness, although considerable investment is needed to improve the medical culture of recording treatment exposure, particularly documentation of ADRs.

Benefits and risks of adjuvant treatment with zoledronic acid in stage II/III breast cancer. 10 years follow-up of the AZURE randomized clinical trial (BIG 01/04).

Adjuvant bisphosphonates improve disease outcomes in postmenopausal early breast cancer (EBC) but the long-term effects are poorly described. The AZURE trial (ISRCTN79831382) was designed to determine whether adjuvant zoledronic acid (ZOL) improves disease outcomes in EBC. Previous analyses showed no effect on overall outcomes but identified benefits in postmenopausal women. Here we present the long-term risks and benefits of adjuvant ZOL with 10-years follow-up. PATIENTS AND METHODS: 3360 patients with stage II/III breast cancer were included in an academic, international, phase III, randomized, open label trial. Patients were followed up on a regular schedule until 10 years. Patients were randomized on a 1:1 basis to standard adjuvant systemic therapy +/- intravenous ZOL 4 mg every 3-4 weeks x6, and then at reduced frequency to complete 5 years treatment. The primary outcome was disease free survival (DFS). Secondary outcomes included invasive DFS (IDFS), overall survival (OS), sites of recurrence, skeletal morbidity and treatment outcomes according to primary tumor amplification of the transcription factor, MAF. Pre-planned subgroup analyses focused on interactions between menopausal status and treatment effects. RESULTS: With a median follow up of 117 months [IQR 70.4-120.4), DFS and IDFS were similar in both arms (HRDFS  = 0.94, 95%CI = 0.84-1.06, p = 0.340; HRIDFS  = 0.91, 95%CI = 0.82-1.02, p = 0.116). However, outcomes remain improved with ZOL in postmenopausal women (HRDFS  = 0.82, 95%CI = 0.67-1.00; HRIDFS  = 0.78, 95%CI = 0.64-0.94). In the 79% of tested women with a MAF FISH negative tumor, ZOL improved IDFS (HRIDFS  = 0.75, 95%CI = 0.58-0.97) and OS HROS  = 0.69, 95%CI = 0.50-0.94), irrespective of menopause. ZOL did not improve disease outcomes in MAF FISH + tumors. Bone metastases as a first DFS recurrence (BDFS) were reduced with ZOL (HRB-DFS  = 0.76, 95%CI = 0.63-0.92, p = 0.005). ZOL reduced skeletal morbidity with fewer fractures and skeletal events after disease recurrence. 30 cases of osteonecrosis of the jaw in the ZOL arm (1.8%) have occurred. CONCLUSIONS: Disease benefits with adjuvant ZOL in postmenopausal early breast cancer persist at 10 years of follow-up. The biomarker MAF identified a patient subgroup that derived benefit from ZOL irrespective of menopausal status.

Hospital resource utilization and treatment cost of skeletal-related events in patients with metastatic breast or prostate cancer: estimation for the Portuguese National Health System.

BACKGROUND: Skeletal-related events (SREs) occur frequently in patients with bone metastases as a result of breast (BC) and prostate (PC) cancers. They increase both morbidity and mortality and lead to extensive health-care resource utilization. METHODS: Health care resource utilization by BC/PC patients with at least one SRE during the preceding 12 months was assessed through retrospective chart review. SRE-treatment costs were estimated using the Portuguese Ministry of Health cost database and analyzed using generalized linear models. RESULTS: This study included 152 patients from nine hospitals. The mean (SD) annual SRE-treatment cost per patient was €5963 (€3646) and €5711 (€4347), for BC (n=121) and PC (n=31) patients, respectively. Mean cost per single episode ranged between €1485 (radiotherapy) and €13,203 (spinal cord compression). Early onset of bone metastasis (P = 0.03) and diagnosis of bone metastases at or after the occurrence of the first SRE (P < 0.001) were associated with higher SRE-treatment costs. CONCLUSION: These results reveal the high hospital SRE-treatment costs, highlighting the need for early diagnosis and treatment, and identify key factors determining the economic value of therapies for patients with skeletal metastases.

Suboptimal survival of male germ-cell tumors in southern Portugal--a population-based retrospective study for cases diagnosed in 1999 and 2000.

BACKGROUND: The outcome of germ-cell tumors (GCTs) is a hallmark of good-quality cancer care. In the Eurocare-4 study, the mean 5-year survival for patients diagnosed from 1995 to 1999 was 95.5%. PATIENTS AND METHODS: We carried out a population-based retrospective chart review study of male patients diagnosed with GCT in 1999 and 2000 in southern Portugal (2 119 065 males). RESULTS: There were 87 GCTs-79 testicular, 2 retroperitoneal, 3 mediastinal, 2 of the central nervous system and 1 of the stomach. For the 81 patients with testicular or retroperitoneal primaries, 35 had stage I, 13 stage II and 30 stage III at presentation (3 unknown). Classification by International Germ Cell Consensus Classification Group criteria, 17 belonged to the poor prognosis group (mediastinal primary 3, liver metastases 11 and very elevated markers 3). With median follow-up of 89 months, the 5-year absolute overall survival was 80% (100% for stage I, 92% for stage II and 53% for stage III disease). CONCLUSIONS: While GCT incidence was similar to neighboring Spain, the 5-year overall survival was lower than that of other European countries. This may result from delays in diagnosis, suggested by high proportion of high-stage and large-burden disease, and poor adherence to recommended treatment algorithms.

[High dose chemotherapy with autologous stem-cell support in germ cell tumors: The Instituto Português de Oncologia de Lisboa Francisco Gentil Series]. / Quimioterapia de alta dose com suporte hematopoiético autólogo em tumores de células germinativas Experiência do Instituto Português de Oncologia de Lisboa Francisco Gentil.

BACKGROUND: High dose chemotherapy with autologous stem cell transplantation (HDCT-ASCT) has been administered to patients with high-risk germ cell tumours (GCT). The role of this treatment for GCT still remains unclear, including the identification of subgroups more likely to benefit from such strategy. METHODS: A retrospective review was conducted of all male patients with gonadal and extra gonadal GCT treated with HDCT-ASCT between 1996 and 2008 at the Instituto Português de Oncologia de Lisboa Francisco Gentil (IPOLFG). RESULTS: Twenty patients were treated with HDCT-ASCT, 17 with primary testicular tumours, two mediastinal and one retroperitoneal GCT. According to the International Germ Cell Cancer Consensus Group (IGCCCG) classification, at diagnosis three patients had good risk, four intermediate, eight poor but for the patients left the risk group could not be ascertained. In eight patients HDCT-ASCT was used upfront, after induction with first-line conventional chemotherapy, and in the remaining 12 for relapsed GCT. One patient had platinum-resistant and another platinum-refractory disease. Only two patients had Beyer score > 2. All but one patient were treated with ICE (Ifosfamide, Carboplatin, Etoposide). Six patients underwent a second HDCT-ASCT course. The 5-year overall survival and progression free survival were respectively 53% and 44%; both patients with mediastinal GCT are long term survivors. CONCLUSION: Randomized studies to date have failed to indicate a survival advantage for HDCT-ASCT in GCT. This series is small and heterogeneous which prevents us from drawing conclusions regarding the benefit of this treatment for GCT. However, we could confirm the lack of benefit of HDCT-ASCT for platinum-resistant GCT and to question the absolute contraindication to this therapeutic modality in mediastinal GCT. HDCT-ASCT should therefore be performed exclusively in experienced centers and, preferably, in the setting of clinical trials.

Statistical classification of road pavements using near field vehicle rolling noise measurements.

Low noise surfaces have been increasingly considered as a viable and cost-effective alternative to acoustical barriers. However, road planners and administrators frequently lack information on the correlation between the type of road surface and the resulting noise emission profile. To address this problem, a method to identify and classify different types of road pavements was developed, whereby near field road noise is analyzed using statistical learning methods. The vehicle rolling sound signal near the tires and close to the road surface was acquired by two microphones in a special arrangement which implements the Close-Proximity method. A set of features, characterizing the properties of the road pavement, was extracted from the corresponding sound profiles. A feature selection method was used to automatically select those that are most relevant in predicting the type of pavement, while reducing the computational cost. A set of different types of road pavement segments were tested and the performance of the classifier was evaluated. Results of pavement classification performed during a road journey are presented on a map, together with geographical data. This procedure leads to a considerable improvement in the quality of road pavement noise data, thereby increasing the accuracy of road traffic noise prediction models.

The effects of adding zoledronic acid to neoadjuvant chemotherapy on tumour response: exploratory evidence for direct anti-tumour activity in breast cancer.

BACKGROUND: Pre-clinical studies have demonstrated synergistic anti-tumour effects of chemotherapy (CT) and zoledronic acid (ZOL). Within the AZURE trial, designed to determine whether the addition of ZOL to neoadjuvant therapy improves disease outcomes, a subgroup received neoadjuvant CT. We report a retrospective evaluation comparing pathological response in the primary tumour between treatment groups. METHODS: In total, 205 patients received neoadjuvant CT+/-ZOL (CT+ZOL, n=102; CT, n=103). The primary end point was pathologically assessed residual invasive tumour size (RITS) at surgery. Secondary end points were pathological complete response (pCR) rate and axillary nodal involvement. Following review of surgical pathology reports (n=195), outcome differences between groups were assessed adjusting for potential response modifiers. RESULTS: Baseline characteristics and CT treatments were similar. In multivariate analysis, allowing for biological and clinical factors known to influence tumour response, the adjusted mean RITS in CT and CT+ZOL groups were 27.4 and 15.5 mm, respectively, giving a difference in means of 12 mm (95% confidence interval: 3.5-20.4 mm; P=0.006). The pCR rate was 6.9% in the CT group and 11.7% in the CT+ZOL group (P=0.146). There was no difference in axillary nodal involvement (P=0.6315). CONCLUSION: These data suggest a possible direct anti-tumour effect of ZOL in combination with CT, warranting formal evaluation in prospective studies.

[Guidelines for prevention of febrile neutropenia]. / Orientações para a prevenção da neutropenia febril em doentes submetidos a quimioterapia.

Neutropenia and febrile neutropenia are common consequences of some cytotoxic chemotherapy regimens. This situation leads to modifications of the therapeutic regimen, conducting to either dose reduction or cycle delays. Granulocyte colony stimulating factors are commonly used to minimize chemotherapy cytotoxic effect on the granulocytic series. The objective of this study is to assess the available evidence in what concerns the efficacy and safety of granulocyte colony stimulating factors, in several settings of their use. An extensive bibliographic review was performed, including clinical trials, observational studies, systematic reviews, and international guidelines for neutropenia prophylaxis, which aims to establish recommendations on their use, in adequacy to the National reality.

The aggressiveness of cancer care in the last three months of life: a retrospective single centre analysis.

BACKGROUND: There is concern that terminally ill cancer patients are over treated with chemotherapy, even when such treatment is unlikely to palliate symptoms. The study objective was to evaluate the use of chemotherapy in the last three months of life in a cohort of adult patients with advanced solid tumours. METHODS: All adult patients with solid tumours who died in our hospital in 2003 and received chemotherapy for advanced cancer, were included. Detailed data concerning chemotherapy and toxicity, in the last three months of life, were collected from patients' clinical charts. RESULTS: A total of 319 patients were included. Median age was 61 years. Median time from diagnosis of metastatic disease to death was 11 months. The proportion of patients who received chemotherapy in the last three months of life was 66% (n = 211), in the last month 37% and in the last two weeks 21%. Among patients who received chemotherapy in the last three months of life, 50% started a new chemotherapy regimen in this period and 14% in the last month. There was an increased probability of receiving chemotherapy in the last three months of life in younger patients and in patients with breast, ovarian and pancreatic carcinomas. CONCLUSION: There was a large proportion of patients who received chemotherapy in the last three months of life, including initiation of a new regimen within the last 30 days. Thus, further study is needed to evaluate if such aggressive attitude results in better palliation of symptoms at the end of life.

Congenital amegakaryocytic thrombocytopenia--report of a new c-mpl gene missense mutation.

A 44-month old girl with congenital amegakaryocytic thrombocytopenia, already with pancytopenia, underwent an unrelated allogeneic cord blood transplantation with recovery of normal blood cell counts. The patient was a compound heterozygote for two c-mpl missense mutations inherited from both parents, one of them, a G578A exon 4 mutation leading to a cysteine to tyrosine replacement of codon 193, previously unreported.

Ancestim (recombinant human stem cell factor, SCF) in association with filgrastim does not enhance chemotherapy and/or growth factor-induced peripheral blood progenitor cell (PBPC) mobilization in patients with a prior insufficient PBPC collection.

Up to a third of autologous transplantation candidates fail to mobilize hematopoietic progenitors into the peripheral blood with chemotherapy and/or growth factor treatment, thus requiring innovative mobilization strategies. In total, 20 cancer patients unable to provide adequate PBPC products after a previous mobilization attempt were treated with ancestim (20 microg/kg/day s.c.) and filgrastim (10 microg/kg/day s.c.). In 16 patients, the pre-study mobilization was with filgrastim alone. Eight patients underwent single large volume leukapheresis (LVL) and 12 multiple standard volume leukaphereses (SVL) in both mobilizations. Pairwise comparison of peripheral blood CD34(+) cell concentrations on the day of first leukapheresis failed to document synergism - median CD34(+)/microl of 3.2 (<0.1 to 15.4) and 4.5 (1-28.56) for the pre-study and on-study mobilizations (P = 0.79, sign test), and 4.2 (<0.1-15.4) and 5 (1-28.56), respectively, for the 16 patients previously mobilized with filgrastim alone (P = 1, sign test). The number of CD34(+) cells/kg collected per unit of blood volume (BV) processed was similar in both mobilizations - median 0.1 x 10(6)/kg/BV and 0.09 x 10(6)/kg/BV, respectively (P = 1, sign test). In this phase II study, the combination of ancestim and filgrastim did not allow adequate PBPC mobilization and collection in patients with a previous suboptimal PBPC collection.

Aumento do torque muscular apos tratamento em esteira com suporte parcial de peso em pacientes com hemiparesia cronica / Muscular torque increase after treadmill training with body weigth support in chronic hemiparesis

A melhora da marcha e a meta mais comum apos um Acidente Vascular Cerebral(AVC), sendo assim, o objetivo deste estudo foi compara o trino da marcha sobre esteira eletrica com suporte parcial de peso corporal por meio da tecnica Kabatm em pacientrs com hemiparesia cronica. Essa comparacao foi feita pela analise das bvariaveis espaco-temporais e do toque muscular em maxima contracao isometrica dos flexores plantares. Doze pacientes com hemiparesia cronica causada por um AVC na regiao da arteria cerebral media completaram o estudo. O sistema A-B-A de analise comparou o trinamento em esteira com suporte parcial de peso corporal (A) a fisioterapia com base no metod Kabat(B), cada fase durando tres semanas. O intervalo minimo apos o AVC foi de seis meses. Observamos que o treinamento em esteira foi mais efetivo no aumento em ciclo (p<0,01), a velocidade da marcha (p<0,01) e torque muscular quando comparado ao metodo Kabat em sujeitos com hemiparesia cronica

Differential expression of polygalacturonase-encoding genes from Penicillium griseoroseum in different carbon sources.

A second polygalacturonase-encoding gene (pgg2) of Penicillium griseoroseum was cloned and consists of an opening reading frame of 1107 bp after removal of two introns. The gene encodes a protein of 369 amino acids with a predicted molecular mass of 38.3 kDa. The deduced protein sequence exhibited high homology with other fungal endopolygalacturonases. A polymerase chain reaction (PCR)-based strategy was used to study the expression patterns of pgg1 and pgg2 genes under different culture conditions and our results show that both genes are regulated by the carbon source at the transcriptional level. The pgg1 transcript was detected at 76 h of fungal growth in pectin while the pgg2 transcript was also induced by sucrose. The addition of yeast extract to glucose medium abolished the repressive effect of glucose, suggesting that the transcription of these genes is controlled by different mechanisms.

Human mammaglobin RT-PCR assay for detection of occult breast cancer cells in hematopoietic products.

BACKGROUND: The aim of this study was: (i) to evaluate the sensitivity and specificity of mammaglobin as a reverse transcriptase polymerase chain reaction (RT-PCR) marker of breast cancer cells; (ii) to determine the incidence of tumor cell contamination of hematopoietic samples from patients with breast cancer. MATERIALS AND METHODS: A nested RT-PCR assay for mammaglobin was developed. Sensitivity was determined by serial dilution assays with breast cancer cell lines, human breast cancers and normal breast tissue. Specificity was evaluated in hematopoietic samples from healthy volunteers and patients with hematological malignancies or solid tumors other than breast cancer. RESULTS: The mammaglobin transcript was detected in all 15 breast cancers, one benign breast tumor and five normal breast tissues studied, as well as in three breast cancer cell lines, in dilutions as low as 10(-8). The transcript was not detected in any of 47 peripheral blood samples, 15 bone marrow aspirates and 28 peripheral blood progenitor cell samples from the three control populations. Mammaglobin mRNA was detected in 19 of 78 peripheral blood samples from patients with breast cancer starting systemic chemotherapy, as well as in five of 30 repeat samples collected before the fourth cycle of treatment. The transcript was also present in six of seven bone marrow aspirates from patients with metastatic disease, two of five with loco-regional disease, but not in the aspirate of two patients with thrombocytopenia and a previous history of breast cancer. CONCLUSIONS: Human mammaglobin mRNA is a sensitive and specific marker of breast cancer cells and should be further studied as a molecular marker of tumor cell contamination of hematopoietic tissues.

Influence of the interval between the administration of doxorubicin and paclitaxel on the pharmacokinetics of these drugs in patients with locally advanced breast cancer.

PURPOSE: The combination of bolus doxorubicin followed by a 3-h infusion of paclitaxel has high antitumor activity in patients with metastatic breast cancer, but is limited by unexpected cardiac toxicity. In contrast, the administration of the two drugs 16 h apart has similar antitumor activity but less cardiac toxicity. The purpose of this study was to compare the pharmacokinetics of these drugs when doxorubicin administration preceded paclitaxel by 30 min or by 24 h. PATIENTS AND METHODS: Women with locally advanced breast cancer were treated with doxorubicin (60 mg/m2 i.v. bolus) followed 24 h later by paclitaxel (200 mg/m2 i.v. over 3 h) for six cycles (four before and two after surgery). In one of the first two cycles doxorubicin preceded paclitaxel by 30 min instead of 24 h, with plasma sampling for pharmacokinetic analysis up to 48 h. Determination of drug levels in plasma was done by HPLC. RESULTS: A total of 28 patients were included. No clinical cardiac toxicity was observed but five patients discontinued doxorubicin-paclitaxel treatment after four cycles because of a decrease in LVEF of at least 15% from baseline or to less than 50%. While paclitaxel pharmacokinetics were not changed, there was a 30% and an 80% increase in the AUC0, 24h for doxorubicin and doxorubicinol, respectively, when the drugs were administered 30 min instead of 24 h apart. Even when paclitaxel was given 24 h after doxorubicin, there was a rebound 240% increase in the plasma concentration of doxorubicinol. CONCLUSIONS: Paclitaxel interferes with the pharmacokinetics of doxorubicin leading to higher systemic exposure to both doxorubicin and doxorubicinol, which is more evident when the plasma concentration of the anthracyclines is higher. This interference may explain the higher incidence of cardiac toxicity observed when the two drugs are administered within a short interval.

Analysis of the effects of CPA and L-histidine on goldfish tested on a conditioned place preference model.

The aim of this study was to investigate whether the histamine H(1)- receptor blocker chlorpheniramine (CPA) and the histaminergic precursor L-histidine have reinforcing effects in goldfish (Carassius auratus). We used a place-preference procedure in a four-compartment aquarium in which, on the first day, the animals were placed for 30 min for habituation. On the second day, the time spent in each chamber was recorded for 10 min in order to determine which was preferred. After 24 h a non-transparent acrylic division was placed in the aquarium to make four identical compartments. The fish were injected intraperotoneally (i.p.) with L-histidine in three doses: 100, 250, and 500 mg/kg of body-weight (n=20) and with vehicle control (n=20). After 30 min the animals were placed in the intermediate preference chamber for another 30 min. The CPA was injected i.p in four doses; 0.4, 1.0, 4.0 or 8.0 mg/kg (n=16) or vehicle (n=16) and after 5 min the animals were placed in the intermediate preference chamber for another 30 min. On the fourth day the procedure of the second day was repeated. The results indicate that the group treated with 500 mg/kg L-histidine spent significantly more time in the paired compartment when compared with the time spent before treatment (Wilcoxon Signed Rank Test, P=0.0172). The same behavior was not observed in other treatment groups (P>0.05). The results indicate a reinforcing effect of L-histidine in this place preference model with goldfish.

Cytokeratin 20 is not a reliable molecular marker for occult breast cancer cell detection in hematological tissues.

Cytokeratins are potential markers for epithelial cell detection in hematological tissues. Thus, we developed a nested reverse transcriptase-polymerase chain reaction (RT-PCR) strategy to detect cytokeratin 20 (CK20) mRNA and studied its sensitivity and specificity as a molecular marker of occult breast cancer cells. In cell dilution experiments with human breast cancer cell lines, the limit of detection was 1 tumor cell in 1,000 hematological cells. In RNA dilution experiments of breast cancer cells' RNA in E. Coli tRNA, the CK20 transcript was only detectable when at least 1 ng of total tumor RNA was present in a total of 1 microg of RNA mixture. In parallel experiments using colorectal cancer specimens, CK20 mRNA was detected with as little as 1 pg of total tumor RNA, suggesting a low level of CK20 mRNA expression in breast cancer cells. The CK20 transcript was detected in all six tumors and five hematological samples of breast cancer patients but in none of nine hematological cell lines. However, CK20 transcript was also detected in unfractionated nucleated cell population of hematological samples from 23 of 31 (74%) healthy volunteers and from 12 of 24 (50%) patients with hematological malignancies. When mononucleated and polymorphonucleated cell populations of hematological samples from these control groups were screened separately, CK20 expression was detected in 94% of polymorphonucleated cell fractions and in 44% of mononucleated cell subpopulations. Thus, we conclude that the low sensitivity and specificity of RT-PCR detection of CK20 mRNA limits its usefulness for breast cancer cell detection in hematological products.