Prediction of Overall Survival by Thymidine Kinase 1 Combined with Prostate-Specific Antigen in Men with Prostate Cancer.

Thymidine kinase 1 (TK1) is an intracellular enzyme involved in DNA-precursor synthesis. Increased serum TK1 levels are used as a biomarker in various malignancies. We combined serum TK1 with PSA and evaluated its capacity to predict overall survival (OS) in 175 men with prostate cancer (PCa), detected by screening in 1988-1989 (n = 52) and during follow-up (median 22.6 years) (n = 123). TK1 was measured in frozen serum, age was stratified into four groups, and dates of PCa diagnosis and dates of death were obtained from Swedish population-based registries. The median concentration of TK1 and PSA was 0.25 and 3.8 ng/ml. TK1 was an independent variable of OS. In the multivariate analysis, PSA was not statistically significant in combination with age whereas the significance remained for TK1 + PSA. Measured once, TK1 + PSA predicted a difference of up to 10 years (depending on patient subgroup) in OS at a median of 9 years before PCa diagnosis. The TK1 concentration in 193 controls without malignancies did not differ from that of the PCa patients, hence TK1 was likely not released from incidental PCa. Thus, TK1 in the blood circulation may indicate the release of TK1 from sources other than cancers, nonetheless associated with OS.

Serum thymidine kinase 1 concentration as a predictive biomarker in prostate cancer.

BACKGROUND: Thymidine kinase 1 (TK1) recycles DNA before cell division. We do not know if baseline blood concentrations of TK1 predict death in prostate cancer within 30 years. Our objective is to determine if there is an association between baseline levels of TK1 and future prostate cancer-specific mortality. METHODS: With a "proof of concept" approach, we performed a nested case-control study among 1782 individuals screened for prostate cancer between 1988 and 1989. The concentration of TK1 was measured in frozen serum from 330 men, 36 of whom have died of prostate cancer. The primary endpoint was prostate cancer-specific mortality and outcomes after 30 years were analyzed using logistic regression modeling odds ratios (Ors). RESULTS: The estimated OR (adjusted for age) for dying from prostate cancer among the men who had a TK1 value in the upper tertile was 2.39 (95% confidence interval 1.02-5.63). The corresponding OR, regardless of the cause of death, was 2.81 (1.24-6.34). CONCLUSIONS: High levels of TK1 predicts death in prostate cancer within 30 years of follow-up.

Early prediction of pathologic response to neoadjuvant treatment of breast cancer: use of a cell-loss metric based on serum thymidine kinase 1 and tumour volume.

BACKGROUND: After neoadjuvant chemotherapy of breast cancer pathologic complete response (pCR) indicates a favorable prognosis. Among non-selected patients, pCR is, however, achieved in only 10-30%. Early evaluation of tumour response to treatment would facilitate individualized therapy, with ineffective chemotherapy interrupted or changed. The methodology for this purpose is still limited. Tumour imaging and analysis of macromolecules, released from disrupted tumour cells, are principal alternatives. OBJECTIVE: To investigate whether a metric of cell-loss, defined as the ratio between serum concentration of thymidine kinase1 (sTK1, ng x ml- 1) and tumour volume, can be used for early prediction of pathologic response. METHODS: One hunred four women with localized breast cancer received neoadjuvant epirubicin/docetaxel in 6 cycles, supplemented with bevacizumab in cycles 3-6. The cell-loss metric was established at baseline (n = 104), 48 h after cycle 2 (n = 104) and prior to cycle 2 (n = 57). The performance of the metric was evaluated by association with pathologic tumour response at surgery 4 months later. RESULTS: Treatment caused a rise in sTK1, a reduction in tumour volume and a marked increase in the cell-loss metric. Patients were subdivided into quartiles according to the baseline cell-loss metric. For these groups, baseline means were 0.0016, 0.0042, 0.0062, 0.0178 units. After subtraction of baselines, means for the quartiles 48 h after treatment 2 were 0.002, 0.011, 0.030 and 0.357 units. pCR was achieved in 24/104, their distribution in the quartiles (11, 11, 23 and 46%) differed significantly (p = 0.01). In 80 patients with remaining tumour, tumour size was inversely related to the metric (p = 0.002). In 57 patients studied before treatment 2, positive and negative predictive values of the metric were 77.8 and 83.3%, compared to 40.5 and 88.7% 48 h after treatment 2. CONCLUSION: A cell-loss metric, based on serum levels of TK1, released from disrupted tumour cells, and tumour volume, reveal tumour response early during neoadjuvant treatment. The metric reflect tumour properties that differ greatly between patients and determine the sensitivity to cytotoxic treatment. The findings point to the significance of cell loss for tumour growth rate. The metric should be considered in personalized oncology and in the evaluation of new therapeutic modalities. TRIAL REGISTRATION: PROMIX (Clinical Trials.govNCT000957125).

Predicting invasiveness and disease-specific survival in upper tract urothelial carcinoma: identifying relevant clinical tumour characteristics.

PURPOSE: The aim of this prospective study was to identify the tumour characteristics that are associated with invasiveness and those that are relevant for disease-specific survival (DSS) in upper tract urothelial carcinoma, UTUC. METHODS: From a prospective consecutive cohort of patients with suspicion of UTUC, those who were diagnosed with UTUC using URS prior to rNU between 2005 and 2012 were included. Tumour characteristics were analysed for prediction of invasiveness and association with DSS. Stages were categorised as superficial (pTa-1 and CIS only) or invasive (≥  pT2). Tumours were graded according to WHO 1999 classification. DSS was analysed regarding possible association with stage, grade, size, multifocality, location, ploidy and rate of proliferation. Associations were tested using Fisher's exact test, Pearson Chi-square or Cox's regression. Kaplan-Meier survival curves were constructed. RESULTS: Forty-five consecutive patients were included, and 43 of them were included in the final analyses because their rNU specimens were available for reassessment. The only tumour characteristics that were significantly associated with stage were tumour grade (P < 0.001), DNA ploidy (P = 0.045) and rate of proliferation (P = 0.004). No association with stage was noted for size, multifocality or location. Grade, stage and rate of proliferation were associated with DSS. CONCLUSIONS: Grade, DNA ploidy and S-phase fraction were the only tumour characteristics associated with stage in our study. However, DNA ploidy was not associated with DSS. The prognostic factors that we identified were tumour grade, stage, and S-phase fraction.

Dynamics of Serum Thymidine Kinase 1 at the First Cycle of Neoadjuvant Chemotherapy Predicts Outcome of Disease in Estrogen-Receptor-Positive Breast Cancer.

Pathologic complete response (pCR) predicts the long-term outcome of neoadjuvantly treated (NAC) breast cancer (BC) but is reached in <10% of hormone-receptor-positive patients. Biomarkers enabling adjustment or interruption of an ineffective therapy are desired. Here, we evaluated whether changes in the serum concentration of thymidine kinase 1 (sTK1) during NAC could be utilized as a biomarker. In the PROMIX trial, women with localized HER2- BC received neoadjuvant epirubicin/docetaxel in six cycles. sTK1 was measured with an ELISA in 54 patients at cycles 1-4 and in an additional 77 patients before and 48 h after treatment 1. Treatment resulted in a 2-fold increase of sTK1 before and a 3-fold increase 48 h after the cycles, except for the first cycle, where half of the patients reacted with a significant decrease and the other half with an increase of sTK1. In Kaplan-Meier estimates of ER+ patients divided by the median of the post/pre-treatment sTK1 ratio at the first treatment cycle, OS was 97.7% and 78% (p = 0.005), and DFS was 90.7% and 68% (p = 0.006), respectively. Thus, the response of sTK1 at the first cycle of chemotherapy could be used both as an early biomarker for the guidance of chemotherapy and for the study of inherent tumor chemo-sensitivity, which could predict long-term outcome prior to therapy.

Diagnostic accuracy of upper tract urothelial carcinoma: how samples are collected matters.

OBJECTIVES: The aims of this study were to determine the accuracy and reliability of cytology, histopathology and ploidy of specimens obtained at ureterorenoscopy, to evaluate the importance of how samples are collected and to determine whether cytology is an alternative to histology of biopsies. METHODS: This prospective study investigated the accuracy of grading of endoscopically taken cytology and histopathology samples from 45 consecutive patients by comparing these with subsequent nephroureterectomy specimens. Histopathology grading was done according to WHO 1999 and 2004 classifications. Ploidy was determined using photospectrometry. RESULTS: Forty-five patients were included. Both cytology and histopathology identified almost all cancers (91% and 94%, respectively) in collected samples. In cytology as well as in histopathology, agreement in grade between barbotage and nephroureterectomy specimens was statistically significant for both 1999 and 2004 WHO classifications. All cancers in the endoscopic biopsies were identified as pathological, although the grading was not correct in all cases. A statistically significant correlation was found between grade and ploidy in grade 1 and grade 3 nephroureterectomy specimens. CONCLUSIONS: Specimens collected at ureterorenoscopy (biopsies for histology, barbotages for cytology and analysis of ploidy) proved to be relevant and useful. Barbotage cytology identified 91% of all cancers, a high rate compared to techniques used in other studies, and was also sensitive in detecting low-grade tumours. Barbotage cytology and biopsy histology were equally efficient in detecting cancer. The authors recommend that both barbotage and biopsy be performed in addition to complete ureterorenoscopy. Moreover, if there is no visible lesion, cytology is the only reliable option.

Molecular prostate cancer pathology: current issues and achievements.

Recent developments in the field of molecular techniques have provided new tools that have led to the discovery of many new promising biomarkers for prostate cancer. These biomarkers may be instrumental in the development of new tests that will have a high specificity for the diagnosis and prognosis of prostate cancer. A biomarker is defined as a molecular test that provides additional information to currently available clinical and pathological tests. Biomarkers should be reproducible (both within and between institutes) and have an impact on clinical management. For diagnostic purposes it is important that potential biomarkers are tested in terms of tissue specificity and their discrimination potential between prostate cancer, normal prostate and benign prostatic hyperplasia. The results of (multiple) biomarker-based assays may enhance the specificity of cancer detection. There is an urgent need for molecular prognostic biomarkers for predicting the biological behavior and outcome of cancer.

Expression of cyclins A, E and topoisomerase II alpha correlates with centrosome amplification and genomic instability and influences the reliability of cytometric S-phase determination.

BACKGROUND: The progression of normal cells through the cell cycle is meticulously regulated by checkpoints guaranteeing the exact replication of the genome during S-phase and its equal division at mitosis. A prerequisite for this achievement is synchronized DNA-replication and centrosome duplication. In this context the expression of cyclins A and E has been shown to play a principal role. RESULTS: Our results demonstrated a correlation between centrosome amplification, cell cycle fidelity and the level of mRNA and protein expression of cyclins A and E during the part of the cell cycle defined as G1-phase by means of DNA content based histogram analysis. It is shown that the normal diploid breast cell line HTB-125, the genomically relatively stable aneuploid breast cancer cell line MCF-7, and the genomically unstable aneuploid breast cancer cell line MDA-231 differ remarkably concerning both mRNA and protein expression of the two cyclins during G1-phase. In MDA-231 cells the expression of e.g. cyclin A mRNA was found to be ten times higher than in MCF-7 cells and about 500 times higher than in HTB-125 cells. Topoisomerase II alpha showed high mRNA expression in MDA compared to MCF-7 cells, but the difference in protein expression was small. Furthermore, we measured centrosome aberrations in 8.4% of the MDA-231 cells, and in only 1.3% of the more stable aneuploid cell line MCF-7. MDA cells showed 27% more incorporation of BrdU than reflected by S-phase determination with flow cytometric DNA content analysis, whereas these values were found to be of the same size in both HTB-125 and MCF-7 cells. CONCLUSIONS: Our data indicate that the breast cancer cell lines MCF-7 and MDA-231, although both DNA-aneuploid, differ significantly regarding the degree of cell cycle disturbance and centrosome aberrations, which partly could explain the different genomic stability of the two cell lines. The results also question the reliability of cytometric DNA content based S-phase determination in genomically unstable tumor cell populations.

Chronic colitis is associated with a reduction of mucosal alkaline sphingomyelinase activity.

BACKGROUND AND AIMS: The hydrolysis of sphingomyelin (SM) generates key molecules regulating cell growth. Animal cancer studies support an inhibitory role for this pathway in the malignant transformation of the colonic mucosa. The activity of a specific intestinal alkaline sphingomyelinase (SMase), which hydrolyzes SM, is reduced in colorectal tumors. In this study we measured alkaline SMase activity in patients with longstanding colitis and assessed if a reduction can be used as a marker in surveillance of high risk patients. METHODS: Alkaline SMase activity was measured in 139 colonic biopsies from 34 patients with longstanding, extensive colitis and from 11 controls. Fifteen patients had earlier diagnosis of dysplasia or DNA aneuploidy. Alkaline SMase activity was related to histologic dysplasia and DNA aneuploidy assessed by flow cytometry, patient age, and duration of disease. RESULTS: Alkaline SMase activity was significantly lower in the patient group with and without dysplasia compared with controls (p = 0.006). In biopsies, an association was not found between alkaline SMase activity, dysplasia, or DNA ploidy. However, alkaline SMase activity decreased with age both in patients and controls (p = 0.008). CONCLUSIONS: Reduction of alkaline SMase activity seen in colorectal cancer and adenomas is also present in patients with chronic colitis. It is not complementary to dysplasia or DNA-aneuploidy in the identification of high risk patients. The age-associated decrease of alkaline SMase activity seems to be a general phenomenon indicating premature senescence of the mucosa in longstanding colitis.

Ursodeoxycholic acid treatment in IBD-patients with colorectal dysplasia and/or DNA-aneuploidy: a prospective, double-blind, randomized controlled pilot study.

BACKGROUND & AIMS: There is an increased risk of colorectal carcinoma (CRC) in patients with longstanding, extensive colonic inflammatory bowel disease (IBD). Primary sclerosing cholangitis, family history of CRC, mucosal dysplasia and DNA-aneuploidy are other risk factors. Recently, results from animal studies have shown that the bile acid ursodeoxycholic acid (UDCA) has a favourable impact on experimentally-induced CRC/neoplasia in rats. The aim of this proof of the concept study was to explore the possible preventive/reverting effects of UDCA in patients with colorectal IBD with existing findings of low grade dysplasia and/or DNA-aneuploidy. PATIENTS AND METHODS: Nineteen patients (13 UC, 6 CD, median age 43 years) with long-standing, extensive IBD (median duration 21 years), with previous findings of low-grade dysplasia and/or DNA-aneuploidy, were randomized to receive either UDCA (500 mg b.i.d) (n=10) or placebo (n=9) in a controlled, double-blind, two-year study. Colonoscopy with multiple biopsies for histopathology and for DNA-flow cytometry was performed at the start and at six-month intervals during the study period. The primary outcome was the need for colectomy due to progression of dysplasia. Changes in dysplasia and DNA-aneuploidy scores were also assessed. RESULTS: There were no significant differences in the overall composed score between the two groups, either at study start or during the study period. In the placebo group one patient had a progression of dysplasia into high-grade and one patient developed DALM with low-grade dysplasia; both had a colectomy. In contrast, no UDCA-treated patient had progression of dysplasia. CONCLUSION: UDCA may prevent further progression of manifest low-grade dysplasia in colorectal IBD. Prolonged treatment or an increased dose may be needed to fully exploit the chemopreventive properties of this compound.

Methodologic aspects of evaluating brush samples from biliary strictures by cytology and DNA flow cytometry.

OBJECTIVE: To present a method of increasing the cell yield from brush samples of the biliary tree for measurement of DNA content by flow cytometry (FCM). STUDY DESIGN: One hundred eight cell specimens from 86 patients were studied by FCM for DNA ploidy and cell cycle composition. All specimens were cytologically classified into benign, suspicious for malignancy and malignant. Two methods for preparation of the cell material were compared. RESULTS: Enzymatic treatment of formalin-fixed brushes for release of cell nuclei was superior to mechanical removal of the cells. The fraction of samples not possible to assess was reduced from 27% to 4%, and good quality histograms increased from 21% to 62%. Aneuploidy was detected in 7% of benign and 57% of suspicious malignant samples. Using DNA analysis in addition to cytology as a diagnostic marker for cancer, the sensitivity increased from 12% to 31%. CONCLUSION: FCM of cells from biliary strictures can be used routinely as an adjunct to cytology for DNA analysis.

Colitis-associated DNA aneuploidy and dysplasia in Crohn's disease and risk of colorectal cancer.

BACKGROUND: There is uncertainty about how patients with Crohn's colitis should be monitored for colorectal cancer (CRC). By analogy to ulcerative colitis, regular colonoscopy with biopsies for dysplasia has been used. We describe the occurrence of dysplasia and DNA aneuploidy in a cohort of patients with Crohn's colitis. METHODS: In all, 245 patients with extensive colitis (225 with a firm diagnosis of Crohn's disease, and 20 diagnosed as indeterminate colitis) at Stockholm Söder Hospital and Karolinska University Hospital, Huddinge were included. They were followed with regular colonoscopies with biopsies both for dysplasia and DNA aneuploidy. The cumulative occurrence of DNA aneuploidy and dysplasia was estimated using Kaplan-Meier curves. Time sequences and interactions between DNA aneuploidy, dysplasia, and CRC were studied using Cox regression analysis, adjusted for age, sex, and age at diagnosis. RESULTS: During a median follow-up time of 9.2 person-years, DNA aneuploidy was found in 53 patients (22%), with 10 patients having multifocal aneuploidy and high S-phase values. Dysplasia was found in 42 patients (17%), 10 having multifocal dysplasia. Relative risk (RR) of dysplasia given DNA aneuploidy was 5.3 (95% confidence interval [CI] 2.3-12). RR of CRC given dysplasia was 10 (95% CI 2-50), and RR of CRC given aneuploidy was 1.5 (95% CI 0.3-9.3). CONCLUSIONS: Dysplasia and DNA aneuploidy including S-phase analysis may complement stratification of patients with Crohn's

The 20-Yr outcome in patients with well- or moderately differentiated clinically localized prostate cancer diagnosed in the pre-PSA era: the prognostic value of tumour ploidy and comorbidity.

OBJECTIVE: This observational cohort study describes the long-term outcome of patients with clinically localized prostate cancer managed with watchful waiting, the prognostic value of tumour ploidy, and the impact of comorbidity. METHODS: A total of 119 patients with clinically localized (T1-2) prostate cancer consecutively diagnosed from 1978 to 1982 were prospectively managed by watchful waiting, with treatment given if progression occurred. RESULTS: Median age was 68 yr. Median observation time was 24 yr+/-6.25 (SD). Of the 112 patients who died, 42 died of prostate cancer. Disease-specific survival rates were 85% (95% CI: 77-93%), 58% (46-70%), and 32% (19-46%) at 10, 15, and 20 yr, respectively. Treatment-free survival rate was 43% (95% CI: 33-54%) at 10 yr. Patients aged 70 yr and over had a statistically significant increased risk of dying from any cause. There was a statistically significant increased risk of dying from prostate cancer for patients with nondiploid tumours. CONCLUSION: In the present series from the pre-PSA era, watchful waiting yielded a relatively high long-term disease-specific survival rate in patients with well- or moderately differentiated clinically localized prostate cancer, and almost half were not treated 10 yr after diagnosis. Watchful waiting may be an option at least for such patients with a 10- to 15-yr life expectancy. Age of 70 yr or more predicted an increased overall mortality. High comorbidity increased the risk (although not statistically significant) for death from any cause and for death from prostate cancer. Patients with nondiploid tumours were at an increased risk to die from prostate cancer.

Double-fluorescence image microscopy for quantitation of prostate-specific antigen in histologic sections of the prostate.

BACKGROUND: Although the assessment of serum prostate-specific antigen (PSA) has become a powerful instrument in the diagnosis and for prognosis of prostate carcinoma, there are few quantitative studies of PSA in tissue sections. METHODS: We developed a technique using double-fluorescence image microscopy for quantifying immunohistochemical reactions in tissue sections. PSA was stained by Texas Red and the cellular DNA was counterstained with 4,6-diamidino-2-phenylindole, dihydrochloride (DAPI). The fluorescence of Texas Red and DAPI was quantified separately after subtraction of background and shading correction. The amount of PSA related to the amount of DNA in identical tissue parts was studied in archival specimens from patients with hyperplasia and prostate carcinoma. RESULTS: The amount of tissue PSA decreased with the increase in tumor grade, Gleason score, and the change from diploid to aneuploid. CONCLUSION: Double-fluorescence image microscopy is a valuable technique for obtaining quantitative information of cellular constituents. For standardization of immunochemical reactions in tissue sections, cellular DNA seems to be most appropriate.

Atrophy and neoplastic transformation of the ileal pouch mucosa in patients with ulcerative colitis and primary sclerosing cholangitis: a case control study.

INTRODUCTION: Patients with ulcerative colitis and primary sclerosing cholangitis have an increased risk of developing carcinoma both in the bile ducts and in the colon. PURPOSE: To investigate whether this patient group also has an increased risk of developing atrophy and neoplasia in the ileal pouch mucosa after construction of a pelvic pouch with an ileoanal anastomosis or a continent Kock ileostomy. METHODS: Flexible video endoscopic examinations of the ileal pouch were performed in 16 patients with ulcerative colitis and primary sclerosing cholangitis and in 16 matched patients with ulcerative colitis without sclerosing cholangitis. Biopsies were sampled from different locations in the pouch for histologic assessment of mucosal atrophy and dysplasia and for flow cytometric DNA analysis assessing chromosomal aberrations. RESULTS: The patients with sclerosing cholangitis developed moderate or severe atrophy in the pouch significantly more often (P < 0.01). Persistent severe mucosal atrophy was revealed in eight patients with sclerosing cholangitis and only in two controls. One patient with sclerosing cholangitis had high-grade dysplasia in multiple locations. Low-grade dysplasia was assessed in three patients with sclerosing cholangitis and in two of the controls. DNA aneuploidy was displayed in three patients, all with sclerosing cholangitis and dysplasia. All patients with neoplastic transformation had a pouch with ileoanal anastomosis and a long pouch duration (> 8 years). CONCLUSION: Patients with ulcerative colitis and primary sclerosing cholangitis with an ileal reservoir are more prone to developing mucosal atrophy in the pouch and seem to have a higher risk of neoplastic transformation in the pouch mucosa than patients with ulcerative colitis without sclerosing cholangitis.

S-phase fraction in superficial urothelial carcinoma of the bladder--a prospective, long-term, follow-up study.

OBJECTIVE: To study, in addition to traditional tumor characteristics at diagnosis, the significance of DNA ploidy and S-phase fraction for tumor progression and tumor-related death in superficial carcinoma of the urinary bladder. MATERIAL AND METHODS: Newly detected superficial bladder carcinomas (stage Ta-T1), from 195 consecutive patients were characterized according to stage, grade, tumor size, multiplicity, growth pattern, cytologic evaluation and random mucosal biopsies, as well as DNA ploidy and S-phase fraction as determined by means of DNA flow cytometry. The outcome of disease was evaluated using hospital charts and death certificates. RESULTS: During a median follow-up period of 98 months (range 1-160 months), 28 patients (14%) progressed to muscle-invasive or metastatic disease and 24 (12%) died from disease. In univariate analysis all factors studied, with the exception of the size and number of tumors at diagnosis, were significantly related to progress and tumor-specific survival. In multivariate analysis, however, S-phase fraction was the most significant prognostic factor. When 21 high-risk patients with T1G3 tumors who underwent early cystectomy were excluded, S-phase fraction remained the most important prognostic factor. DNA ploidy failed as an independent predictor of survival. CONCLUSIONS: High S-phase fraction at diagnosis of superficial urothelial carcinoma of the bladder identifies patients at high risk of progression and death from disease.

Diagnostic DNA-flow- vs. -image-cytometry in effusion cytology.

AIMS: To determine the sensitivity and specificity of flow- and image-cytometry for the detection of DNA-aneuploidy as a marker for malignant cells in effusions. METHODS: 200 effusions (80 tumor cell-positive, 74 negative and 46 cytologically equivocal) were stained with DAPI-SR for DNA-flow- and with Feulgen-Pararosaniline for -image-cytometry. They were measured using a PAS-flow-cytometer and an AutoCyte-QUIC-DNA-workstation according to the ESACP consensus reports for DNA-flow- and -image-cytometry, respectively [7,23,29,49]. RESULTS: Sensitivity of DNA-aneuploidy for the identification of malignant cells was 32.1% for DNA-flow- and 75.0% for -image-cytometry, specificity of -euploidy in benign cells was 100.0% for both methods. Positive predictive value of DNA-aneuploidy for the identification of malignant cells was 100.0% for both techniques, negative predictive value of DNA-euploidy was 48.6% for DNA-flow- and 72.0% for -image-cytometry. CONCLUSIONS: Searching for DNA-aneuploidy as a diagnostic marker for neoplastic cells in serous effusions image-cytometry revealed superior sensitivity as compared with monoparametric flow cytometry.