Optimising surgical accuracy in palpable breast cancer with intra-operative breast ultrasound--feasibility and surgeons' learning curve.

AIMS: To evaluate if intra-operative guidance with ultrasonography (US) could improve surgical accuracy of palpable breast cancer excision, and to evaluate the performance of surgeons during training for US-guided excision. MATERIALS AND METHODS: Thirty female patients undergoing breast-conserving surgery for palpable T1-T2 invasive breast cancer were recruited. Three individual breast surgeons, assisted by US, targeted and excised the tumours. The main objective was to obtain adequate resection margins with optimal resection volumes. The specimen volume, tumour diameter and histological margin status were recorded. The specimen volume was divided by the optimal resection volume, defined as the spherical tumour volume plus a 1.0-cm margin. The resulting calculated resection ratio (CRR) indicated the amount of excess tissue resected. RESULTS: All tumours were correctly identified during surgery, 29 of 30 tumours (96.7%) were removed with adequately negative margins, and one tumour was removed with focally positive margins. The median CRR was 1.0 (range, 0.4-2.8), implying optimal excision volume. For all breast surgeons, CRR improved during the training period. By the 8th procedure, all surgeons showed proficiency in performing intra-operative breast US. CONCLUSION: Surgeons can easily learn the skills needed to perform intra-operative US for palpable breast tumour excision. The technique is non-invasive, simple, safe and effective for obtaining adequate resection margins. Within the first two cases, resections reached optimal volumes, thereby, presumably resulting in improved cosmetic outcomes. In a multicentre, randomised, clinical trial, intra-operative US guidance for palpable breast tumours will be evaluated for oncological and cosmetic outcomes.

Brain atrophy and lesion load as explaining parameters for cognitive impairment in multiple sclerosis.

Multiple sclerosis (MS) is a multifocal demyelinating disease of the central nervous system, with lesions widespread through the brain and spinal cord. An important manifestation is cognitive impairment, which, though difficult to measure, may have a major social impact. To better understand the relationship between structural tissue damage and cognitive impairment, we examined the extent and spatial distribution of brain lesions, as measured by magnetic resonance imaging (MRI), in relation to abnormal cognitive performance as measured by the Brief Repeatable Battery (BRB) in 82 MS patients. Possible confounders, like fatigue, pain and depression were also assessed. Brain MR image analysis included hyperintense T2 and hypointense T1 lesion load in the whole brain and the four lobes separately, as well as whole brain volume measurements. Cognitive impairment (defined as more than two abnormal tests) was found in 67% of the patients. Moderately strong correlations were found between the subtests of the BRB and the lesion loads in the brain regions hypothesized to be associated with that cognitive test, although these correlations were in general not much stronger than those between the subtests and the overall lesion load (due to strong interrelationships). The Spatial Recall Test correlated best with parietal lesion load; the Symbol Digit Modalities Test, the Paced Auditory Serial Addition Task (PASAT) and the Word List Generation best with frontal, parietal and temporal lesion load; while the Verbal List Generation Test Index correlated only with atrophy. Atrophy and lesion load were the main factors determining the test scores, explaining 10-25% of the variance in the test results, and were more important than fatigue, pain and depression; only depression had a minor, but significant, additional effect on the PASAT. In conclusion, cognitive impairment in MS is moderately dependent on amount (and distribution) of structural brain damage, especially in the more physically impaired patients group.

Spinal cord abnormalities in recently diagnosed MS patients: added value of spinal MRI examination.

OBJECTIVE: The most recent diagnostic criteria for multiple sclerosis (MS) ascertain that findings from spinal cord MRI can be used to demonstrate dissemination in space. Because little is known about the prevalence and characteristics of cord lesions early in the disease, the authors studied the prevalence of spinal cord abnormalities in patients with early-stage MS and assessed their impact on diagnostic classification. METHODS: The brains and spinal cords of 104 recently diagnosed patients with MS were examined. Median interval between first symptom and diagnosis was 18.4 months. The brain MRI protocol included before and after gadolinium axial T1-weighted conventional spin-echo sequences and dual-echo spin-echo images. For spinal cord MRI, sagittal cardiac-triggered dual-echo T2-weighted and sagittal T1-weighted spin-echo images were included. Clinical assessment for each patient included age, sex, clinical signs for spinal cord involvement, and Expanded Disability Status Scale. RESULTS: Abnormal cord MRIs were found in 83% of patients, usually with only focal lesions. Diffuse cord abnormalities were found in 13% of patients, although in isolation they were found in only three patients. Focal cord lesions were often multiple (median number, 3.0), small (median, 0.8 vertebral segments), and primarily (56.4%) situated in the cervical spinal cord. In 68 of 104 patients (65.4%), two or more focal lesions were visible on spinal cord images. The criteria for dissemination in space, as defined in the McDonald criteria for the brain, were met in only 66.3% of the patients. This percentage increased to 84.6% when spinal cord MRI abnormalities were also included. CONCLUSION: Spinal cord abnormalities are prevalent in patients with early-stage MS, have distinct morphologic characteristics, and help to determine dissemination in space at time of diagnosis.

The effect of the neuroprotective agent riluzole on MRI parameters in primary progressive multiple sclerosis: a pilot study.

Progressive axonal loss is the most likely pathologic correlate of irreversible neurologic impairment in primary progressive multiple sclerosis. In a run-in versus treatment trial, we show that the neuroprotective agent riluzole seems to reduce the rate of cervical cord atrophy and the development of hypointense T1 brain lesions on magnetic resonance imaging.

Axonal damage in the spinal cord of MS patients occurs largely independent of T2 MRI lesions.

OBJECTIVE: To determine the degree of axonal damage in relationship to signal abnormalities on T2-weighted high-resolution MRI in spinal cord tissue of patients with MS. METHODS: Spinal cord specimens of nine patients with MS and four controls were imaged at high resolution (4.7 T) in an axial plane and scored for lesions with increased signal intensity (SI). Histopathologic sections were cut and immunostained with NE14 (neurofilament marker) and Luxol fast blue (myelin stain). For each area, axonal density and diameter were quantified; axonal irregularity, NE14 axonal staining intensity, and myelin content were semiquantitatively scored. Included were 209 areas from MS cases and 109 areas from control cases distributed over lateral, posterior, and anterior columns. RESULTS: In control cases, no SI changes were found, average density of axons was 26,989/mm(2), average diameter was 1.1 micro m, and all scores for axonal irregularity, NE14 staining intensity, and myelin were normal. In MS cases, areas with increased SI were found, average axonal density was 11,807/mm(2) (p < 0.0001), and average axonal diameter 2.0 micro m (p = 0.001). Areas with high SI on MRI had lowest axonal density (average count: 10,504/mm(2); range: 3,433 to 26,325/mm(2)), largest diameter (average: 2.3 micro m; range: 1.0 to 4.0 micro m), and highest axonal irregularity and NE14 staining intensity compared to normal appearing cord tissue (NACT). However, NACT of MS cases also had lower axonal density (14,158/mm(2)) and higher average axonal diameter (1.6 micro m) than controls. CONCLUSIONS: Marked axonal loss occurs in MS spinal cords, largely independent of the degree of signal abnormality on T2-weighted MRI.

Optimizing the association between disability and biological markers in MS.

OBJECTIVE: Axonal damage is an important feature of MS pathology and the likely substrate of development of progressive disability. Brain volume measurement on MRI can be used as an overall marker of tissue damage and axonal loss. The authors studied the relation of brain volume measurements with the MS Functional Composite (MSFC) in an attempt to improve the clinico-radiologic association. METHODS: In 137 patients with MS (80 relapsing-remitting [RR], 36 secondary progressive [SP], and 21 primary progressive [PP]) and 12 healthy controls, a brain MRI scan was obtained. Patients also underwent MSFC and Expanded Disability Status Scale (EDSS) assessments. MRI analysis included determination of hypointense T1- and hyperintense T2-weighted lesion load, and two brain volume measurements: 1) the parenchymal fraction (PF): whole brain parenchyma/intracranial volume; and 2) the ventricular fraction (VF): ventricular volume/whole brain parenchyma. RESULTS: The median PF was smaller and the median VF larger in the patient group (0.81 for PF and 0.029 for VF) than in the control group (0.87 for PF, p < 0.001; and 0.013 for VF, p < 0.01). For the patient population, moderate correlations were found between brain volume measurements and MSFC (0.36 for PF and -0.40 for VF). Patients with short disease duration showed a correlation of MSFC with both brain and lesion volume measurements on MRI, whereas patients with long disease duration only showed a correlation with brain volume measurements. CONCLUSION: Brain volume measurements are correlated with disability as assessed by the MSFC. Although in the early phase of the disease the amount of focal demyelination is important, the residual brain volume seems to be more relevant in determining disability in later phases of the disease.

Post-mortem MRI-guided sampling of multiple sclerosis brain lesions: increased yield of active demyelinating and (p)reactive lesions.

Macroscopic sampling of multiple sclerosis lesions in the brain tends to find chronic lesions. For a better understanding of the dynamics of the multiple sclerosis disease process, research into new and developing lesions is of great interest. As MRI in vivo effectively demonstrates lesions in multiple sclerosis patients, we have applied it to unfixed post-mortem brain slices to identify abnormalities, in order to obtain a higher yield of active lesions. The Netherlands Brain Bank organized the rapid autopsy of 29 multiple sclerosis patients. The brain was cut in 1 cm coronal slices. One or two slices were subjected to T(1)- and T(2)-weighted MRI, and then cut at the plane of the MRI scan into 5 mm thick opposing sections. Areas of interest were identified based on the MRI findings and excised. One half was fixed in 10% formalin and paraffin-embedded, and the corresponding area in the adjacent half was snap-frozen in liquid nitrogen. In total, 136 out of 174 brain tissue samples could be matched with the abnormalities seen on T(2)-weighted MRIs. The stage of lesional development was determined (immuno) histochemically. For 54 MRI-detectable samples, it was recorded whether they were macroscopically detectable, i.e. visible and/or palpable. Histopathological analysis revealed that 48% of the hyperintense areas seen on T(2)-weighted images represented active lesions, including lesions localized in the normal appearing white matter, without apparent loss of myelin but nevertheless showing a variable degree of oedema, small clusters of microglial cells with enhanced major histocompatibility complex class II antigen, CD45 and CD68 antigen expression and a variable number of perivascular lymphocytes around small blood vessels [designated as (p)reactive lesions]. From the macroscopically not-visible/not-palpable MRI-detected abnormalities, 58% were (p)reactive lesions and 21% contained active demyelinating lesions. In contrast, visible and/or palpable brain tissue samples mainly contained chronic inactive lesions. We conclude that MRI-guided sampling of brain tissue increases the yield of active multiple sclerosis lesions, including active demyelinating and (p)reactive lesions.

Post-mortem high-resolution MRI of the spinal cord in multiple sclerosis: a correlative study with conventional MRI, histopathology and clinical phenotype.

We used high-resolution MRI to study the post-mortem appearance of spinal cord multiple sclerosis in relation to histopathology and low-resolution images. Fifty-nine 3 cm long formalin-fixed spinal cord specimens from 19 multiple sclerosis patients and three controls were studied. Clinical characteristics of each patient were reviewed. High-field MRI consisted of proton-density weighted spin-echo imaging with an in-plane resolution of 80 microm. Specimens were also imaged at 1.0 T, with 1 mm pixel resolution. After MRI, the specimens were cut at 5 mm intervals and stained for myelin (Luxol fast blue/cresyl violet) and axons (Bodian method). Two observers scored the MRIs for abnormalities and divided them into (i) well-delineated areas of high signal intensity (SI) and (ii) poorly defined areas of mildly increased SI. Abnormalities were scored semiquantitatively, white matter and grey matter separately. In 81 sections the total area of abnormalities per section was measured on both histopathology sections and on matched high-field MRIs. Abnormalities ranged from just a few abnormal areas to complete involvement of the spinal cord specimen. Patients with an aggressive disease course had more abnormalities than patients with a mild or intermediate disease course. Areas of mildly increased SI were seen in all specimens, and were often found around focal high-SI lesions. However, in six patients, areas of mildly increased SI were the predominant finding on the MRIs, correlating with a primary progressive disease course. Histopathologically, high-SI areas correlated with complete demyelination, while mildly increased SI corresponded with partial demyelination. All areas scored as abnormal by the neuropathologist were also found on the MRIs, and sizes measured using both methods correlated well (r = 0.85, P<0.01). On conventional MRIs, abnormalities could be recognized fairly well. However, better differentiation could be made between high-SI and mildly increased SI abnormalities on the 4.7 T images. In conclusion, high-resolution MRI revealed a great range of abnormalities in spinal cord multiple sclerosis, which related to disease course during life. Furthermore, we found very good correlation between the extent of abnormalities shown by histopathology and the SI changes on proton-density MRIs, mainly relating to demyelination revealed histopathologically.

Comparison of a conventional cardiac-triggered dual spin-echo and a fast STIR sequence in detection of spinal cord lesions in multiple sclerosis.

The current optimal imaging protocol in spinal cord MR imaging in patients with multiple sclerosis includes a long TR conventional spin-echo (CSE) sequence, requiring long acquisition times. Using short tau inversion recovery fast spin-echo (fast STIR) sequences both acquisition time can be shortened and sensitivity in the detection of multiple sclerosis (MS) abnormalities can be increased. This study compares both sequences for the potential to detect both focal and diffuse spinal abnormalities. Spinal cords of 5 volunteers and 20 MS patients were studied at 1.0 T. Magnetic resonance imaging included cardiac-gated sagittal dual-echo CSE and a cardiac-gated fast STIR sequence. Images were scored regarding number, size, and location of focal lesions, diffuse abnormalities and presence/hindrance of artifacts by two experienced radiologists. Examinations were scored as being definitely normal, indeterminate, or definitely abnormal. Interobserver agreement regarding focal lesions was higher for CSE (kappa = 0.67) than for fast STIR (kappa = 0.57) but did not differ significantly. Of all focal lesions scored in consensus, 47% were scored on both sequences, 31% were only detected by fast STIR, and 22% only by dual-echo CSE (n.s.). Interobserver agreement for diffuse abnormalities was lower with fast STIR (kappa = 0.48) than dual-echo CSE (kappa = 0.65; n. s.). After consensus, fast STIR showed in 10 patients diffuse abnormalities and dual-echo CSE in 3. After consensus, in 19 of 20 patients dual-echo CSE scans were considered as definitely abnormal compared with 17 for fast STIR. The fast STIR sequence is a useful adjunct to dual-echo CSE in detecting focal abnormalities and is helpful in detecting diffuse MS abnormalities in the spinal cord. Due to the frequent occurrence of artifacts and the lower observer concordance, fast STIR cannot be used alone.

Spinal cord magnetic resonance imaging in suspected multiple sclerosis.

We examined the value of spinal cord magnetic resonance imaging (MRI) in the diagnostic work-up of multiple sclerosis (MS). Forty patients suspected of having MS were examined within 24 months after the start of symptoms. Disability was assessed, and symptoms were categorized as either brain or spinal cord. Work-up further included cerebrospinal fluid analysis and standard proton-density, T2-, and T1-weighted gadolinium-enhanced brain and spinal cord MRI. Patients were categorized as either clinically definite MS (n = 13), laboratory-supported definite MS (n = 14), or clinically probable MS (n = 4); four patients had clinically probable MS, and in nine MS was suspected. Spinal cord abnormalities were found in 35 of 40 patients (87.5 %), consisting of focal lesions in 31, only diffuse abnormalities in two, and both in two. Asymptomatic spinal cord lesions occurred in six patients. All patients with diffuse spinal cord abnormality had clear spinal cord symptoms and a primary progressive disease course. In clinically definite MS, the inclusion of spinal imaging increased the sensitivity of MRI to 100 %. Seven patients without a definite diagnosis had clinically isolated syndromes involving the spinal cord. Brain MRI was inconclusive, while all had focal spinal cord lesions which explained symptoms and ruled out other causes. Two other patients had atypical brain abnormalities suggesting ischemic/vascular disease. No spinal cord abnormalities were found, and during follow-up MS was ruled out. Spinal cord abnormalities are common in suspected MS, and may occur asymptomatic. Although diagnostic classification is seldom changed, spinal cord imaging increases diagnostic sensitivity of MRI in patients with suspected MS. In addition, patients with primary progressive MS may possibly be earlier diagnosed. Finally, differentiation with atypical lesions may be improved.

Prognostic value of DNA ploidy using flow cytometry in 1301 breast cancer patients: results of the prospective Multicenter Morphometric Mammary Carcinoma Project.

The literature on breast cancer reports conflicting prognostic results with respect to DNA ploidy of flow cytometric DNA histograms. This might result from different DNA ploidy classification methods. Our study evaluated the prognostic power of DNA ploidy, using different classification methods, in a large prospective group (n = 1301) of breast cancer patients. Flow cytometric DNA histograms obtained from fresh frozen material were interpreted with use of a commercially available computer program. On the basis of the number of stemlines and the DNA Index, we classified the DNA ploidy by different methods. In all of the cases, the classification method "DNA diploid versus DNA nondiploid" provided the best prognostic significance for overall survival (OS) (Mantel-Cox (MC) = 5.4, P = .02; relative risk (RR) = 1.3, P = .05) and for disease-free survival (DFS) (MC = 11.8, P = .0006; RR = 1.3, P < .05). This was also true for the OS of the lymph node-positive (but not the lymph node-negative) subgroup (MC = 4.1, P = .04; RR = 1.3, P = .05). In subgroups classified on the basis of tumor size, DNA ploidy showed prognostic significance for DFS only in the subgroup of tumors smaller than 2 cm and larger than 5 cm. In multivariate analysis, DNA ploidy showed no additional prognostic power to lymph node status and tumor size. The classification "DNA diploid versus DNA nondiploid" was mostly consistent with respect to prognostic power for OS and DFS, especially in small or lymph node-positive tumors. The RR of DNA nondiploid patients was only marginally higher, however, so large study groups are required to reach statistical significance. This could partly explain the disagreements in the literature. Therefore, DNA ploidy seems to be of little clinical importance in breast cancer patients, compared with other prognostic parameters.

Reliable DNA histogram interpretation. Number of nuclei requiring measurement with flow cytometry.

OBJECTIVE: The reasons for conflicting prognostic results as to DNA ploidy and cell cycle variables (DNA index, percent S (%S) phase) may be found mainly at different levels of the flow cytometric methodology used. The present study concentrated on how many nuclei have to be measured with flow cytometry for reliable DNA histogram interpretation. STUDY DESIGN: Twenty-three samples of fresh frozen and 22 samples of paraffin-embedded material from different sites were used. For each sample, 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 (x1,000) events were cumulatively measured. The resulting DNA histograms were analyzed with the MultiCycle computer program using a highly reproducible interpretation protocol. RESULTS: No disagreements about DNA ploidy classification were found in samples with 10,000 or more events for the fresh frozen and 40,000 or more events for the paraffin-embedded material. Excluding DNA ploidy disagreements, the DNA index was stable in all cases. To obtain a %S-phase cell measurement within 20% of the reference value, at least 20,000 and 40,000 events were needed for, respectively, DNA diploid and DNA nondiploid cases for the fresh frozen material and, respectively, 40,000 and 50,000 events for the paraffin-embedded material. CONCLUSION: For reliable combined determination of DNA ploidy, DNA index and %S-phase cells, at least 40,000 and 50,000 events are necessary for fresh frozen and paraffin-embedded material, respectively.

Prognostic implications of different cell cycle analysis models of flow cytometric DNA histograms of 1,301 breast cancer patients: results from the Multicenter Morphometric Mammary Carcinoma Project (MMMCP).

Conflicting prognostic results with regard to DNA flow cytometric cell cycle variables have been reported for breast cancer patients. An important reason for this may be related to differences in the interpretation of DNA histograms. Several computer programs based on different cell cycle fitting models are available resulting in significant variations in percent S-phase and other cell cycle variables. Our present study evaluated the prognostic value of percent S-phase cells obtained using 5 different cell cycle analysis models. Flow cytometric DNA histograms obtained from 1,301 fresh frozen breast cancer samples were interpreted with 5 different cell cycle analysis models using a commercially available computer program. Model 1 used the zero order S-phase calculation and "sliced nuclei" debris correction, model 2 added fixed G2/M- to G0/G1-phase ratio, and model 3 added correction for aggregates. Model 4 applied the first-order S-phase calculation and sliced debris correction. Model 5 fixed the coefficients of variation CVs of the G0/G1- and G2/M-phases in addition to applying the sliced nuclei debris correction and zero order S-phase calculation. The different models yielded clearly different prognostic results. The average percent S-phase cells of the aggregate correction model (model 3) provided the best prognostic value in all cases for overall survival (OS) as well as disease-free survival (DFS) (OS: p < 0.0001; DFS: p < 0.0001), in lymph node-positive cases (OS: p < 0.0001; DFS: p = 0.004) and in DNA-diploid subgroups (OS: p = 0.004; DFS: p = 0.001). For the lymph node negative and DNA-non-diploid subgroups, the percent S-phase of the second cell cycle reached slightly better prognostic significance than the average percent S-phase cells. In multivariate analysis, the average percent S-phase of the aggregate correction model had the best additional prognostic value to tumor size and lymph node status. In conclusion, different cell cycle analysis models yield clearly different prognostic results for invasive breast cancer patients. The most important prognostic percent S-phase variable was the average percent S-phase cells when aggregate correction was included in cell cycle analysis.

Comparison of five cell cycle analysis models applied to 1414 flow cytometric DNA histograms of fresh frozen breast cancer.

Conflicting prognostic results with regard to DNA flow cytometric variables have been reported for breast cancer patients. Reasons for this can be found mainly on the different levels of methodology, including the interpretation of the DNA-histograms. Several computer programs based on different fitting models are available for cell cycle analyses which result in different %S-phase calculations. The present study evaluated the influence of 5 different cell cycle analysis models on several cell cycle variables (%S-phase, %G2M-phase, %diploid cells, DNA-index, %debris) derived from flow cytometric DNA-histograms obtained from breast cancers. DNA-histograms obtained from 1414 fresh frozen breast cancers were interpreted using 5 different cell cycle analysis models using the computer program MultiCycle AV. Model 1 used the zero order S-phase calculation and "sliced nuclei" debris correction, model 2 added fixed G0/G1 and G2/M-phase ratio, and model 3 added correction for aggregates. Model 4 applied the first order S-phase calculation and sliced nuclei debris correction. Model 5 fixed the CVs of the G0/G1 and G2/M-phase in addition to applying the sliced nuclei debris correction and zero-order S-phase calculation. Using all cases, it was shown that when the aggregates correction was included (model 3) in the analysis, on average, significantly lower mean values were obtained for %S-phase cells, and %debris, and %G2M-phase cells of the first cell cycle. No significant differences were observed for the other variables. Analyzing the DNA-diploid, tetraploid, and aneuploid cases separately, similar results were obtained. Linear regression analysis showed only moderately strong correlations for the %S-phase and %G2M-phase variables between the different models, indicating that for individual DNA-histograms the cell cycle analysis results may vary. In conclusion, quite different values can be obtained for especially the %S-phase cells using different cell cycle analysis models in individual cases. Correction for aggregates results on average in significantly lower %S-phase values. This clearly has implications for comparing %S-phase results from studies using aggregate correction or not, especially with regard to prognostic thresholds. Large follow-up studies are necessary to derive at the prognostically best model.

The influence of fixation delay on mitotic activity and flow cytometric cell cycle variables.

Proliferation variables such as mitotic activity and the percentage of S-phase cells have been shown to be of prognostic value in many tumors, especially in breast cancer. However, some studies reported a decrease in mitotic activity caused by delay in fixation of the tissue. In contrast, other studies showed that the identifiability of mitotic figures decreases after fixation delay, but the total number of mitotic figures and also the percentage of S-phase cells remain unchanged. Most studies have been done on small numbers of experimental tumors, thus introducing the risk of selection bias. The aim of this study was to reinvestigate the influence of fixation delay on mitotic activity and cell cycle variables assessed by flow cytometry in an adequate number of resected human tissues to reach firmer conclusions. Resection specimens of 19 and 21 cases, respectively, for the mitotic activity estimate and the flow cytometric percentage of S-phase calculation were collected directly from the operating theater using lung, breast, and intestinal cancers and normal intestinal mucosa. The tissues were cut in pieces, and from each specimen, pieces were fixed in 4% buffered formaldehyde (for mitosis counting) as well as snap frozen (for flow cytometry) immediately after excision, as well as after a fixation delay of 1, 2, 4, 6, 8, 18, and 24 hours. Moreover, during the fixation delay, one series from each specimen was kept in the refrigerator and the second at room temperature. Thus, a total of 304 (19 X 16) and 336 (21 X 16) specimens were investigated for the mitotic activity estimate and the percentage of S-phase cells calculation, respectively. With regard to the estimation of the mitotic activity, both clear and doubtful mitotic figures were registered separately, obtaining an "uncorrected" and "corrected" (for doubtful mitotic figures) mitotic activity estimate. The percentage of S-phase cells was obtained by cell cycle analysis of flow cytometric DNA-histograms. The results showed that the quality of the material decreased during the fixation delay, as reflected by poorer cellular morphology in the hematoxylin-and-eosin-stained slides, resulting in more difficult identification of mitotic figures and a more time-consuming procedure with regard to the mitosis counts, but not in a worse intraobserver and interobserver reproducibility, which was acceptable. The reduction in quality of the tissues also was shown by the flow cytometric measurements because the coefficient of variation and percentage of debris increased after 4 hours or more of fixation delay. However, the mean values of the "uncorrected" mitotic activity and the "corrected" mitotic activity showed no decreasing trend; neither did the average percentage of S-phase cells. In conclusion, within the time investigated, fixation delay has no clear influence on the proliferation features studied. Because of the decreasing quality of the histological sections, resulting in more difficult identification of mitoses and interpretation of DNA histograms, fixation delay should be kept as short as possible, keeping the tissue at 4 degrees C until fixation.

Tumour heterogeneity of DNA cell cycle variables in breast cancer measured by flow cytometry.

AIM/BACKGROUND: Conflicting results have been reported concerning the prognostic value of DNA flow cytometric variables (DNA ploidy, DNA index, %S phase fraction) in breast cancer. Selection bias and differences in treatment may have contributed to these conflicting prognostic results. Differences in tissue processing, the number of nuclei measured, DNA histogram/cell cycle analysis, and intra-tumour heterogeneity may also have played a role. The aim of the present study was to assess intra-tumour heterogeneity of DNA flow cytometric variables in breast cancer. METHODS: Fresh frozen specimens (n = 274) (0.3 x 0.3 x 0.3 cm) of 17 breast cancers and 167 slices, 50 microns thick, of 58 paraffin wax embedded blocks of 21 breast cancers were studied. All samples were prepared individually for DNA flow cytometry. DNA histograms were interpreted by semi-automated cell cycle analysis (MultiCycle) by two observers to avoid biased interpretation. An artificial averaged DNA histogram of each case was composed to simulate a sample prepared from whole tumour tissue. RESULTS: With regard to DNA ploidy, classified as diploid or aneuploid, the fresh frozen and paraffin wax embedded breast cancers showed intra-tumour heterogeneity in 53% and 38% of cases, respectively. For fresh frozen and paraffin wax embedded material, respectively, six samples had to be measured to detect the highest DNA ploidy class in 71% and 86% of cases. Averaged DNA histograms showed a loss of DNA aneuploidy in 36% and 6% of fresh frozen and paraffin wax embedded samples, respectively. High intra-tumour heterogeneity (wide ranges) was found for the %S phase fraction. Average %S phase fraction and average aneuploid %S phase fraction had the widest ranges at 9.5-31.6% and 0.0-62.7%, respectively. There was no correlation between the number of stemlines and intra-tumour %S phase variability on the one hand and tumour size and grade on the other. CONCLUSIONS: High intra-tumour heterogeneity for breast cancer was found for DNA ploidy, the DNA index and %S phase fraction as measured by flow cytometry, which may explain the conflicting prognostic results reported in the literature. To detect aneuploid cells, six samples may have to be prepared and measured separately. Measurement of these variables may be more reliable in paraffin wax sections because the thick slices provide a more representative sample. Prospective studies are required to determine whether the highest %S phase fraction value or the average value is more useful in the clinical context.

Cell cycle analysis of 932 flow cytometric DNA histograms of fresh frozen breast carcinoma material. Correlations between flow cytometric, clinical, and pathologic variables. MMMCP Collaborative Group. Multicenter Morphometric Mammary Carcinoma Project Collaborative Group.

BACKGROUND: Confusing data have been presented for breast cancer patients on correlations between DNA ploidy and the percentage of S-phase cells and other prognostic variables. The aim of this study was to compare DNA ploidy classification and cell cycle variables with clinical, classic, and quantitative pathologic variables and clinical variables in a large group of patients. METHODS: DNA ploidy and cell cycle variables were extracted from MultiCycle (Phoenix Flow Systems, San Diego, CA) interpreted flow cytometric DNA histograms of fresh frozen material from 932 breast cancer patients and compared with clinical (age, hormonal status), classic pathology (lymph node status, tumor size and type), and quantitative pathologic variables (steroid receptor status, mitotic activity index [MAI], mean nuclear area [MNA]). RESULTS: The DNA ploidy correlated significantly with MAI, MNA steroid receptor status, and tumor type. No significant correlations were found with tumor size, lymph node status, age, and hormonal status. The first DNA index correlated significantly with MAI, MNA, and steroid receptor status. The percentage of S-phase cells significantly correlated with MAI, MNA, steroid receptor status, and lymph mode status. CONCLUSIONS: DNA index and DNA ploidy, as markers of genetic instability, correlated well with differentiation and proliferation markers and less well with lymph node status and tumor size as markers of metastatic potential and duration of disease. The percentage of S-phase cells was not independent of the percentage of differentiation markers and did not correlate strongly with mitotic activity. This indicates that the percentage of S-phase cells and the mitotic activity partially reflect different proliferative properties.

Interlaboratory reproducibility of semiautomated cell cycle analysis of flow cytometry DNA-histograms obtained from fresh material of 1,295 breast cancer cases.

Conflicting prognostic results have been published as to the DNA variables, such as DNA ploidy, DNA index, and % S-phase cells for breast cancer patients. These variables can be obtained by interpreting DNA histograms by cell cycle analysis. Explanations for these conflicting results might be found on the level of the interpretation of the DNA histograms. In a previous study, the semi automated cell cycle analysis computer program MultiCycle (Phoenix Flow Systems, San Diego, CA) showed high intralaboratory reproducibility. However, what types of DNA histograms may cause disagreements was still unclear. The aim of this study was to determine the interlaboratory reproducibility of MultiCycle-based cell cycle analysis of 1,295 flow cytometric DNA histograms derived from fresh frozen breast cancer material and to clarify potential sources of interobserver variation when analyzing DNA histograms. DNA ploidy classification into diploid, hyperdiploid, tetraploid, hypertetraploid, and multiploid showed an interlaboratory agreement of 94% (kappa value = 0.92). The 6% discrepancies (n = 74) were caused by tetraploid peaks, as established in one laboratory, which shifted outside the tetraploid region on reanalysis by the other laboratory (37%), shoulders sometimes interpreted as peaks (24%), small peaks not always recognized as such (24%), fitting failures (10%), and overlooking of tetraploid peaks (5%). Furthermore, the cell cycle analysis variables showed variable reproducibility. The % S-phase cells of the first, second, and third cell cycle showed overall a moderate reproducibility (0.62 < or = R < or = 0.79), but the average % S-phase cells and the average aneuploid % S-phase cells were more reproducible with correlation coefficients of 0.89 and 0.81, respectively. The coefficient of variation of the G0/G1 peak of the first cell cycle, the DNA indices and the % diploid cells were highly reproducible (R > or = 0.94), and the % G2/M-phase cells of the first, second, and third cell cycle were poorly reproducible (0.22 < or = R < or = 0.68). When a cut-point was used at the mean value of 7% for the average % S-phase cells, the number of "threshold discrepancy cases" was 6%. Sources of variation for cell cycle analysis were variations in the debris correction procedures, disagreement about the modes of the aneuploid peaks, disagreement about small peaks, shoulders sometimes interpreted as peaks, and overlooking of tetraploid peaks.

Reproducibility of semi-automated cell cycle analysis of flow cytometric DNA-histograms of fresh breast cancer material.

DNA-variables such as DNA-ploidy, DNA-index and %S-phase cells have been proven to have prognostic value for breast cancer patients. These variables can be obtained by interpreting DNA-histograms by cell cycle analysis. Since there are a number of potential error sources, the aim of this study was to determine the intra- and inter-observer reproducibility of semi-automated cell cycle analysis with the emphasis on DNA ploidy and %S-phase assessments. The 149 DNA-histograms we used were randomly selected from the Multicentre Morphometric Mammary Carcinoma Project, a nationwide prospective study in The Netherlands on the reproducibility and prognostic power of quantitative assessments. These DNA-histograms were obtained by flow cytometry of fresh frozen breast cancer material. Cell cycle analysis was performed according to a strict protocol with the semi-automated computer program 'MultiCycle', using the background correction option; 68 histograms were analyzed in duplicate by the same observer, and 81 histograms were analyzed by two observers. Assessment of DNA-ploidy showed an intra-observer concordance of 99% (kappa-value 0.98) and an inter-observer concordance of 94% (kappa-value 0.91). The disagreement could be attributed to overlooking a DNA-tetraploid cell cycle in one case, some difficult histograms and varying opinions about small peaks between the observers in a few cases. Intra-observer %S-phase correlation coefficients varied between 0.72 for the %S-phase of the second aneuploid cell cycle and 0.99 for the %S-phase of the diploid cell cycle. Inter-observer correlation coefficients varied between 0.81 for the %S-phase of the second cell cycle and 0.95 for the %S-phase of the diploid cell cycle and the average %S-phase cells. As for DNA-index, intra- and inter-observer correlation coefficients were 0.97 and 0.94, respectively. In conclusion, intra- and inter-observer reproducibility of semi-automated cell cycle analysis of flow cytometric DNA-histograms from fresh breast cancer material using the Multi-Cycle program, following a strict protocol, is in principle high. The results of this study may help us to decide which of the different %S-phases provided by cell cycle analysis software should be used in daily practice.

Quantitative microscopical and confocal laser scanning microscopy for intermediate endpoint biomarkers in breast cancer: potential and reproducibility.

Diagnostic quantitative pathological (QP) determinations are increasingly used in our hospital. The number of requests for QP for reference materials is rising rapidly. This is understandable; quantitative assessments have a strong prognostic value and can be very reproducible, depending on the care taken with a number of factors including cell and tissue processing, application of the appropriate stains, and the measurement protocol used. As to the latter, systematic random sampling gives the best intra- and interobserver agreement (with correlation coefficients between observers for certain features > or = 0.94). Flow cytometric determinations are often regarded as more reproducible than interactive morphometry due to the high speed of the assessments, the large number of objects measured per specimen, and the lack of observer interaction. Indeed, flow cytometrically assessed DNA ploidy is very reproducible, even though the % S-phase fraction is much more variable. Unlike image cytometry (ICM), visual inspection of cells is not easily accomplished with flow cytometry (FCM). With ICM, the fully automated measurement of DNA in thousands of cells is possible in 3-5 minutes, with a very low coefficient of variation (< or = 2% for the diploid and tetraploid peak of liver cell nuclei). ICM also allows measurement of texture features. However, quantitative immunohisto/cytochemical determinations may not always be as reproducible as sometimes believed. Recently, we found large variations in the measurements, made by a commercially available image processing instrument, of the estrogen and progesterone receptors, Ki-67, cathepsin D, and neu protein overexpression in breast cancer.(ABSTRACT TRUNCATED AT 250 WORDS)