External validation and clinical utility assessment of PREDICT breast cancer prognostic model in young, systemic treatment-naïve women with node-negative breast cancer.

BACKGROUND: The validity of the PREDICT breast cancer prognostic model is unclear for young patients without adjuvant systemic treatment. This study aimed to validate PREDICT and assess its clinical utility in young women with node-negative breast cancer who did not receive systemic treatment. METHODS: We selected all women from the Netherlands Cancer Registry who were diagnosed with node-negative breast cancer under age 40 between 1989 and 2000, a period when adjuvant systemic treatment was not standard practice for women with node-negative disease. We evaluated the calibration and discrimination of PREDICT using the observed/expected (O/E) mortality ratio, and the area under the receiver operating characteristic curve (AUC), respectively. Additionally, we compared the potential clinical utility of PREDICT for selectively administering chemotherapy to the chemotherapy-to-all strategy using decision curve analysis at predefined thresholds. RESULTS: A total of 2264 women with a median age at diagnosis of 36 years were included. Of them, 71.2% had estrogen receptor (ER)-positive tumors and 44.0% had grade 3 tumors. Median tumor size was 16 mm. PREDICT v2.2 underestimated 10-year all-cause mortality by 33% in all women (O/E ratio:1.33, 95%CI:1.22-1.43). Model discrimination was moderate overall (AUC10-year:0.65, 95%CI:0.62-0.68), and poor for women with ER-negative tumors (AUC10-year:0.56, 95%CI:0.51-0.62). Compared to the chemotherapy-to-all strategy, PREDICT only showed a slightly higher net benefit in women with ER-positive tumors, but not in women with ER-negative tumors. CONCLUSIONS: PREDICT yields unreliable predictions for young women with node-negative breast cancer. Further model updates are needed before PREDICT can be routinely used in this patient subset.

Network Meta-analysis for the Diagnostic Approach to Pathologic Nipple Discharge.

Pathologic nipple discharge (PND) is one of the most common breast-related complaints for referral because of its supposed association with breast cancer. The aim of this network meta-analysis (NMA) was to compare the diagnostic efficacy of ultrasound, mammogram, cytology, magnetic resonance imaging (MRI), and ductoscopy in patients with PND, as well as to determine the best diagnostic strategy to assess the risk of malignancy as cause for PND. Cochrane Library, PubMed, and Embase were searched to collect relevant literature from the inception of each of the diagnostic methods until January 27, 2020. The search yielded 1472 original citations, of which 36 studies with 3764 patients were finally included for analysis. Direct and indirect comparisons were performed using an NMA approach to evaluate the combined odd ratios and to determine the surface under the cumulative ranking curves (SUCRA) of the diagnostic value of different imaging methods for the detection of breast cancer in patients with PND. Additionally, a subgroup meta-analysis comparing ductoscopy to MRI when conventional imaging was negative was also performed. According to this NMA, sensitivity for detection of malignancy in patients with PND was highest for MRI (83%), followed by ductoscopy (58%), ultrasound (50%), cytology (38%), and mammogram (22%). Specificity was highest for mammogram (93%) followed by ductoscopy (92%), cytology (90%), MRI (76%), and ultrasound (69%). Diagnostic accuracy was the highest for ductoscopy (88%), followed by cytology (82%), MRI (77%), mammogram (76%), and ultrasound (65%). Subgroup meta-analysis (comparing ductoscopy to MRI when ultrasound and mammogram were negative) showed no significant difference in sensitivity, but ductoscopy was statistically significantly better with regard to specificity and diagnostic accuracy. The results from this NMA indicate that although ultrasound and mammogram may remain low-cost useful first choices for the detection of malignancy in patients with PND, ductoscopy outperforms most imaging techniques (especially MRI) and cytology.

Assessment of tumour proliferation by use of the mitotic activity index, and Ki67 and phosphohistone H3 expression, in early-stage luminal breast cancer.

AIMS: Phosphohistone H3 (PhH3) has been proposed as a novel proliferation marker in breast cancer. This study compares the interobserver agreement for assessment of the mitotic activity index (MAI), Ki67 expression, and PhH3 in a cohort of oestrogen receptor (ER)-positive breast cancer patients. METHODS AND RESULTS: Tumour samples of 159 luminal breast cancer patients were collected. MAI and PhH3 scores were assessed by three breast cancer pathologists. Ki67 scores were assessed separately by two of the three pathologists. PhH3-positive cells were counted in an area of 2 mm2 , with a threshold of ≥13 positive cells being used to discriminate between low-proliferative and high-proliferative tumours. Ki67 expression was assessed with the global scoring method. Ki67 percentages of <20% were considered to be low. The intraclass correlation coefficient (ICC) and Cohen's κ statistics were used to evaluate interobserver agreement. The impact on histological grading of replacing the MAI with PhH3 was assessed. Counting PhH3-positive cells was highly reproducible among all three observers (ICC of 0.86). The κ scores for the categorical PhH3 count (κ = 0.78, κ = 0.68, and κ = 0.80) reflected substantial agreement among all observers, whereas agreement for the MAI (κ = 0.38, κ = 0.52, and κ = 0.26) and Ki67 (κ = 0.55) was fair to moderate. When PhH3 was used to determine the histological grade, agreement in grading increased (PhH3, κ = 0.52, κ = 0.48, and κ = 0.52; MAI, κ = 0.43, κ = 0.35, and κ = 0.32), and the proportion of grade III tumours increased (14%, 18%, and 27%). CONCLUSION: PhH3 seems to outperform Ki67 and the MAI as a reproducible means to measure tumour proliferation in luminal-type breast cancer. Variation in the assessment of histological grade might be reduced by using PhH3, but would result in an increase in the proportion of high-grade cancers.

Luminal A versus luminal B breast cancer: MammaTyper mRNA versus immunohistochemical subtyping with an emphasis on standardised Ki67 labelling-based or mitotic activity index-based proliferation assessment.

AIMS: Proliferation assessment by the use of Ki67 is a crucial component in intrinsic subtyping of luminal breast cancers (BCs), but suffers from variability between laboratories, observers, and methods. MammaTyper is a quantitative molecular tool that measures mRNA levels of ERBB2, ESR1, PGR and MKI67 in BC, and interprets the results according to the St Gallen 2013 consensus recommendations. We compared MammaTyper with immunohistochemistry (IHC)-based subtypes, with a focus on standardised proliferation assessment. METHODS AND RESULTS: We analysed the agreement in assigning subtypes between MammaTyper and receptor IHC in 101 unifocal luminal HER2-negative early BCs of no special type. Two Ki67 counting protocols, Ki67-Global (Ki67-G) and Ki67-HotSpot (Ki67-H), recommended by the International Ki67 in BC Working Group, and the mitotic activity index (MAI) were used for proliferation assessment. The proportions of BCs identified as luminal A and as luminal B were 55% and 45% for MammaTyper, 55% and 45% for IHC + Ki67-G, 36% and 64% for IHC + Ki67-H, and 56% and 44% for IHC + MAI. The levels of agreement between MammaTyper-based and IHC-based subtyping were 84% (κ = 0.679) for IHC + Ki67-G, 72% (κ = 0.462) for IHC + Ki67-H, and 89% (κ = 0.779) for IHC + MAI. CONCLUSIONS: High rates of agreement between mRNA-based and IHC-based intrinsic subtyping of luminal HER2-negative BC can be achieved. However, the agreement between IHC-based and MammaTyper-based luminal subtypes depends on the proliferation assessment method, and was highest when the MAI was used. Further comparative clinical studies are needed to determine which method is to be preferred, including analysis of cost-effectiveness.

Hormone- and HER2-receptor assessment in 33,046 breast cancer patients: a nationwide comparison of positivity rates between pathology laboratories in the Netherlands.

PURPOSE: Patient management of invasive breast cancer (IBC) is to a large extent based on hormone- and HER2-receptor assessment. High-quality, reliable receptor assessment is of key importance as false results may lead to under- or overtreatment of patients. Surveillance of case-mix adjusted positivity rates has been suggested as a tool to identify laboratories with insufficient testing assays, as this covers the whole process of receptor assessment and enables laboratories to benchmark their positivity rates against other laboratories. We studied laboratory-specific variation in hormone- and HER2 positivity rates of 33,046 breast cancer patients using real-life nationwide data. METHODS: All synoptic pathology reports of IBC resection-specimens, obtained between 2013 and 2016, were retrieved from the nationwide Dutch pathology registry (PALGA). Absolute and case-mix adjusted receptor positivity rates were compared to the mean national proportion and presented in funnel plots in separate analyses for estrogen (ER), progesterone (PR) and HER2. Case-mix adjustment was performed by multivariable logistic regression. RESULTS: 33,794 IBC lesions from 33,046 patients of 39 pathology laboratories were included. After case-mix adjustment, mean positivity rates were 87.2% for ER (range 80.4-94.3), 71.3% for PR (62.5-77.5%), and 9.9% for HER2 (5.5-12.7%). Overall, 14 (35.9%), 17 (43.6%) and 11 (28.2%) laboratories showed positivity rates outside the 95% confidence interval for ER, PR and HER2, respectively. CONCLUSION: This nationwide study shows that absolute variation in hormone- and HER2-receptor positivity rates between Dutch pathology laboratories is limited. Yet, the considerable number of outlying laboratories shows that there is still need for improvement. Continuous monitoring and benchmarking of positivity rates may help to realize this.

Evaluating the benefits of digital pathology implementation: time savings in laboratory logistics.

BACKGROUND: The benefits of digital pathology for workflow improvement and thereby cost savings in pathology, at least partly outweighing investment costs, are being increasingly recognised. Successful implementations in a variety of scenarios have started to demonstrate the cost benefits of digital pathology for both research and routine diagnosis, contributing to a sound business case encouraging further adoption. To further support new adopters, there is still a need for detailed assessment of the impact that this technology has on the relevant pathology workflows, with an emphasis on time-saving. AIMS: To assess the impact of digital pathology adoption on logistic laboratory tasks (i.e. not including pathologists' time for diagnosis-making) in the Laboratorium Pathologie Oost Nederland, a large regional pathology laboratory in The Netherlands. METHODS AND RESULTS: To quantify the benefits of digitisation, we analysed the differences between the traditional analogue and new digital workflows, carried out detailed measurements of all relevant steps in key analogue and digital processes, and compared the time spent. We modelled and assessed the logistic savings in five workflows: (i) routine diagnosis; (ii) multidisciplinary meeting; (iii) external revision requests; (iv) extra stainings; and (v) external consultation. On average, >19 working hours were saved on a typical day by working digitally, with the highest savings in routine diagnosis and multidisciplinary meeting workflows. CONCLUSIONS: By working digitally, a significant amount of time could be saved in a large regional pathology laboratory with a typical case mix. We also present the data in each workflow per task and concrete logistic steps to allow extrapolation to the context and case mix of other laboratories.

Assessment of HER2 status in breast cancer biopsies is not affected by accelerated tissue processing.

AIMS: To establish whether core needle biopsy (CNB) specimens processed with an accelerated processing method with short fixation time can be used to determine accurately the human epidermal growth factor receptor 2 (HER2) status of breast cancer. METHODS AND RESULTS: A consecutive case-series from two high-volume breast clinics was created. We compared routine HER2 immunohistochemistry (IHC) assessment between accelerated processing CNB specimens and routinely processed postoperative excision specimens. Additional amplification-based testing was performed in cases with equivocal results. The formalin fixation time was less than 2 h and between 6 and 72 h, respectively. Fluorescence in-situ hybridisation and multiplex ligation-dependent probe amplification were used for amplification testing. One hundred and forty-four cases were included, 15 of which were HER2-positive on the routinely processed excision specimens. On the CNB specimens, 44 were equivocal on IHC and required an amplification-based test. Correlation between the CNB specimens and the corresponding excision specimens was high for final HER2 status, with an accuracy of 97% and a kappa of 0.85. CONCLUSIONS: HER2 status can be determined reliably on CNB specimens with accelerated processing time using standard clinical testing methods. Using this accelerated technology the minimum 6 h of formalin fixation, which current guidelines consider necessary, can be decreased safely. This allows for a complete and expedited histology-based diagnosis of breast lesions in the setting of a one-stop-shop, same-day breast clinic.

St Gallen 2015 subtyping of luminal breast cancers: impact of different Ki67-based proliferation assessment methods.

Ki67 has been proposed as prognostic proliferation marker in luminal breast cancer (BC), but little is known on the influence of Ki67 assessment methods on subtyping into luminal A- and B-like tumors. Our aim was to study the influence of different Ki67-labeling index (Ki67-LI) assessment methods on the proportion of BCs classified as luminal A-like. 280 early BCs were subtyped according to the St Gallen 2015 definitions into 71 % luminal (HER2 negative), 6 % luminal B-like (HER2 positive), 13 % triple negative, 1 % HER2 positive (nonluminal), and 9 % special type. Digitized whole slides were counted manually on the screen. We used nine defined counting methods to assess the Ki67-LI (including the International Ki67 in Breast Cancer Working Group recommendations), and compared the resulting medians and the proportions of cancers classified as luminal A-like according to the formerly used cut-off <20 %. Methods assessing hot spots and tumor periphery resulted in significantly higher Ki67-LI medians than those measuring an average proliferation (27.45 % vs 16.96 %, p < 0.0001). Substantially lower median Ki67-LI were found when assessing 1020 compared to counting 100, 200, 300 cells (17.65 vs 33 %, vs 28 %, vs 24.33 %, respectively; p < 0.0001), or 510 cells (20.59 %, p = 0.019). Applying a standard Ki67-LI cut-off <20 % to define low proliferation for all methods, the proportion of luminal A-like cancers varied between 13 and 44 %. The proportion of BCs classified as luminal A-like is highly influenced by the Ki67-LI assessment method. As a consequence, the selection of a specific Ki67-LI assessment method may have a direct effect on the proportion of patients considered having low-risk disease and thus influence therapeutic decision making. This calls for a standardized assessment method.

High Prevalence of MRI-Detected Contralateral and Ipsilateral Malignant Findings in Patients With Invasive Ductolobular Breast Cancer: Impact on Surgical Management.

INTRODUCTION: Invasive breast cancer comprises a spectrum of histologic changes with purely lobular and purely ductal cancer on either side and mixed lesions in between. Our aim was to evaluate to what extent preoperative magnetic resonance imaging (MRI) leads to the finding of additional malignancies and the effect on surgical management in the subcategory of women with invasive ductolobular disease. PATIENTS AND METHODS: From 2007 to 2012, 109 patients diagnosed with breast cancer containing a lobular component underwent preoperative MRI. The MRI findings were compared with the findings from mammography and ultrasonography. Clinically relevant additional MRI findings were verified histologically. The histologic slides were reviewed, and the percentage of the lobular component was determined. In a multidisciplinary setting, the TNM classification and surgical policy were determined using the conventional imaging findings and as a scenario that included preoperative MRI. RESULTS: MRI revealed additional malignant foci in 28 of 109 patients (26%). More extensive disease was seen in 25 patients (23%). The preoperative MRI findings changed the TNM classification in 42% of the patients and altered the surgical policy in 37%. No correlation was found between the lobular component and the probability of detecting additional malignant foci, more extensive disease, or the frequency of a change in TNM classification or surgical policy. According to the final pathology report, the change in surgical policy was justified in 85% of the patients. CONCLUSION: In patients with breast cancer presenting with lobular differentiation at biopsy, preoperative MRI can lead to the detection of additional malignancies and clinically relevant changes in surgical policy in a high percentage of patients, irrespective of the lobular component. The use of MRI as a part of the standard workup of such patients deserves consideration.

Assessment of algorithms for mitosis detection in breast cancer histopathology images.

The proliferative activity of breast tumors, which is routinely estimated by counting of mitotic figures in hematoxylin and eosin stained histology sections, is considered to be one of the most important prognostic markers. However, mitosis counting is laborious, subjective and may suffer from low inter-observer agreement. With the wider acceptance of whole slide images in pathology labs, automatic image analysis has been proposed as a potential solution for these issues. In this paper, the results from the Assessment of Mitosis Detection Algorithms 2013 (AMIDA13) challenge are described. The challenge was based on a data set consisting of 12 training and 11 testing subjects, with more than one thousand annotated mitotic figures by multiple observers. Short descriptions and results from the evaluation of eleven methods are presented. The top performing method has an error rate that is comparable to the inter-observer agreement among pathologists.

Brief fixation does not affect assessment of hormone receptor expression in invasive breast carcinoma biopsies: paving the road for same-day tissue diagnostics.

In patients with invasive breast carcinoma, estrogen receptor α (ERα) and progesterone receptor (PR) expressions need to be assessed in core-needle biopsies (CNBs) before the start of neoadjuvant systemic treatment. Current guidelines recommend a minimum formalin fixation time of 6 hours. Considering the increasing demand for same-day diagnostics in oncology, more rapid tissue processing with shorter fixation times is required. To identify whether brief fixation (<6 h) of CNBs compared with conventionally fixed resection specimens provides for reliable immunohistochemical assessment of ERα and PR expression, 78 consecutive patients diagnosed with invasive breast carcinoma were included through the same-day diagnostics programme of the UMC Utrecht. Paraffin-embedded CNBs fixed for approximately 45 minutes were retrieved. Immunohistochemistry for ERα and PR was compared between the briefly fixed CNBs and conventionally fixed resection specimens. All slides were reviewed by means of consensus scoring by 2 blinded observers. Overall agreement between CNB and resections was 73/74 (98.6%) for ERα (κ=0.85; 95% confidence interval [CI]=0.56-1.00) and 69/75 (92.0%) for PR (κ=0.81; 95% CI=0.66-0.96). For ERα, positive and negative predictive values were 98.6% (95% CI=0.91-0.99) and 100.0% (95% CI=0.31-1.00), respectively. For PR, positive and negative predictive values were 100.0% (95% CI=0.91-1.00) and 76.0% (95% CI=0.54-0.90). In conclusion, analysis of hormone receptor expression in briefly fixed CNB seems comparable to results from conventionally fixed resection specimens of the same tumor.

Discrepancy between routine and expert pathologists' assessment of non-palpable breast cancer and its impact on locoregional and systemic treatment.

Histopathological parameters are essential for deciding on adjuvant treatment following breast cancer surgery. We assessed the impact of inter-observer variability on treatment strategy in patients operated for clinically node negative, non-palpable breast carcinomas. In the context of a multicenter randomised controlled trial, clinical and histological data of 310 patients with clinically node negative non-palpable invasive breast cancer were prospectively collected. Histological assessment of the primary tumour and sentinel nodes was first performed in a routine setting, subsequently central review took place. In case of discordance between local en central assessments, we determined the impact on locoregional and systemic treatment strategy. Discordance between local and central review was observed in 13% of the patients for type (kappa 0.60, 95% CI 0.50-0.71), in 12% for grade (k=0.796, 95% CI 0.73-0.86), in 1% for ER status (k=0.898, 95% CI 0.80-1.0), in 2% for PR status (k=0.940 95% CI 0.89-0.99). Discrepancy in the assessment of the sentinel node(s) was seen in 2% of the patients (k=0.954, 95% CI 0.92-0.98). Applying current Dutch Guidelines, central review would have affected locoregional treatment in 2% (7/310), systemic treatment in 5% (16/310) and both in 1% (2/310) of the patients. For the 9 (3%) patients in whom central review would have led to additional systemic treatment, Adjuvant! predicted 10 years mortality and recurrence rate would have decreased with a median of 4.6% and 15%, respectively. Discordance between routine histological assessment and central review of non-palpable breast carcinoma specimens and sentinel nodes was observed in 24% of patients. This inter-observer variation would have impacted locoregional and/or systemic treatment strategies in 8% of the patients.

Validation of a gene expression signature for assessment of lymph node metastasis in oral squamous cell carcinoma.

PURPOSE: Current assessment of lymph node metastasis in patients with head and neck squamous cell carcinoma is not accurate enough to prevent overtreatment. The aim of this study was validation of a gene expression signature for distinguishing metastasizing (N+) from nonmetastasizing (N0) squamous cell carcinoma of the oral cavity (OSCC) and oropharynx (OPSCC) in a large multicenter cohort, using a diagnostic DNA microarray in a Clinical Laboratory Improvement Amendments/International Organization for Standardization-approved laboratory. METHODS: A multigene signature, previously reported as predictive for the presence of lymph node metastases in OSCC and OPSCC, was first re-evaluated and trained on 94 samples using generic, whole-genome, DNA microarrays. Signature genes were then transferred to a dedicated diagnostic microarray using the same technology platform. Additional samples (n=222) were collected from all head and neck oncologic centers in the Netherlands and analyzed with the diagnostic microarray. Human papillomavirus status was determined by real-time quantitative polymerase chain reaction. RESULTS: The negative predictive value (NPV) of the diagnostic signature on the entire validation cohort (n=222) was 72%. The signature performed well on the most relevant subset of early-stage (cT1-T2N0) OSCC (n=101), with an NPV of 89%. CONCLUSION: Combining current clinical assessment with the expression signature would decrease the rate of undetected nodal metastases from 28% to 11% in early-stage OSCC. This should be sufficient to enable clinicians to refrain from elective neck treatment. A new clinical decision model that incorporates the expression signature is therefore proposed for testing in a prospective study, which could substantially improve treatment for this group of patients.

[Chemoprevention of breast cancer: exemestane ready to be tested in study setting]. / Chemopreventie van borstkanker: exemestaan toepassen in studieverband.

Prevention of breast cancer is difficult, as most risk factors are difficult to control. Therefore, we have not been able to stop breast cancer incidence rates from rising over past decades. Chemoprevention using aromatase inhibitors, such as exemestane, may change this situation. Aromatase inhibitors are very effective and safe in preventing recurrent disease in postmenopausal breast cancer. A recent randomised placebo-controlled trial showed that in healthy women at moderately increased risk of breast cancer, use of exemestane over a 3-year period reduced the risk of breast cancer by 65%. The reduction was from 0.55% to 0.19% per year, with no adverse events or measurable effect on quality of life. If confirmed by larger studies with longer duration of follow-up, preventive use of exemestane may turn out to be the first acceptable alternative for women at increased risk of developing breast cancer. On a population level, implementation of prevention using aromatase inhibitors has the potential to reduce the incidence of breast cancer. However, currently in the Dutch population, 140 women would need to use exemestane for three years in order to prevent one invasive breast tumour. Therefore, large-scale implementation of exemestane is not advised, but the evidence is sufficient to start studies with exemestane.

Do we see what we think we see? The complexities of morphological assessment.

Reliable pathological interpretation is vital to so many aspects of tissue-based research as well as being central to patient care. Understanding the complex processes involved in decision-making is the starting point to improve both diagnostic reproducibility and the definition of diagnostic groups that underpin our experiments. Unfortunately, there is a paucity of research in this field and it is encouraging to see The Journal of Pathology publishing work in this area. This review attempts to highlight the opportunities that exist in this field and the technologies that are now available to support this type of research. Key amongst these are the use of decision analysis tools such as inference networks, and virtual microscopy that allows us to simulate diagnostic decision-making. These tools have roles, not only in studying the subtleties of diagnostic decision-making, but also in delivering new methods of training and proficiency testing. Research which helps us to better understand what we see, why we see it, and standardizing interpretative reasoning in pathological classification is essential for improving the wide range of activities that pathologists support, including clinical diagnosis, teaching, training, and experimental research.

HER-2/neu amplification testing in breast cancer by multiplex ligation-dependent probe amplification in comparison with immunohistochemistry and in situ hybridization.

BACKGROUND: Assessment of HER-2/neu status in invasive breast cancer is crucial to establish eligibility for trastuzumab and taxane based chemotherapy. Next to immunohistochemistry (IHC) to evaluate protein overexpression, a second line gene amplification test is required for cases with equivocal protein expression. This study aimed to validate a new PCR based test, called Multiplex Ligation-dependent Probe Amplification (MLPA), as a simple and quick method to assess HER-2/neu gene amplification status in invasive breast cancer. METHODS: MPLA results were compared with gene amplification status assessed by fluorescence in situ hybridization (FISH) and chromogenic in situ hybridization (CISH) as gold standard, and with protein overexpression by IHC in 518 breast carcinoma patients. RESULTS: About 10% of cases overexpressed HER-2/neu at the protein level (IHC), and 11% of cases showed gene-amplification by MLPA. A high concordance was found between FISH and CISH, MLPA and IHC, and MLPA and CISH. MLPA showed amplification in 7/36 (19%) of the equivocal IHC 2+ cases. However, of the IHC 0/1+ cases, 6/434 (1.4%) were also amplified by MLPA, and amplification was confirmed in all of these cases by FISH/CISH. On the other hand, one of the 48 (2%) IHC 3+ cases was normal by MLPA and lack of amplification was confirmed by FISH/CISH. CONCLUSION: MLPA is a fast, accurate and cheap method to detect breast cancer HER-2/neu amplification in small quantities of DNA extracted from paraffin blocks, and thereby a reliable alternative to FISH and CISH.

Development of 3D chromatin texture analysis using confocal laser scanning microscopy.

INTRODUCTION: Analysis of nuclear texture features as a measure of nuclear chromatin changes has been proven to be useful when measured on thin (5-6 microm) tissue sections using conventional 2D bright field microscopy. The drawback of this approach is that most nuclei are not intact because of those thin sections. Confocal laser scanning microscopy (CLSM) allows measurements of texture in 3D reconstructed nuclei. The aim of this study was to develop 3D texture features that quantitatively describe changes in chromatin architecture associated with malignancy using CLSM images. METHODS: Thirty-five features thoughtfully chosen from 4 categories of 3D texture features (discrete texture features, Markovian features, fractal features, grey value distribution features) were selected and tested for invariance properties (rotation and scaling) using artificial images with a known grey value distribution. The discriminative power of the 3D texture features was tested on artificially constructed benign and malignant 3D nuclei with increasing nucleolar size and advancing chromatin margination towards the periphery of the nucleus. As a clinical proof of principle, the discriminative power of the texture features was assessed on 10 benign and 10 malignant human prostate nuclei, evaluating also whether there was more texture information in 3D whole nuclei compared to a single 2D plane from the middle of the nucleus. RESULTS: All texture features showed the expected invariance properties. Almost all features were sensitive to variations in the nucleolar size and to the degree of margination of chromatin. Fourteen texture features from different categories had high discriminative power for separating the benign and malignant nuclei. The discrete texture features performed less than expected. There was more information on nuclear texture in 3D than in 2D. CONCLUSION: A set of 35 3D nuclear texture features was used successfully to assess nuclear chromatin patterns in 3D images obtained by confocal laser scanning microscopy, and as a proof of principle we showed that these features may be clinically useful for analysis of prostate neoplasia.

Confocal 3D DNA cytometry: assessment of required coefficient of variation by computer simulation.

BACKGROUND: Confocal Laser Scanning Microscopy (CLSM) provides the opportunity to perform 3D DNA content measurements on intact cells in thick histological sections. So far, sample size has been limited by the time consuming nature of the technology. Since the power of DNA histograms to resolve different stemlines depends on both the sample size and the coefficient of variation (CV) of histogram peaks, interpretation of 3D CLSM DNA histograms might be hampered by both a small sample size and a large CV. The aim of this study was to analyze the required CV for 3D CLSM DNA histograms given a realistic sample size. METHODS: By computer simulation, virtual histograms were composed for sample sizes of 20000, 10000, 5000, 1000, and 273 cells and CVs of 30, 25, 20, 15, 10 and 5%. By visual inspection, the histogram quality with respect to resolution of G0/1 and G2/M peaks of a diploid stemline was assessed. RESULTS: As expected, the interpretability of DNA histograms deteriorated with decreasing sample sizes and higher CVs. For CVs of 15% and lower, a clearly bimodal peak pattern with well distinguishable G0/1 and G2/M peaks were still seen at a sample size of 273 cells, which is our current average sample size with 3D CLSM DNA cytometry. CONCLUSIONS: For unambiguous interpretation of DNA histograms obtained using 3D CLSM, a CV of at most 15% is tolerable at currently achievable sample sizes. To resolve smaller near diploid stemlines, a CV of 10% or better should be aimed at. With currently available 3D imaging technology, this CV is achievable.