Jonathan Henriksen

Quantitative comparison of immunohistochemical staining measured by digital image analysis versus pathologist visual scoring

AbstractImmunohistochemical (IHC) assays performed on formalin-fixed paraffin-embedded (FFPE) tissue sections traditionally have been semi-quantified by pathologist visual scoring of staining. IHC is useful for validating biomarkers discovered through genomics methods as large clinical repositories of FFPE specimens support the construction of tissue microarrays (TMAs) for high throughput studies. Due to the ubiquitous availability of IHC techniques in clinical laboratories, validated IHC biomarkers may be translated readily into clinical use. However, the method of pathologist semi-quantification is costly, inherently subjective, and produces ordinal rather than continuous variable data. Computer-aided analysis of digitized whole slide images may overcome these limitations. Using TMAs representing 215 ovarian serous carcinoma specimens stained for S100A1, we assessed the degree to which data obtained using computer-aided methods correlated with data obtained by pathologist visual scoring. To evaluate computer-aided image classification, IHC staining within pathologist annotated and software-classified areas of carcinoma were compared for each case. Two metrics for IHC staining were used: the percentage of carcinoma with S100A1 staining (%Pos), and the product of the staining intensity (optical density [OD] of staining) multiplied by the percentage of carcinoma with S100A1 staining (OD*%Pos). A comparison of the IHC staining data obtained from manual annotations and software-derived annotations showed strong agreement, indicating that software efficiently classifies carcinomatous areas within IHC slide images. Comparisons of IHC intensity data derived using pixel analysis software versus pathologist visual scoring demonstrated high Spearman correlations of 0.88 for %Pos (p < 0.0001) and 0.90 for OD*%Pos (p < 0.0001). This study demonstrated that computer-aided methods to classify image areas of interest (e.g., carcinomatous areas of tissue specimens) and quantify IHC staining intensity within those areas can produce highly similar data to visual evaluation by a pathologist.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1649068103671302

Multisite assessment of NIA-AA guidelines for the neuropathologic evaluation of Alzheimer's disease

Neuropathologic assessment is the current “gold standard” for evaluating the Alzheimers disease (AD), but there is no consensus on the methods used.

Time trends in fungal infections as a cause of death in hematopoietic stem cell transplant recipients: an autopsy study.

Infectious complications remain an important cause of death in hematopoietic stem cell transplant (HSCT) recipients. We undertook a 20-year (1988-2007) retrospective review of all autopsies performed on HSCT recipients in our institution, with emphasis on infections, especially fungal infections, as the cause of death. Of the 2,943 autopsies performed in our institution from 1988 to 2007, 395 (13.4%) involved HSCT recipients (117 pediatric; 278 adult). Of the patients, 298 had received allogeneic, 46 autologous, 41 umbilical cord blood, and 3 autologous plus allogeneic types, and 7 were unknown HSCT types. The most common causes of death were pulmonary complications, occurring in 247 (62.5%) of 395 cases. In 178 cases (45.1%), microorganisms (viral, bacterial, and/or fungal) were documented at autopsy in one or more organs and contributed to the cause of death. Fungal infections were found in 23.5% of cases, but their frequency as a cause of death decreased throughout this study, from 30.3% in the 1988-1992 period to 10.9% in the 2003-2007 period.

Development of multigene expression signature maps at the protein level from digitized immunohistochemistry slides

Molecular classification of diseases based on multigene expression signatures is increasingly used for diagnosis, prognosis, and prediction of response to therapy. Immunohistochemistry (IHC) is an optimal method for validating expression signatures obtained using high-throughput genomics techniques since IHC allows a pathologist to examine gene expression at the protein level within the context of histologically interpretable tissue sections. Additionally, validated IHC assays may be readily implemented as clinical tests since IHC is performed on routinely processed clinical tissue samples. However, methods have not been available for automated n-gene expression profiling at the protein level using IHC data. We have developed methods to compute expression level maps (signature maps) of multiple genes from IHC data digitized on a commercial whole slide imaging system. Areas of cancer for these expression level maps are defined by a pathologist on adjacent, co-registered H&E slides, allowing assessment of IHC statistics and heterogeneity within the diseased tissue. This novel way of representing multiple IHC assays as signature maps will allow the development of n-gene expression profiling databases in three dimensions throughout virtual whole organ reconstructions.

Detection of Prostate Cancer: Quantitative Multiparametric MR Imaging Models Developed Using Registered Correlative Histopathology

Purpose To develop multiparametric magnetic resonance (MR) imaging models to generate a quantitative, user-independent, voxel-wise composite biomarker score (CBS) for detection of prostate cancer by using coregistered correlative histopathologic results, and to compare performance of CBS-based detection with that of single quantitative MR imaging parameters. Materials and Methods Institutional review board approval and informed consent were obtained. Patients with a diagnosis of prostate cancer underwent multiparametric MR imaging before surgery for treatment. All MR imaging voxels in the prostate were classified as cancer or noncancer on the basis of coregistered histopathologic data. Predictive models were developed by using more than one quantitative MR imaging parameter to generate CBS maps. Model development and evaluation of quantitative MR imaging parameters and CBS were performed separately for the peripheral zone and the whole gland. Model accuracy was evaluated by using the area under the receiver operating characteristic curve (AUC), and confidence intervals were calculated with the bootstrap procedure. The improvement in classification accuracy was evaluated by comparing the AUC for the multiparametric model and the single best-performing quantitative MR imaging parameter at the individual level and in aggregate. Results Quantitative T2, apparent diffusion coefficient (ADC), volume transfer constant (K(trans)), reflux rate constant (kep), and area under the gadolinium concentration curve at 90 seconds (AUGC90) were significantly different between cancer and noncancer voxels (P < .001), with ADC showing the best accuracy (peripheral zone AUC, 0.82; whole gland AUC, 0.74). Four-parameter models demonstrated the best performance in both the peripheral zone (AUC, 0.85; P = .010 vs ADC alone) and whole gland (AUC, 0.77; P = .043 vs ADC alone). Individual-level analysis showed statistically significant improvement in AUC in 82% (23 of 28) and 71% (24 of 34) of patients with peripheral-zone and whole-gland models, respectively, compared with ADC alone. Model-based CBS maps for cancer detection showed improved visualization of cancer location and extent. Conclusion Quantitative multiparametric MR imaging models developed by using coregistered correlative histopathologic data yielded a voxel-wise CBS that outperformed single quantitative MR imaging parameters for detection of prostate cancer, especially when the models were assessed at the individual level. (©) RSNA, 2016 Online supplemental material is available for this article.

Correlation between HER2 determined by fluorescence in situ hybridization and reverse transcription-polymerase chain reaction of the oncotype DX test.

The human epidermal growth factor receptor 2 (HER2) gene is amplified and its protein product overexpressed in about 20% of invasive breast cancers. Despite more than a decade of efforts to standardize HER2 testing, controversy persists regarding the most optimal testing method. Recently, Oncotype DX reports have begun including HER2 results in addition to the previously reported Recurrence Score. We compared HER2 results obtained by fluorescence in situ hybridization (FISH) in our laboratories with HER2 results obtained by reverse transcription-polymerase chain reaction (RT-PCR) as documented in the Oncotype DX report. We then sought to identify potentially significant characteristics in the discrepant cases. We identified breast cancer patients with estrogen receptor-positive, lymph node–negative tumors who had Oncotype DX testing performed between September 2008 and March 2012. Patient and tumor characteristics including HER2 FISH and Oncotype DX test results were recorded. Image analysis was performed on cases with discrepancy between the HER2 FISH and Oncotype DX HER2 results to determine the relative proportion of invasive tumor. Eight of 194 (4.1%) cases showed discrepancy between HER2 FISH and Oncotype DX RT-PCR results. Although the overall percent agreement (96%) and percent negative agreement (100%) were high, percent positive agreement was only 50%. Three of 8 (38%) discrepant cases showed heterogeneous amplification by FISH. Seven of 8 (88%) discrepant cases had <50% invasive tumor in the Oncotype DX tissue block. Percent positive agreement between HER2 FISH and Oncotype DX RT-PCR is low. Multiple factors may contribute to this discrepancy including a suboptimal microdissection and possibly heterogeneous amplification of HER2 gene in some cases.

Visual estimates of nucleus-to-nucleus ratios: can we trust our eyes to use the Bethesda ASCUS and LSIL size criteria?

Apart from several subjective criteria, ASCUS and LSIL are defined by nuclear enlargement of 2.5× to 3× and ≥3× the area of a normal intermediate squamous cell nucleus, respectively. The aim of this study was to assess the ability of observers with various degrees of experience to estimate nuclear area ratios.

Elevated hyaluronan and hyaluronan‐mediated motility receptor are associated with biochemical failure in patients with intermediate‐grade prostate tumors

The clinical course of prostate cancer (PCa) measured by biochemical failure (BF) after prostatectomy remains unpredictable in many patients, particularly in intermediate Gleason score (GS) 7 tumors, suggesting that identification of molecular mechanisms associated with aggressive PCa biology may be exploited for improved prognostication or therapy. Hyaluronan (HA) is a high molecular weight polyanionic carbohydrate produced by synthases (HAS1 through HAS3) and fragmented by oxidative/nitrosative stress and hyaluronidases (HYAL1 through HYAL4, SPAM1) common in PCa microenvironments. HA and HA fragments interact with receptors CD44 and hyaluronan‐mediated motility receptor (HMMR), resulting in increased tumor aggressiveness in experimental PCa models. This study evaluated the association of HA‐related molecules with BF after prostatectomy in GS7 tumors.

Safe and effective sarcoma therapy through bispecific targeting of EGFR and uPAR

Sarcomas differ from carcinomas in their mesenchymal origin. Therapeutic advancements have come slowly, so alternative drugs and models are urgently needed. These studies report a new drug for sarcomas that simultaneously targets both tumor and tumor neovasculature. eBAT is a bispecific angiotoxin consisting of truncated, deimmunized Pseudomonas exotoxin fused to EGF and the amino terminal fragment of urokinase. Here, we study the drug in an in vivo “ontarget” companion dog trial as eBAT effectively kills canine hemangiosarcoma and human sarcoma cells in vitro. We reasoned the model has value due to the common occurrence of spontaneous sarcomas in dogs and a limited lifespan allowing for rapid accrual and data collection. Splenectomized dogs with minimal residual disease were given one cycle of eBAT followed by adjuvant doxorubicin in an adaptive dose-finding, phase I–II study of 23 dogs with spontaneous, stage I–II, splenic hemangiosarcoma. eBAT improved 6-month survival from <40% in a comparison population to approximately 70% in dogs treated at a biologically active dose (50 μg/kg). Six dogs were long-term survivors, living >450 days. eBAT abated expected toxicity associated with EGFR targeting, a finding supported by mouse studies. Urokinase plasminogen activator receptor and EGFR are targets for human sarcomas, so thorough evaluation is crucial for validation of the dog model. Thus, we validated these markers for human sarcoma targeting in the study of 212 human and 97 canine sarcoma samples. Our results support further translation of eBAT for human patients with sarcomas and perhaps other EGFR-expressing malignancies. Mol Cancer Ther; 16(5); 956–65. ©2017 AACR.

Expression of FAP, ADAM12, WISP1, and SOX11 is heterogeneous in aggressive fibromatosis and spatially relates to the histologic features of tumor activity

Aggressive fibromatosis (AF) represents a group of tumors with a variable and unpredictable clinical course, characterized by a monoclonal proliferation of myofibroblastic cells. The optimal treatment for AF remains unclear. Identification and validation of genes whose expression patterns are associated with AF may elucidate biological mechanisms in AF, and aid treatment selection. This study was designed to examine the protein expression by immunohistochemistry (IHC) of four genes, ADAM12, FAP, SOX11, and WISP1, that were found in an earlier study to be uniquely overexpressed in AF compared with normal tissues. Digital image analysis was performed to evaluate inter‐ and intratumor heterogeneity, and correlate protein expression with histologic features, including a histopathologic assessment of tumor activity, defined by nuclear chromatin density ratio (CDR). AF tumors exhibited marked inter‐ and intratumor histologic heterogeneity. Pathologic assessment of tumor activity and digital assessment of average nuclear size and CDR were all significantly correlated. IHC revealed protein expression of all four genes. IHC staining for ADAM12, FAP, and WISP1 correlated with CDR and was higher, whereas SOX11 staining was lower in tumors with earlier recurrence following excision. All four proteins were expressed, and the regional variation in tumor activity within and among AF cases was demonstrated. A spatial correlation between protein expression and nuclear morphology was observed. IHC also correlated with the probability of recurrence following excision. These proteins may be involved in AF pathogenesis and the corresponding pathways could serve as potential targets of therapy.