Comparison of bias and resolvability in single-cell and single-transcript methods.

Single-cell and single-transcript measurement methods have elevated our ability to understand and engineer biological systems. However, defining and comparing performance between methods remains a challenge, in part due to the confounding effects of experimental variability. Here, we propose a generalizable framework for performing multiple methods in parallel using split samples, so that experimental variability is shared between methods. We demonstrate the utility of this framework by performing 12 different methods in parallel to measure the same underlying reference system for cellular response. We compare method performance using quantitative evaluations of bias and resolvability. We attribute differences in method performance to steps along the measurement process such as sample preparation, signal detection, and choice of measurand. Finally, we demonstrate how this framework can be used to benchmark different methods for single-transcript detection. The framework we present here provides a practical way to compare performance of any methods.

Review of the current medical literature and assessment of current utilization patterns regarding human papillomavirus in situ hybridization and immunohistochemistry in dermatopathology.

Human papillomaviruses have been implicated in many cutaneous diseases. Practicing dermatopathologists often consider using immunohistochemistry and in situ hybridization to help clarify the histologic diagnosis, particularly in cases with borderline or nondiagnostic features. We reviewed the current evidence behind the use of these two techniques in dermatopathology. We identified only two studies utilizing the currently available immunohistochemical antibodies. We found more evidence regarding the use of in situ hybridization; however, the majority of this evidence focuses on diagnosing condylomas and other lesions of the genital skin. We also assessed current utilization patterns of attendees of the American Society of Dermatopathology annual meeting (Chicago, 2016) which revealed a wide spectrum of current utilization ranging from no use to regular use more than once per month. Two-thirds of respondents utilized these tests primarily when requested by the submitting clinician and one-third of the respondents utilize these tests reflexively in specific clinical scenarios.

Eficacia de los inhibidores de ALK en dos casos con evaluación positiva por inmunohistoquímica y negativa por FISH / Efficacy of ALK inhibitors in two cases with positive evaluation by immunohistochemistry and negative by FISH

En el carcinoma de pulmón de células no pequeñas (CPCNP), la activación de ALK se produce por la formación de genes de fusión. El perfil clínico donde ocurre con más frecuencia corresponde a pacientes jóvenes, mayormente mujeres, no fumadores, histología de adenocarcinoma y ausencia de mutaciones de EGFR y KRAS. Su presencia se describe en el 3-10% de los CPCNP. La importancia de la determinación de ALK radica en que identifica un subgrupo de pacientes con un comportamiento biológico diferente, en los cuales el tratamiento con inhibidores específicos, como el crizotinib, ceritinib o alectinib, es más eficaz que los convencionales. Las alteraciones moleculares de ALK pueden identificarse por hibridación in situ (ISH), por inmunohistoquímica (IHQ) y por RT-PCR, aunque el FISH es el procedimiento diagnóstico de referencia a nivel clínico. Se examinaron 308 casos de CPCNP y se compararon los resultados por FISH e IHQ. De los 8 (3%) identificados con expresión positiva, sólo 6 presentaron el rearreglo de ALK. Se presentan dos casos clínicos con ALK positivo por IHC y FISH negativo, uno presentó respuesta al tratamiento dirigido y otro no. A pesar de que el FISH es el gold standard, se acepta el uso de IHQ ya sea para definir conducta como único test o para screening y ulterior confirmación por FISH en los casos positivos. Estos dos casos con distinta respuesta al tratamiento con IHQ positiva pero FISH negativo, indican la ausencia de pautas, requiriendo de más conocimiento en el futuro para optimizar las conductas médicas (AU)

In non-small cell lung cancer, ALK activation is produced by gene fusion. The clinical scenario where this type of tumor appears more frequently is in young, female patients, without smoking history, adenocarcinoma histology and with no EGFR or KRAS mutation. It is described as 3 to 10% of non- small cell lung cancer cases. The importance of ALK determinations lies in the identification of a subgroup of patients with a different biological behavior and sensible tumor to target therapy with ALK inhibitors. Molecular alterations of ALK can be determined by in situ hybridization, immunohistochemistry (IHC) and RT-PCR, FISH is the reference diagnostic procedure in clinical applications. Were evaluated 308 cases of non-small cell lung cancer, and FISH and IHC results were compared. Eight (3%) cases presented positive expression, but only 6 of them presented ALK rearrangements. These two clinical cases of patients with IHC positive but FISH negative for ALK are presented, observing good clinical response in only one of them. Although FISH is considered the gold standard technique, IHC use is accepted for treatment decisions as a lone procedure or as screening with FISH confirmation in positive cases. These two particular cases express the absence of guidelines in this infrequent scenario, needing more knowledge in the future in order to take better medical decisions (AU)

Use of in situ hybridization for HPV in head and neck tumors: experience from a national reference laboratory.

The human papillomavirus (HPV) status of head and neck squamous cell carcinomas (SCCs) is a frequent request for Anatomic Pathology labs. However, prognostic value of HPV status is limited to identification of high risk HPV in oropharyngeal SCCs. The purpose of this study is to investigate the ordering practices of in situ hybridization (ISH) for HPV in head and neck tissues at our national reference laboratory. All testing orders for low risk, high risk, and combined low and high risk HPV-ISH tests requested at ARUP Laboratories between January 2010 and November 2013 had their results reviewed and were grouped by anatomic location of the tested tissue. The H&E and HPV-ISH slides from a sample of the most recent 123 tests were reviewed by two pathologists. A total of 1,128 HPV-ISH tests were ordered during the study period. Testing for combined low and high risk HPV was the most commonly ordered test. The positivity rate for high risk HPV was highest in oropharyngeal tissues. 49 of 123 reviewed cases had testing requested on non-malignant tissue, 11 of which were non-neoplastic. Unnecessary HPV-ISH ordering is prevalent in head and neck tissues. Dual testing for low and high risk HPV, frequent testing outside of the oropharynx, and testing non-neoplastic tissues appear to be common practices.

HER2/neu testing in gastric cancer by immunohistochemistry: assessment of interlaboratory variation.

CONTEXT: Immunohistochemical (IHC) testing for HER2/neu is becoming the standard of care for guiding adjuvant treatment of gastric carcinoma with trastuzumab. OBJECTIVE: To assess interlaboratory variation in IHC staining and interpretation across multiple laboratories. DESIGN: A tissue microarray consisting of 45 cores from 28 gastric cancers was distributed to 37 laboratories for HER2/neu assessment. The IHC results were compared against expert scores at an academic institution and correlated with in situ hybridization results from the originating specimen. Interlaboratory agreement was calculated using Cohen κ statistic. RESULTS: The survey demonstrated several variations in IHC methods, including the primary antibodies in use. There was excellent agreement among laboratories in HER2/neu(+) (IHC 3(+)) cases (κ = 0.80 ± 0.01) and very good agreement among laboratories in HER2/neu(-) (IHC 0 or 1(+)) cases (κ = 0.58 ± 0.01). Less agreement was observed among laboratories when scoring equivocal (IHC 2(+)) cases (κ = 0.22 ± 0.01). Sensitivity and specificity of HER2/neu IHC were 99% and 100%, respectively, when measured against expert review and consensus score as a reference standard. CONCLUSIONS: There is substantial interlaboratory agreement in the interpretation of HER2/neu IHC despite variability in protocols. Although HER2/neu IHC is a highly sensitive and specific test, primary antibody selection may significantly affect IHC results. Furthermore, gastric tumors require a unique scoring system and expertise in interpretation. Intratumoral heterogeneity has a significant effect on HER2/neu scoring by IHC. Ongoing quality assurance exercises among laboratories will help ensure optimized HER2/neu testing.

Statistical evaluation of methods for quantifying gene expression by autoradiography in histological sections.

BACKGROUND: In situ hybridisation (ISH) combined with autoradiography is a standard method of measuring the amount of gene expression in histological sections, but the methods used to quantify gene expression in the resulting digital images vary greatly between studies and can potentially give conflicting results. RESULTS: The present study examines commonly used methods for analysing ISH images and demonstrates that these methods are not optimal. Image segmentation based on thresholding can be subject to floor-effects and lead to biased results. In addition, including the area of the structure or region of interest in the calculation of gene expression can lead to a large loss of precision and can also introduce bias. Finally, converting grey level pixel intensities to optical densities or units of radioactivity is unnecessary for most applications and can lead to data with poor statistical properties. A modification of an existing method for selecting the structure or region of interest is introduced which performs better than alternative methods in terms of bias and precision. CONCLUSION: Based on these results, suggestions are made to reduce bias, increase precision, and ultimately provide more meaningful results of gene expression data.

Molecular testing for somatic mutations in common cancers: the views of UK oncologists.

BACKGROUND: Somatic mutations are important determinants of cancer behaviour and response to therapy. However, molecular testing in this context has a relatively low profile within the clinical community, despite publicity surrounding targeted therapies such as Herceptin. AIMS: As the testing process affects many stakeholders, especially oncologists, this paper examines current test request patterns and views of such testing. METHODS: A postal questionnaire was mailed to 582 UK oncologists and haematologists, achieving a 20% response rate. RESULTS: The survey revealed that immunohistochemistry and fluorescent in situ hybridisation are the most commonly requested tests (used by 70% and 55% of respondents, respectively), especially for breast cancer. Availability of suitable treatment options is the main factor influencing the decision to test (selected by 62% of respondents). Respondents were generally positive about future demand for immunohistochemistry, fluorescent in situ hybridisation, microarray analysis and DNA-based tests, but uncertain about the prospects for microsatellite instability and ploidy testing. CONCLUSIONS: Overall, respondents thought that somatic mutation testing could have a significant and positive effect on oncology and haematology departments and patient care, especially with better treatment and tumour classification. However, lack of supportive scientific evidence and funding were considered key barriers to widespread testing. Further research is clearly required on both the resource implications of this increase in demand and the best model of service delivery to ensure the most efficient use of health service resources.

In situ-synthesized novel microarray optimized for mouse stem cell and early developmental expression profiling.

Applications of microarray technologies to mouse embryology/genetics have been limited, due to the nonavailability of microarrays containing large numbers of embryonic genes and the gap between microgram quantities of RNA required by typical microarray methods and the miniscule amounts of tissue available to researchers. To overcome these problems, we have developed a microarray platform containing in situ-synthesized 60-mer oligonucleotide probes representing approximately 22,000 unique mouse transcripts, assembled primarily from sequences of stem cell and embryo cDNA libraries. We have optimized RNA labeling protocols and experimental designs to use as little as 2 ng total RNA reliably and reproducibly. At least 98% of the probes contained in the microarray correspond to clones in our publicly available collections, making cDNAs readily available for further experimentation on genes of interest. These characteristics, combined with the ability to profile very small samples, make this system a resource for stem cell and embryogenomics research.

Northern blot and in situ hybridization analyses of MARCKS mRNA expression in the cerebral cortex of the macaque monkey.

Myristoylated alanine-rich C-kinase substrate (MARCKS) is a major substrate for protein kinase C, and is involved in synaptic plasticity. Using both Northern blot and in situ hybridization techniques, we investigated whether MARCKS expression varied according to the cerebral region, including the hippocampal formation, or according to the type of neuron. Northern blot analysis showed that the MARCKS mRNA level was higher in the association areas than in the primary sensory and motor areas of the cerebral neocortex. MARCKS mRNA levels in the hippocampus and the amygdala were as high as those in the association areas. The in situ hybridization experiments confirmed the Northern blot results and showed the distribution and characteristics of MARCKS mRNA-positive neurons. In the association areas of the neocortex, prominent signals were observed in neurons in layers II-VI. In the primary areas, prominent signals were restricted to neurons in layers IV-VI. In the hippocampus, the most intense hybridization signals were observed in neurons in the granule cell layer of the dentate gyrus. The observed region-specific expression might reflect functional specialization for plasticity in individual regions of the monkey cerebral cortex.

Genome profiling of breast cancer cells selected against in vitro shows copy number changes.

About 20% of breast carcinomas show no clonal chromosome abnormalities when analyzed after short-term culturing. An interesting question is whether this subset of breast carcinomas really is karyotypically normal or if selection for normal cells occurred in vitro. To address this issue, 26 breast carcinomas that had shown no cytogenetic changes by chromosome banding analysis were examined by comparative genomic hybridization (CGH), a technique that does not require culturing or tumor metaphase cells. All but one case showed copy number changes by CGH (median, four). A comparison of these findings with those of a karyotypically abnormal series analyzed using the same CGH protocol found that the cytogenetically "normal" cases were typically genetically less complex (median, four and eight, respectively; P = 0.0058). Although largely the same alterations were found in both series, some differences with respect to the frequencies of specific imbalances were seen. Gains of 3p and 6q and losses of 10q, 14q, and 17p more often were found in the cytogenetically abnormal series than in the normal tumors. We conclude that in most instances cells found to be normal by chromosome banding analysis after short-term culture do not belong to the tumor parenchyma. Furthermore, when we compared the distribution of the number of imbalances detected by CGH in the total data set according to the mitotic index in vivo (scored from 1 to 3), the median values were three, seven, and 18, respectively (P < 0.001). These data indicate not only that karyotypically normal breast carcinomas may represent a genetically simpler subgroup that grows poorly in vitro but also that this subset of tumors already has a slow growth rate in vivo.

Systematic determination of patterns of gene expression during Drosophila embryogenesis.

BACKGROUND: Cell-fate specification and tissue differentiation during development are largely achieved by the regulation of gene transcription. RESULTS: As a first step to creating a comprehensive atlas of gene-expression patterns during Drosophila embryogenesis, we examined 2,179 genes by in situ hybridization to fixed Drosophila embryos. Of the genes assayed, 63.7% displayed dynamic expression patterns that were documented with 25,690 digital photomicrographs of individual embryos. The photomicrographs were annotated using controlled vocabularies for anatomical structures that are organized into a developmental hierarchy. We also generated a detailed time course of gene expression during embryogenesis using microarrays to provide an independent corroboration of the in situ hybridization results. All image, annotation and microarray data are stored in publicly available database. We found that the RNA transcripts of about 1% of genes show clear subcellular localization. Nearly all the annotated expression patterns are distinct. We present an approach for organizing the data by hierarchical clustering of annotation terms that allows us to group tissues that express similar sets of genes as well as genes displaying similar expression patterns. CONCLUSIONS: Analyzing gene-expression patterns by in situ hybridization to whole-mount embryos provides an extremely rich dataset that can be used to identify genes involved in developmental processes that have been missed by traditional genetic analysis. Systematic analysis of rigorously annotated patterns of gene expression will complement and extend the types of analyses carried out using expression microarrays.

Increased transcription of the tyrosine hydroxylase gene in individual locus coeruleus neurons following footshock stress.

Footshock-evoked change in transcriptional activity of tyrosine hydroxylase in neurons of the locus coeruleus was examined using an intron-specific in situ hybridization histochemical technique. A significant increase in the cellular concentration of tyrosine hydroxylase primary transcripts was found in locus coeruleus neurons 3h following 30 min of intermittent footshock. However, the footshock-induced increase in tyrosine hydroxylase transcription was not homogeneously expressed in locus coeruleus neurons. Similarly, administration of the alpha(2)-adrenergic receptor antagonist idazoxan produced a significant increase in the cellular concentration of tyrosine hydroxylase primary transcripts that was heterogeneously distributed among locus coeruleus neurons. Both footshock and idazoxan significantly increased the regional levels of tyrosine hydroxylase messenger RNA in the locus coeruleus. The time-course of changes in tyrosine hydroxylase transcription rate and messenger RNA levels in the locus coeruleus was examined after a 15 min exposure to footshock. A robust increase in tyrosine hydroxylase transcription rate was found at the end of 15 min of footshock, which remained elevated for 6h and was back to the control levels by 24h. In contrast, in response to a 15 min period of footshock tyrosine hydroxylase messenger RNA concentrations in the locus coeruleus did not increase until 6h and remained elevated at 24h. These findings demonstrate that transcription of the tyrosine hydroxylase gene in locus coeruleus neurons in response to footshock stress occurs rapidly, is sustained for many hours and is heterogeneously distributed. These data also suggest that the increase in tyrosine hydroxylase messenger RNA following footshock is mediated, at least in part, by an increase in tyrosine hydroxylase gene transcription.

A highly sensitive nonisotopic detection method for in situ hybridization.

In situ hybridization is a technique that allows detection of specific DNA and RNA sequences in tissue sections. Nonisotopic techniques are fast and give a precise localization of the hybridization product, but a drawback is the low sensitivity. However, the sensitivity is dependent on the detection system used. To evaluate a sensitive in situ hybridization method with nonradioactive probes we compared three different detection systems, using biotin-labeled human papillomavirus (HPV) 16 probes. The three detection systems included (i) STAV-FITC method (streptavidin-fluorescein isothiocyanate/alkaline phosphatase anti-FITC), (ii) APAAP method (mouse anti-biotin/anti-mouse IgG/alkaline phosphatase mouse anti-alkaline phosphatase), and (iii) tyramide signal amplification (TSA) method (STAV-horseradish peroxidase (HRP)/biotinyl tyramide/STAV-HRP). The in situ hybridization methods were tested on CaSki and SiHa cells and two cervical carcinomas known to be HPV16 positive. The cells and tissues and been fixed in 4% buffered formalin and paraffin embedded. The three different detection systems gave satisfactory nuclear staining in CaSki cells (CaSki cells contain > 500 copies of HPV16 DNA) and the two cervical carcinomas. However, demonstration of HPV16 DNA in SiHa cells (SiHa cells contain one to two HPV16 genome copies) was possible only by use of the APAAP method. It was concluded that the APAAP method provides the best sensitivity among the nonisotopic detection systems and can detect single viral copies in formalin-fixed and paraffin-embedded material.

[Relationship between tissue inhibitors of metalloproteinase and metastasis and prognosis in breast cancer].

OBJECTIVE: To investigate the clinical significance of tissue inhibitors of metalloproteinase (TIMP) -3 and TIMP-4 in human breast cancer. METHODS: The levels of TIMP-3 and TIMP-4 mRNA in paraffin-embedded breast carcinoma specimens from 70 patients with primary invasive breast cancer who had undergone radical mastectomy from January of 1984 to December of 1985 were assessed by in situ hybridization assay. Intratumoral microvessel density (MVD) and vascular endothelial growth factor (VEGF) expression were detected by the standard streptavidin biotin peroxides method. RESULTS: Both TIMP-3 and TIMP-4 mRNA expressions were higher in patients with axillary lymph node metastasis than in those without metastasis (P < 0.01), and patients with higher expression of any of these two genes had a better prognosis than those with lower expression (P < 0.01). We introduced lymph node status, TNM staging, MVD and VEGF expression, plus TIMP-3 and TIMP-4 to the Cox's proportional hazards regression model for significant analysis, and found that the TIMP-3, TNM staging and MVD play an important role in prognosis of this group. CONCLUSIONS: TIMP-3 and TIMP-4 are correlated with metastasis in breast cancer, and TIMP-3 demonstrates an independent prognostic factor for the patients. These results provide a valuable clue for developing new, practical prognosticators in breast cancer patients.

Specific primer design and exonuclease III treatment for the reduction of nonspecific staining in direct in situ PCR.

Although direct in situ PCR is more rapid than indirect method for the in situ identification of low copy number genes, several reports indicate serious nonspecific signals with this method. Some procedures have been reported in an effort to eliminate the nonspecific signals, but the results have not been satisfactory. Exonuclease III can progressively digest blunt or recessed 3' termini of double-stranded DNA whereas DNA with 3' overhanging end is resistant to digestion. DNA fragments amplified by PCR with primers incorporating the recognition site for Sph I generate 3' four bases extensions at both end after digestion. These fragments are expected to be resistant to exonuclease III digestion. It is also expected that nonspecifically incorporated digoxigenin would be released by treatment with exonuclease III thereby reducing background. We succeeded in digesting selectively with the samples after standard PCR by Sph I and exonuclease III treatment. However, we failed to eliminate the nonspecific signals of direct in situ PCR. Southern blotting revealed that the amount of nonspecific incorporation was so huge that exonuclease III was unable to release all of nonspecifically incorporated digoxigenin and maintain specific incorporated digoxigenin simultaneously. Direct in situ PCR is a sensitive method. However, its specificity has a significant problem.

Detection of proviral DNA of bovine immunodeficiency virus in bovine tissues by polymerase chain reaction (PCR) and PCR in situ hybridization.

In this study, experiments were designed to investigate the distribution of bovine immunodeficiency virus (BIV) proviral DNA in the tissues and cells of infected calves by solution-phase polymerase chain reaction (SP-PCR) and PCR in situ hybridization (PCR-ISH). Total DNA samples extracted from tissues of 10 BIV-infected and 5 uninfected calves were amplified by SP-PCR with the primers directed to the BIV conserved pol gene segment. The identity of the SP-PCR product was confirmed by Southern hybridization with a BIV pol gene cDNA probe. SP-PCR results demonstrated that BIV proviral DNA was present predominantly in neural tissues and some lymphoid tissues in BIV-infected calves. It also was detected frequently in other tissues including lung, heart, esophagus, and pancreas. Further investigation on cell location of BIV proviral DNA was performed by in situ amplification of DNA on formalin-fixed tissue sections. The amplified DNA was subjected to in situ hybridization with an internal biotinylated probe and detected with streptavidin-gold followed by silver enhancement. Specific BIV proviral DNA signals were observed in neurons, microglial cells, lymphocytes, septal macrophages, smooth muscle cells, and endothelial cells. On the basis of these results, we conclude that BIV replicates in a variety of bovine tissues in vivo and has a broad cell tropism.

Analysis of TCRAV and TCRBV repertoires in healthy individuals by microplate hybridization assay.

We have developed an adaptor ligation PCR-based microplate hybridization assay (MHA) for analysis of T cell receptor alpha chain variable region (TCRAV) and T cell receptor beta chain variable region (TCRBV) repertoires. Forty three TCRAV and thirty eight TCRBV-specific probes were immobilized onto microplate wells in water-soluble carbodiimide. After hybridization of 5'-biotinylated PCR products, quantitative ELISA was carried out and followed by automated colorimetric reading. The conditions for immobilization and hybridization were optimized using representative TCRBV-specific probes. The sensitivity of MHA allows us to detect as low as 40 pg of biotinylated PCR products. The frequencies of individual V segments obtained by MHA were consistent with those obtained by FACS analysis and reverse dot blot assays. Analysis of the entire TCRAV and TCRBV repertoires could be done using a single 96-well plate, and completed in less than 6 h. Simplicity and reproducibility of this method make it suitable for routine laboratory use. The expression of TCRAV and TCRBV segments was next studied in peripheral blood mononuclear cells (PBMC) of 14 healthy donors using the newly developed MHA method. TCRAV8S1, TCRAV23S1, TCRBV2S1, TCRBV3S1, TCRBV4S1, and TCRBV6S5 were highly expressed in PBMC. Further, the TCRAV repertoires among individuals were less variable compared to the TCRBV repertoires. Interestingly, considerable variations in the expression levels of BV3S1, BV4S1, and BV17S1 were observed among individuals. One polymorphic site was found at the coding region of BV4S1, and there were two alleles. These results suggest that variable expression among individuals may be associated with unknown allelic polymorphism in coding and/or regulatory regions of these TCRBV segments, or with disparity in HLA genes.

In vitro cellular aging stimulates interleukin-1 beta production in stretched human periodontal-ligament-derived cells.

Although the severity of periodontal disease is known to be affected by host age, the pathological role of aging in periodontal disease, and especially that attributable to trauma from occlusion, has not been well-characterized. Interleukin (IL)-1 beta is a key mediator involved in periodontal diseases, a potent stimulator of bone resorption. Furthermore, it is produced by human periodontal ligament (PDL) cells in response to mechanical stress. To investigate the age-related changes in the biosynthetic capacity of IL-1 beta in PDL cells, we examined the effects of in vitro cellular aging with mechanical stress on IL-1 beta protein and gene expression by human PDL cells. Human PDL cells (young = 5th or 6th passage; old = 18-20th passage) were cultured on flexible-bottomed culture plates, and the cells were deformed at 6 cycles per min at 2 steps of tension force for 1 to 5 days. We found a two-fold increase in IL-1 beta production by old PDL cells subjected to mechanical tension compared with that by young PDL cells, although the constitutive levels of IL-1 beta were similar in both the young and old PDL cells. This increase was tension-dependent. IL- 1 beta mRNA was also detected in both cell types under basal conditions, and its expression was further enhanced by application of mechanical tension by use of reverse-transcription-polymerase chain-reaction (RT-PCR) and in situ hybridization methods. The increase in signal rate was higher in the old cells than in the young cells. IL-1 beta-converting enzyme mRNA remained unchanged. It is possible that a large amount of IL- 1 beta produced by PDL cells from an aged host in response to mechanical force may be positively related to the acceleration of alveolar bone resorption.