- Basal cell carcinoma (BCC) is the most common human malignant neoplasm and is a frequently encountered diagnosis in dermatopathology. Although BCC may be locally destructive, it rarely metastasizes. Many diagnostic entities display morphologic and immunophenotypic overlap with BCC, including nonneoplastic processes, such as follicular induction over dermatofibroma; benign follicular tumors, such as trichoblastoma, trichoepithelioma, or basaloid follicular hamartoma; and malignant tumors, such as sebaceous carcinoma or Merkel cell carcinoma. Thus, misdiagnosis has significant potential to result in overtreatment or undertreatment.- To review key features distinguishing BCC from histologic mimics, including current evidence regarding immunohistochemical markers useful for that distinction.- Review of pertinent literature on BCC immunohistochemistry and differential diagnosis.- In most cases, BCC can be reliably diagnosed by histopathologic features. Immunohistochemistry may provide useful ancillary data in certain cases. Awareness of potential mimics is critical to avoid misdiagnosis and resulting inappropriate management.
Abstract Quantitative measurement of BCR-ABL fusion gene transcripts in whole blood by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) is a clinically validated method to monitor tyrosine kinase inhibitor drug response in patients diagnosed with chronic myelogenous leukemia (CML). Monitoring of BCR-ABL expression in blood of cML patients under treatment with a tyrosine kinase inhibitor of BCR-ABL is considered standard of care. A three log or greater reduction in BCR-ABL expression from baseline is considered a Maximum Molecular Response (MMR). A subsequent 0.5 log increase in BCR-ABL from MMR is considered a sign of treatment failure sufficient to trigger change in treatment. The challenge is to develop a test with sufficient analytical performance characteristics and quality control to reliably measure BCRABL in MMR samples in which there may be less than 10 molecules in 300 of RNA extracted from whole blood. A method validation protocol was developed using the International Committee on Harmonization (ICH) Q2(R1) international validation guidelines. BCRABL variants b2a2 and b3a2 as well as the housekeeping gene GUSB were measured in KCL22 cell line RNA and Stratagene Universal Human Reference RNA test articles in extreme linear dilution assay, robustness testing conditions, and in multiple laboratories. Quality controls were developed to enable loading as much whole blood RNA into the assay as possible without assay interference. These included a reverse transcription (RT) standardization mixture comprising External RNA Control Consortium (ERCC) reagents to control for RT interference, and controls for genomic DNA contamination, RNA integrity, and PCR interference. Results: This test for b2a2 and b3a2 variants of BCR-ABL meets ICHQ2(R1) guidelines for specificity, linearity, accuracy, LOD and LOQ, imprecision, robustness, and reproducibility. Each analyte comprised by the test was linear (R2 > 0.97) over more than 3 logs10, and demonstrated inter-experimental and inter-laboratory imprecision low enough to enable measurement of a 3-fold difference as significant (P <0.05) from a value as low as five molecules/assay. Experiments are underway to quantify the amount of whole blood RNA that can be loaded into RT reaction without interference. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2232. doi:10.1158/1538-7445.AM2011-2232
Whole transcriptome RNA-sequencing is a powerful tool, but is costly and yields complex data sets that limit its utility in molecular diagnostic testing. A targeted quantitative RNA-sequencing method that is reproducible and reduces the number of sequencing reads required to measure transcripts over the full range of expression would be better suited to diagnostic testing. Toward this goal, we developed a competitive multiplex PCR-based amplicon sequencing library preparation method that a) targets only the sequences of interest and b) controls for inter-target variation in PCR amplification during library preparation by measuring each transcript native template relative to a known number of synthetic competitive template internal standard copies. To determine the utility of this method, we intentionally selected PCR conditions that would cause transcript amplification products (amplicons) to converge toward equimolar concentrations (normalization) during library preparation. We then tested whether this approach would enable accurate and reproducible quantification of each transcript across multiple library preparations, and at the same time reduce (through normalization) total sequencing reads required for quantification of transcript targets across a large range of expression. We demonstrate excellent reproducibility (R2 = 0.997) with 97% accuracy to detect 2-fold change using External RNA Controls Consortium (ERCC) reference materials; high inter-day, inter-site and inter-library concordance (R2 = 0.97–0.99) using FDA Sequencing Quality Control (SEQC) reference materials; and cross-platform concordance with both TaqMan qPCR (R2 = 0.96) and whole transcriptome RNA-sequencing following “traditional” library preparation using Illumina NGS kits (R2 = 0.94). Using this method, sequencing reads required to accurately quantify more than 100 targeted transcripts expressed over a 107-fold range was reduced more than 10,000-fold, from 2.3×109 to 1.4×105 sequencing reads. These studies demonstrate that the competitive multiplex-PCR amplicon library preparation method presented here provides the quality control, reproducibility, and reduced sequencing reads necessary for development and implementation of targeted quantitative RNA-sequencing biomarkers in molecular diagnostic testing.
Harmful algal blooms are increasing globally and pose serious health concerns releasing cyanotoxins. Microcystin-LR (MC-LR), one of the most frequently produced cyanotoxins, has recently been detected in aerosols generated by the normal motions of affected bodies of water. MC-LR aerosol exposure has been linked to a pro-inflammatory influence on the airways of mice, however little is understood about the underlying mechanism or the potential consequences. This study aimed to investigate the pro-inflammatory effects of aerosolized MC-LR on murine airways. C57BL/6 and BALB/c mice were exposed to MC-LR aerosols, as these strains are predisposed to type 1/type 17 and type 2 immune responses, respectively. Exposure to MC-LR induced granulocytic inflammation in C57BL/6 but not BALB/c mice, as observed by increased expression of cytokines MIP-1α, CXCL1, CCL2, and GM-CSF compared with their respective vehicle controls. Furthermore, the upregulation of interleukins IL-17A and IL-12 is consistent with Th1 and Th17 driven type 1/type 17 inflammation. Histological analysis confirmed inflammation in the C57BL/6 lungs, with elevated neutrophils and macrophages in the bronchoalveolar lavage fluid and increased pro-inflammatory oxidized lipids. In contrast, BALB/c mice showed no significant airway inflammation. These results highlight the ability of aerosolized MC-LR to trigger harmful airway inflammation, requiring further research.
Harmful algal blooms are increasing globally and pose serious health concerns releasing cyanotoxins. Microcystin-LR (MC-LR), one of the most frequently produced cyanotoxins, has recently been detected in aerosols generated by the normal motions of affected bodies of water. MC-LR aerosol exposure has been linked to a pro-inflammatory influence on the airways of mice; however, little is understood about the underlying mechanism or the potential consequences. This study aimed to investigate the pro-inflammatory effects of aerosolized MC-LR on murine airways. C57BL/6 and BALB/c mice were exposed to MC-LR aerosols, as these strains are predisposed to type 1/type 17 and type 2 immune responses, respectively. Exposure to MC-LR induced granulocytic inflammation in C57BL/6 but not BALB/c mice, as observed by increased expression of cytokines MIP-1α, CXCL1, CCL2, and GM-CSF compared with their respective vehicle controls. Furthermore, the upregulation of interleukins IL-17A and IL-12 is consistent with Th1- and Th17-driven type 1/type 17 inflammation. Histological analysis confirmed inflammation in the C57BL/6 lungs, with elevated neutrophils and macrophages in the bronchoalveolar lavage fluid and increased pro-inflammatory and pro-resolving oxidized lipids. In contrast, BALB/c mice showed no significant airway inflammation. These results highlight the ability of aerosolized MC-LR to trigger harmful airway inflammation, requiring further research, particularly into populations with predispositions to type 1/type 17 inflammation.
Abstract Measurement of BCR-ABL fusion transcripts in whole blood by quantitative, reverse-transcriptase PCR is a clinically validated method to monitor treatment response in patients with chronic myelogenous leukemia. For example, achieving a three-log or greater reduction in BCR-ABL expression from baseline is considered a Maximum Molecular Response (MMR). A 0.5-log increase from MMR signifies treatment failure sufficient to motivate change in treatment. The purpose of this study was to develop a BCR-ABL test with improved analytical performance characteristics including adequate quality control for RNA degradation or reverse transcription interference and reliable comparison of results across testing sites. To test whether RNA degradation could mask a 0.5-log increase in expression, an RNA degradation model was established by incubating whole blood from each of six individuals with K562 cells at room temperature for various times post-venipuncture. When data from six subjects were combined, the BCR-ABL/103 GUSB value trended down at 24h but was not significantly decreased until 48h (51% decrease, p = 0.004). To quantify the amount of whole blood RNA that can be loaded into an RT reaction without reducing RT efficiency, six different amounts of total RNA extracted from whole blood mixed with K562 cells were added into RT reactions. To measure RT efficiency, an RT Standards Mixture (RTSM) containing known copy numbers of External RNA Control Consortium (ERCC) 171 RNA and ERCC 113 cDNA was added to each RT reaction. RT efficiency was measured as the yield of PCR product from ERCC 171 RNA relative to ERCC 113 cDNA. Based on data from three subjects, compared to RT efficiency at reference concentration of 30 ng/µl RNA in RT, there was a 35% decrease (p = 0.043) at 167 ng/μl RNA and 82% reduction at 1000 ng/μl RNA. The maximum yield of BCR-ABL (molecules/μL cDNA) was observed at 300 ng/µl RNA/RT with a 3-fold increase (p=0.003) compared to 30 ng/µl RNA. Greater than 300 ng/µl RNA/RT did not further increase yield of BCR-ABL molecules/μL cDNA, likely due to reduced RT efficiency. We conclude that BCR-ABL/103 GUSB should be measured within 24 hours following blood collection. Because decreasing RT efficiency is associated with increasing RNA input into RT reactions, including 300 ng/µl RNA in RT reactions provides the maximum sensitivity in measuring BCR-ABL expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5538. doi:1538-7445.AM2012-5538
Whole transcriptome RNA-sequencing is a powerful tool, but is costly and yields complex data sets that limit its utility in molecular diagnostic testing.A targeted quantitative RNA-sequencing method that is reproducible and reduces the number of sequencing reads required to measure transcripts over the full range of expression would be better suited to diagnostic testing.Toward this goal, we developed a competitive multiplex PCR-based amplicon sequencing library preparation method that a) targets only the sequences of interest and b) controls for inter-target variation in PCR amplification during library preparation by measuring each transcript native template relative to a known number of synthetic competitive template internal standard copies.To determine the utility of this method, we intentionally selected PCR conditions that would cause transcript amplification products (amplicons) to converge toward equimolar concentrations (normalization) during library preparation.We then tested whether this approach would enable accurate and reproducible quantification of each transcript across multiple library preparations, and at the same time reduce (through normalization) total sequencing reads required for quantification of transcript targets across a large range of expression.We demonstrate excellent reproducibility (R 2 = 0.997) with 97% accuracy to detect 2-fold change using External RNA Controls Consortium (ERCC) reference materials; high inter-day, inter-site and inter-library concordance (R 2 = 0.97-0.99)using FDA Sequencing Quality Control (SEQC) reference materials; and cross-platform concordance with both TaqMan qPCR (R 2 = 0.96) and whole transcriptome RNA-sequencing following ''traditional'' library preparation using Illumina NGS kits (R 2 = 0.94).Using this method, sequencing reads required to accurately quantify more than 100 targeted transcripts expressed over a 10 7 -fold range was reduced more than 10,000-fold, from 2.3610 9 to 1.4610 5 sequencing reads.These studies demonstrate that the competitive multiplex-PCR amplicon library preparation method presented here provides the quality control, reproducibility, and reduced sequencing reads necessary for development and implementation of targeted quantitative RNA-sequencing biomarkers in molecular diagnostic testing.
Aim Primary cutaneous neuroendocrine carcinoma, or Merkel cell carcinoma ( MCC ), cannot be distinguished morphologically from small‐cell neuroendocrine carcinomas (Sm CC ) from other sites. Immunohistochemistry is required to confirm cutaneous origin, and is also used for detection of sentinel lymph node ( SLN ) metastases of MCC . Cytokeratin 20 ( CK 20) expression is commonly used for these purposes, but is negative in some MCC cases, and has unclear specificity. We evaluated immunohistochemistry for neurofilament and CK 20 in MCC compared with Sm CC from other sites. Methods and results We evaluated neurofilament expression in 55 MCC specimens from 39 unique patients, including nine CK 20‐negative MCC tumours. Neurofilament expression was observed in 42 of 55 (76.4%) MCC cases, including seven of nine (77.8%) CK 20‐negative MCC cases. Neurofilament was expressed in nine of 12 (75%) Merkel cell polyomavirus‐positive tumours and five of 10 (50%) virus‐negative tumours. Compared to a standard immunohistochemical panel (cytokeratin cocktail and CK 20), neurofilament was 87.5% sensitive for detecting SLN metastases. Neurofilament and CK 20 expression was also assessed in 61 extracutaneous Sm CC from 60 unique patients, with primary sites including lung (27), bladder (18), cervix (3), gastrointestinal tract (3), sinonasal tract (2) and other sites (7). The specificity of neurofilament and CK 20 for MCC versus non‐cutaneous Sm CC was 96.7% and 59.0%, respectively. Conclusions Neurofilament has superior specificity to CK 20 in distinguishing MCC from non‐cutaneous Sm CC . Neurofilament is frequently expressed in CK 20‐ and virus‐negative MCC tumours. Limitations of neurofilament immunohistochemistry include lower sensitivity than CK 20 and subtle staining in some tumours. However, our findings indicate that neurofilament is useful for excluding non‐cutaneous Sm CC .
