Swiss digital pathology recommendations: results from a Delphi process conducted by the Swiss Digital Pathology Consortium of the Swiss Society of Pathology.

Integration of digital pathology (DP) into clinical diagnostic workflows is increasingly receiving attention as new hardware and software become available. To facilitate the adoption of DP, the Swiss Digital Pathology Consortium (SDiPath) organized a Delphi process to produce a series of recommendations for DP integration within Swiss clinical environments. This process saw the creation of 4 working groups, focusing on the various components of a DP system (1) scanners, quality assurance and validation of scans, (2) integration of Whole Slide Image (WSI)-scanners and DP systems into the Pathology Laboratory Information System, (3) digital workflow-compliance with general quality guidelines, and (4) image analysis (IA)/artificial intelligence (AI), with topic experts for each recruited for discussion and statement generation. The work product of the Delphi process is 83 consensus statements presented here, forming the basis for "SDiPath Recommendations for Digital Pathology". They represent an up-to-date resource for national and international hospitals, researchers, device manufacturers, algorithm developers, and all supporting fields, with the intent of providing expectations and best practices to help ensure safe and efficient DP usage.

Digital image analysis and artificial intelligence in pathology diagnostics-the Swiss view.

Digital pathology (DP) is increasingly entering routine clinical pathology diagnostics. As digitization of the routine caseload advances, implementation of digital image analysis algorithms and artificial intelligence tools becomes not only attainable, but also desirable in daily sign out. The Swiss Digital Pathology Consortium (SDiPath) has initiated a Delphi process to generate best-practice recommendations for various phases of the process of digitization in pathology for the local Swiss environment, encompassing the following four topics: i) scanners, quality assurance, and validation of scans; ii) integration of scanners and systems into the pathology laboratory information system; iii) the digital workflow; and iv) digital image analysis (DIA)/artificial intelligence (AI). The current article focuses on the DIA-/AI-related recommendations generated and agreed upon by the working group and further verified by the Delphi process among the members of SDiPath. Importantly, they include the view and the currently perceived needs of practicing pathologists from multiple academic and cantonal hospitals as well as private practices.

Extrafollicular proliferation of B-blasts: Morphologic correlate to Spikevax-induced lymphadenopathy.

A 30-year-old male developed a PET-positive left-sided cervical lymphadenopathy that was suspected representing metastasis of a known right-sided papillary thyroid cancer. First-dose-application of Spikevax three weeks ago was neither reflected, nor reported to the pathologists. Diagnostic lymphadenectomy was performed showing extrafollicular proliferation of B-blasts, likely attributable to the vaccine application.

The time-resolved hard X-ray diffraction endstation KMC-3 XPP at BESSY†II.

The time-resolved hard X-ray diffraction endstation KMC-3 XPP for optical pump/X-ray probe experiments at the electron storage ring BESSY II is dedicated to investigating the structural response of thin film samples and heterostructures after their excitation with ultrashort laser pulses and/or electric field pulses. It enables experiments with access to symmetric and asymmetric Bragg reflections via a four-circle diffractometer and it is possible to keep the sample in high vacuum and vary the sample temperature between ∼15 K and 350 K. The femtosecond laser system permanently installed at the beamline allows for optical excitation of the sample at 1028 nm. A non-linear optical setup enables the sample excitation also at 514 nm and 343 nm. A time-resolution of 17 ps is achieved with the `low-α' operation mode of the storage ring and an electronic variation of the delay between optical pump and hard X-ray probe pulse conveniently accesses picosecond to microsecond timescales. Direct time-resolved detection of the diffracted hard X-ray synchrotron pulses use a gated area pixel detector or a fast point detector in single photon counting mode. The range of experiments that are reliably conducted at the endstation and that detect structural dynamics of samples excited by laser pulses or electric fields are presented.

The expression of PD-L1 in salivary gland carcinomas.

Objective was to analyze the role of PD-L1 and its relation to demographic, patho-clinical and outcome parameters in salivary gland carcinoma (SGC) patients. Patients treated for salivary gland carcinomas between 1994 and 2010 were included. A retrospective chart review for baseline characteristics, pathohistological, clinical and outcome data was performed. Immunohistochemistry for PD-L1 was performed using tissue microarrays. PD-L1 expression was assessed in tumor cells and tumor-infiltrating immune cells (TIIC) and statistical analysis with regard to baseline and outcome data was performed. Expression of PD-L1 (by means ≥1% of the cells with PD-L1 positivity) was present in the salivary gland carcinoma cells of 17%, in the TIIC of 20% and in both tumor cells and TIIC of 10% the patients. PD-L1 expression in tumor cells and both tumor cells and TIIC was related to tumor grading (p = 0.035 and p = 0.031, respectively). A trend towards higher grading was also seen for PD-L1 expression in TIICs (p = 0.058). Patients with salivary duct carcinomas and PD-L1 expressing TIICs showed a significantly worse DFS and OS (p = 0.022 and p = 0.003, respectively), those with both tumor cells and TIIC expressing PD-L1 a significantly worse DFS (p = 0.030). PD-L1 expression is present in 17% and 20% of salivary gland carcinoma cells and TIIC. Ten percent of the patient showed a PD-L1 positivity in both tumor cells and TIIC. This is related to high tumor grading and therefore might be a negative prognostic factor.

PD-L1 testing of non-small cell lung cancer using different antibodies and platforms: a Swiss cross-validation study.

With the approval of pembrolizumab for first- and second-line treatment of PD-L1+ non-small cell lung cancer (NSCLC), PD-L1 testing by immunohistochemistry (IHC) has become a necessity. However, the DAKO autostainer ASL48 for the FDA approved DAKO 22C3 pharmDx assay is not broadly available in Switzerland and other parts of Europe. The primary goal of this study was to cross-validate the 22C3 anti-PD-L1 antibody on Benchmark Ultra (VBMU) and Leica Bond (LBO) immunostainers. IHC protocols were developed for 22C3 on both platforms with the 22C3phDx using ASL48 as reference. A tissue microarray (TMA) was constructed from 23 NSCLC specimens with a range of PD-L1 staining results. Empty TMA sections and the 22C3 antibody were distributed to 16 participants for staining on VBMU (8 centers) and/or LBO (12 centers) using the centrally developed protocols. Additionally the performance of the Ventana SP263 assay was tested in five centers. IHC scoring was performed centrally. Categorical PD-L1 staining (0-49% vs. 50-100%) did not significantly differ between centers using VBMU, whereas data from LBO were highly variable (p < 0.001). The SP263 assay was well concordant with 22C3 on VBMU and with 22C3 pharmDx. PD-L1 IHC using a standardized 22C3 protocol on VBMU provides satisfactory results in most centers. The SP263 assay is confirmed as a valid alternative to 22C3 pharmDx. 22C3 PD-L1 IHC on LBO shows major staining variability between centers, highlighting the need for local validation and adjustment of protocols.

Cytology smears as excellent starting material for next-generation sequencing-based molecular testing of patients with adenocarcinoma of the lung.

BACKGROUND: Molecular testing of lung adenocarcinomas (ADCs) is crucial for therapy stratification of patients. Because of the often limited diagnostic material, the authors aimed to explore the suitability of cytology smears for next-generation sequencing (NGS) and compared the results with concurrent histological specimens or cell blocks. METHODS: A total of 16 formalin-fixed paraffin-embedded (FFPE) ADCs with known genetic alterations were used as the first cohort for targeted DNA and RNA sequencing. In the second cohort of 8 cases, 8 cytological smears were compared with matching histological specimens or cell blocks for the study. For NGS library amplification, commercially available panels for DNA and RNA sequencing were applied. The Ion Torrent Personal Genome Machine and the Ion Reporter workflow (version 5.0) were used for sequencing. RESULTS: All DNA libraries derived from FFPE and non-formalin-fixed cytological smear samples produced acceptable quality metrics, thereby enabling successful targeted DNA sequencing (100% performance). Targeted RNA sequencing failed in 1 FFPE case and 1 cytology probe by not reaching enough mapped fusion reads (92% performance rate). All previously detected mutations and gene rearrangements could be confirmed (sensitivity of 100%), whereas specificity of the DNA-based NGS assay reached 96%. CONCLUSIONS: The results of the current study demonstrated the suitability of non-formalin cytology specimens for the simultaneous NGS testing of lung ADCs using amplicon resequencing panels. These assays allowed for the input of cytological smears equal to concurrent histology or cell blocks and proved to be accurate in the detection of therapeutically actionable somatic mutations and gene rearrangements. Cancer Cytopathol 2017;125:30-40. © 2016 American Cancer Society.

Expression of CK19 is an independent predictor of negative outcome for patients with squamous cell carcinoma of the tongue.

OBJECTIVES: To explore the prognostic role of CK19 expression in squamous cell carcinomas within a well-defined cohort of oral tongue cancer patients. METHODS: In our retrospective study, we investigated 129 patients with tongue cancer that had suitable material for inclusion in a tissue microarray (TMA). Where possible, samples were taken from central and peripheral regions of the tumor to generate a representative sample of the tumor. The expression level of CK19 was assessed by immunohistochemical staining. RESULTS: Expression of CK19 in squamous cell carcinoma of the tongue was identified as a negative predictor for overall survival (OS; p<0.000) and disease specific survival (DSS; p=0.001). No significant difference could be shown for disease free survival (DFS) between patients with positive and negative CK19 staining (p=.094). CONCLUSION: This is the first description of the highly significant role of CK19 in a selective, organ specific head and neck cancer cohort. Our results are of special importance against the background that CK19 positive carcinomas revealed a significantly poorer prognosis and therefore emphasize its prognostic and possible diagnostic role in tongue cancer.

Interleukin-33 Expression Indicates a Favorable Prognosis in Malignant Salivary Gland Tumors.

The cytokine interleukin-33 (IL-33) is abundantly expressed in epithelial barrier tissues such as salivary glands. Here, we characterized nuclear IL-33 protein expression by immunohistochemistry in benign and malignant salivary gland tumors and associated it with disease outcome. Most benign salivary gland tumors expressed IL-33, and all Warthin's tumors showed strong and consistent IL-33 expression in the basally oriented cells of their bilayered epithelium. In the malignant group of neoplasms, nuclear IL-33 expression was limited to specific tumor entities-for example, to epithelial-myopepithelial carcinomas (n = 9/11), acinic cell carcinomas (n = 13/27), and oncocytic carcinomas (n = 2/2). IL-33 expression in the combined group of malignant salivary gland neoplasms was significantly associated with favorable histological parameters, lack of metastasis, and longer overall survival, compared with IL-33-negative tumors. We conclude that IL-33 expression is a novel prognostic marker for malignant salivary gland tumors with potential use in clinical diagnostics.

Perineural Invasion in Squamous Cell Carcinoma of the Oral Cavity: Histology, Tumor Stage, and Outcome.

OBJECTIVES/HYPOTHESIS: To analyze the impact of different types of perineural invasion (PNI) in squamous cell carcinoma (SCC) of the oral cavity on overall survival and recurrence rate, with a special focus on histologic subtypes and tumor stage. STUDY DESIGN: Retrospective case-control study with clinicopathological analysis. METHODS: Seventeen patients who received primary surgical treatment for SCC of the oral cavity with PNI were matched to a control group. In a histologic review, PNI was classified into subtypes according to an adapted Liebig classification. The term type A was used to describe tumor invasion into the nerve, whereas type B was used to describe circumferential growth around the nerve. Clinical charts were reviewed, and a Kaplan-Meier survival analysis was performed. RESULTS: The recurrence-free survival rates were 47.1% versus 80.4% (PNI vs. matched control group, P < 0.05), 60.0% versus 94.1% (PNI in stage I and II disease vs. matched control group, P < 0.05) and 41.7% versus 73.5% (PNI in stage III and IV disease vs. matched control group, P < 0.05). In most cases (n = 9) of PNI, both histologic subtypes (type A and type B) were present. Five cases exclusively showed type A, and three cases exclusively showed type B. CONCLUSIONS: Perineural invasion in early disease oral carcinoma has a particularly high impact on survival. Both histologic subtypes showed a significantly worse recurrence-free survival rate when compared to the control group. LEVEL OF EVIDENCE: 3.

Healthcare-associated prosthetic heart valve, aortic vascular graft, and disseminated Mycobacterium chimaera infections subsequent to open heart surgery.

AIMS: We identified 10 patients with disseminated Mycobacterium chimaera infections subsequent to open-heart surgery at three European Hospitals. Infections originated from the heater-cooler unit of the heart-lung machine. Here we describe clinical aspects and treatment course of this novel clinical entity. METHODS AND RESULTS: Interdisciplinary care and follow-up of all patients was documented by the study team. Patients' characteristics, clinical manifestations, microbiological findings, and therapeutic measures including surgical reinterventions were reviewed and treatment outcomes are described. The 10 patients comprise a 1-year-old child and nine adults with a median age of 61 years (range 36-76 years). The median duration from cardiac surgery to diagnosis was 21 (range 5-40) months. All patients had prosthetic material-associated infections with either prosthetic valve endocarditis, aortic graft infection, myocarditis, or infection of the prosthetic material following banding of the pulmonary artery. Extracardiac manifestations preceded cardiovascular disease in some cases. Despite targeted antimicrobial therapy, M. chimaera infection required cardiosurgical reinterventions in eight patients. Six out of 10 patients experienced breakthrough infections, of which four were fatal. Three patients are in a post-treatment monitoring period. CONCLUSION: Healthcare-associated infections due to M. chimaera occurred in patients subsequent to cardiac surgery with extracorporeal circulation and implantation of prosthetic material. Infections became clinically apparent after a time lag of months to years. Mycobacterium chimaera infections are easily missed by routine bacterial diagnostics and outcome is poor despite long-term antimycobacterial therapy, probably because biofilm formation hinders eradication of pathogens.

Prolonged Outbreak of Mycobacterium chimaera Infection After Open-Chest Heart Surgery.

BACKGROUND: Invasive Mycobacterium chimaera infections were diagnosed in 2012 in 2 heart surgery patients on extracorporeal circulation. We launched an outbreak investigation to identify the source and extent of the potential outbreak and to implement preventive measures. METHODS: We collected water samples from operating theaters, intensive care units, and wards, including air samples from operating theaters. Mycobacterium chimaera strains were characterized by randomly amplified polymorphic DNA polymerase chain reaction (RAPD-PCR). Case detection was performed based on archived histopathology samples and M. chimaera isolates since 2006, and the patient population at risk was prospectively surveyed. RESULTS: We identified 6 male patients aged between 49 and 64 years with prosthetic valve endocarditis or vascular graft infection due to M. chimaera, which became clinically manifest with a latency of between 1.5 and 3.6 years after surgery. Mycobacterium chimaera was isolated from cardiac tissue specimens, blood cultures, or other biopsy specimens. We were able also to culture M. chimaera from water circuits of heater-cooler units connected to the cardiopulmonary bypass, and air samples collected when the units were in use. RAPD-PCR demonstrated identical patterns among M. chimaera strains from heater-cooler unit water circuits and air samples, and strains in 2 patient clusters. CONCLUSIONS: The epidemiological and microbiological features of this prolonged outbreak provided evidence for the airborne transmission of M. chimaera from contaminated heater-cooler unit water tanks to patients during open-heart surgery.

Papillary lesions of the breast: outcomes of 156 patients managed without excisional biopsy.

Papillomas of the breast are benign epithelial neoplasms. Because of the low, but continued potential for malignancy, the treatment options after initial diagnosis remain controversial. The aim of this study was to analyze the clinical course of patients with papilloma who were managed by active surveillance following initial diagnosis by core needle biopsy or vacuum-assisted biopsy. This retrospective study analyzed 174 patients with 180 papillomas that were diagnosed by core needle biopsy (113 cases) or vacuum-assisted biopsy (67 cases) at the Breast Center Seefeld Zurich between February 2002 and May 2011. We excluded 24 cases that underwent excisional biopsy for removal of the lesion. Over a mean follow-up of 3.5 years, 13 further events occurred in 156 cases (8%). These events included two cases of ductal carcinoma in situ (one after 4 and one after 6 years), one case of atypical ductal hyperplasia, one radial scar, eight cases of papilloma, and one case of flat epithelial atypia. No invasive carcinomas occurred during the follow-up period. Conservative management of 156 papillary lesions with removal by vacuum-assisted biopsy and surveillance was not associated with invasive cancer over a median follow-up of 3.5 years. Therefore, this approach seems to be a safe option for the clinical management of papillary lesions.

Epithelial cyst in the posterior triangle of the neck: atypical branchial cyst or cystic lymph node metastasis?

We report the case of a 66-year-old man with a cervical neck mass located behind the left sternocleidomastoid muscle. To exclude malignancy, a full workup, including clinical, radiological, and cytological examination, was performed but failed to provide a definitive diagnosis. Histological analysis following excisional biopsy revealed a benign epithelial cyst, consistent with an atypically located branchial cyst. We describe an approach to the management of these neck masses and discuss several theories of the etiology of branchial cysts and how they may come to be abnormally located.

Value of immunohistochemistry in the detection of BRAF(V600E) mutations in fine-needle aspiration biopsies of papillary thyroid carcinoma.

BACKGROUND: Fine-needle aspiration biopsy (FNAB) is important in the diagnostic establishment of suspicious thyroid nodules. In thyroid neoplasms, mutation of the BRAF gene occurs rather exclusively in papillary thyroid carcinoma (PTC) and results in>98% of the cases in V600E amino acid substitution. In the current study, the authors investigated the diagnostic value of a recently described monoclonal antibody that detects this specific mutation on FNAB specimens from patients with PTC. METHODS: BRAF(V600E) status of FNAB cell blocks from 55 patients with PTC was analyzed by immunohistochemistry (IHC) with the new BRAF(V600E) antibody (clone VE1) and by Sanger sequencing (SaS). In discrepant cases, ultra-deep sequencing was also performed. Available corresponding histological specimens were investigated by IHC and, in selected cases, with SaS as well. RESULTS: All cases yielded evaluable IHC staining results of the cell block sections with good interobserver agreement (kappa value, 0.650). Ten tumors (18.2%) demonstrated no staining, 10 tumors (18.2%) demonstrated equivocal staining, 25 tumors (45.4%) demonstrated moderate staining, and 10 tumors (18.2%) demonstrated strong staining. SaS was able to be performed in 48 cases. Nineteen cases demonstrated wild-type BRAF and 29 cases were found to have the BRAF(V600E) mutation. After performing ultra-deep sequencing 1 false-positive and 2 false-negative VE1 IHC cases remained, resulting in a sensitivity of 93.8% and a specificity of 93.8%. CONCLUSIONS: BRAF(V600E) mutations in FNAB specimens from patients with PTC can be reliably detected in most cases by IHC with a new mutation-specific antibody. Interpretation of VE1 IHC staining results on cell block slides of PTC can be difficult in some cases.

ASPP2 suppresses squamous cell carcinoma via RelA/p65-mediated repression of p63.

Squamous cell carcinoma (SCC) is highly malignant and refractory to therapy. The majority of existing mouse SCC models involve multiple gene mutations. Very few mouse models of spontaneous SCC have been generated by a single gene deletion. Here we report a haploinsufficient SCC mouse model in which exon 3 of the Tp53BP2 gene (a p53 binding protein) was deleted in one allele in a BALB/c genetic background. Tp53BP2 encodes ASPP2 (ankyrin repeats, SH3 domain and protein rich region containing protein 2). Keratinocyte differentiation induces ASPP2 and its expression is inversely correlated with p63 protein in vitro and in vivo. Up-regulation of p63 expression is required for ASPP2(Δexon3/+) BALB/c mice to develop SCC, as heterozygosity of p63 but not p53 prevents them from developing it. Mechanistically, ASPP2 inhibits ΔNp63 expression through its ability to bind IκB and enhance nuclear Rel/A p65, a component of the NF-κB transcription complex, which mediates the repression of p63. Reduced ASPP2 expression associates with tumor metastasis and increased p63 expression in human head and neck SCCs. This study identifies ASPP2 as a tumor suppressor that suppresses SCC via inflammatory signaling through NF-κB-mediated repression of p63.

Ultra-deep sequencing confirms immunohistochemistry as a highly sensitive and specific method for detecting BRAF V600E mutations in colorectal carcinoma.

The activating BRAF (V600) mutation is a well-established negative prognostic biomarker in metastatic colorectal carcinoma (CRC). A recently developed monoclonal mouse antibody (clone VE1) has been shown to detect reliably BRAF (V600E) mutated protein by immunohistochemistry (IHC). In this study, we aimed to compare the detection of BRAF (V600E) mutations by IHC, Sanger sequencing (SaS), and ultra-deep sequencing (UDS) in CRC. VE1-IHC was established in a cohort of 68 KRAS wild-type CRCs. The VE1-IHC was only positive in the three patients with a known BRAF (V600E) mutation as assessed by SaS and UDS. The test cohort consisted of 265 non-selected, consecutive CRC samples. Thirty-nine out of 265 cases (14.7%) were positive by VE1-IHC. SaS of 20 randomly selected IHC negative tumors showed BRAF wild-type (20/20). Twenty-four IHC-positive cases were confirmed by SaS (24/39; 61.5%) and 15 IHC-positive cases (15/39; 38.5%) showed a BRAF wild-type by SaS. UDS detected a BRAF (V600E) mutation in 13 of these 15 discordant cases. In one tumor, the mutation frequency was below our threshold for UDS positivity, while in another case, UDS could not be performed due to low DNA amount. Statistical analysis showed sensitivities of 100% and 63% and specificities of 95 and 100% for VE1-IHC and SaS, respectively, compared to combined results of SaS and UDS. Our data suggests that there is high concordance between UDS and IHC using the anti-BRAF(V600E) (VE1) antibody. Thus, VE1 immunohistochemistry is a highly sensitive and specific method in detecting BRAF (V600E) mutations in colorectal carcinoma.

EGFR expression and copy number changes in low T-stage oral squamous cell carcinomas.

AIMS: EGFR-directed therapies are used to treat patients with advanced head and neck squamous cell carcinoma (SCC). As it is still unclear whether or not EGFR amplification represents an early or late event in head and neck SCC progression, we aimed to determine the frequency of abnormalities of EGFR protein and gene copy numbers in early oral SCC. METHODS AND RESULTS: A tissue microarray of cancer tissue from 120 patients with pT1/2 oral SCC was constructed. We investigated EGFR protein expression by immunohistochemistry. EGFR gene copy enumeration was performed using fluorescence in-situ hybridization (FISH) and the novel automated silver in-situ hybridization (SISH) technology. Of early oral SCC, 19.3% showed high, 57.1% moderate and 23.6% low EGFR expression. EGFR amplification/polysomy was identified in 8% and 9% of cases by FISH and SISH, respectively. EGFR-SISH had a high concordance with EGFR-FISH (kappa value = 1.0), and both methods showed high conformity with EGFR immunohistochemistry (P = 0.001 and P = 0.006, respectively). No correlation was found of EGFR protein expression or gene amplification status with pT or pN stage. CONCLUSIONS: Only a small subgroup of early oral SCC is characterized by EGFR amplification, which can be identified reliably using EGFR-SISH technology. This finding suggests that EGFR gene amplification mostly occurs in advanced stages of oral SCC.

HBME-1 expression in hyalinizing trabecular tumours of the thyroid gland.

AIMS: Hyalinizing trabecular tumour (HTT) is a rare thyroid neoplasm with a trabecular growth pattern, marked intratrabecular hyalinization and nuclear features of papillary thyroid carcinoma (PTC). Immunohistochemical HBME-1 expression was reported recently in PTC, but not in HTT. To clarify further the value of HBME-1 expression as a tool in differential diagnosis, we investigated the immunophenotype of HTT. METHODS AND RESULTS: Eight HTT diagnosed from 1997 to 2012 were reviewed on H&E-stained tissue sections and analysed for HBME-1, galectin-3, CK19 and Ki67 expression by immunohistochemistry. Three of eight HTTs (37.5%) were HBME-1 positive, with staining of tumour cells as well as of intratrabecular hyaline matrix material. All cases were CK19 negative. Galectin-3 was expressed weakly in four of eight cases (50%). Five of eight cases (62.5%) showed weak-to-moderate cytoplasmic Ki67 positivity. CONCLUSIONS: Immunohistochemical HBME-1 expression is present in HTT and may not serve as a reliable marker in differentiating HTT from PTC. The HBME-1 positivity of the hyaline matrix suggests that this material is partly of cytoplasmic origin.

Prosthetic valve endocarditis and bloodstream infection due to Mycobacterium chimaera.

Prosthetic valve endocarditis (PVE) due to fast-growing nontuberculous mycobacteria (NTM) has been reported anecdotally. Reports of PVE with slowly growing NTM, however, are lacking. We present here one case of PVE and one case of bloodstream infection caused by Mycobacterium chimaera. Randomly amplified polymorphic DNA (RAPD)-PCR indicated a relatedness of the two M. chimaera strains. Both patients had heart surgery 2 years apart from each other. A nosocomial link was not detected.