During the past years, many studies have shown that infrared spectral histopathology (SHP) can distinguish different tissue types and disease types independently of morphological criteria. In this manuscript, we report a comparison of immunohistochemical (IHC), histopathological and spectral histopathological results for colon cancer tissue sections. A supervised algorithm, based on the "random forest" methodology, was trained using classical histopathology, and used to automatically identify colon tissue types, and areas of colon adenocarcinoma. The SHP images subsequently were compared to IHC-based images. This comparison revealed excellent agreement between the methods, and demonstrated that label-free SHP detects compositional changes in tissue that are the basis of the sensitivity of IHC.

By integration of FTIR imaging and a novel trained random forest classifier, lung tumour classes and subtypes of adenocarcinoma are identified in fresh-frozen tissue slides automated and marker-free. The tissue slices are collected under standard operation procedures within our consortium and characterized by current gold standards in histopathology. In addition, meta data of the patients are taken. The improved standards on sample collection and characterization results in higher accuracy and reproducibility as compared to former studies and allows here for the first time the identification of adenocarcinoma subtypes by this approach. The differentiation of subtypes is especially important for prognosis and therapeutic decision.

A feasibility study using a quantum cascade laser-based infrared microscope for the rapid and label-free classification of colorectal cancer tissues is presented. Infrared imaging is a reliable, robust, automated, and operator-independent tissue classification method that has been used for differential classification of tissue thin sections identifying tumorous regions. However, long acquisition time by the so far used FT-IR-based microscopes hampered the clinical translation of this technique. Here, the used quantum cascade laser-based microscope provides now infrared images for precise tissue classification within few minutes. We analyzed 110 patients with UICC-Stage II and III colorectal cancer, showing 96% sensitivity and 100% specificity of this label-free method as compared to histopathology, the gold standard in routine clinical diagnostics. The main hurdle for the clinical translation of IR-Imaging is overcome now by the short acquisition time for high quality diagnostic images, which is in the same time range as frozen sections by pathologists.

In recent years spectral histopathology (SHP) has been established as a label-free method to identify cancer within tissue. Herein, this approach is extended. It is not only used to identify tumour tissue with a sensitivity of 94% and a specificity of 100%, but in addition the tumour grading is determined. Grading is a measure of how much the tumour cells differ from the healthy cells. The grading ranges from G1 (well-differentiated), to G2 (moderately differentiated), G3 (poorly differentiated) and in rare cases to G4 (anaplastic). The grading is prognostic and is needed for the therapeutic decision of the clinician. The presented results show good agreement between the annotation by SHP and by pathologists. A correlation matrix is presented, and the results show that SHP provides prognostic values in colon cancer, which are obtained in a label-free and automated manner. It might become an important automated diagnostic tool at the bedside in precision medicine.

Diffuse malignant mesothelioma (DMM) is a heterogeneous malignant neoplasia manifesting with three subtypes: epithelioid, sarcomatoid and biphasic. DMM exhibit a high degree of spatial heterogeneity that complicates a thorough understanding of the underlying different molecular processes in each subtype. We present a novel approach to spatially resolve the heterogeneity of a tumour in a label-free manner by integrating FTIR imaging and laser capture microdissection (LCM). Subsequent proteome analysis of the dissected homogenous samples provides in addition molecular resolution. FTIR imaging resolves tumour subtypes within tissue thin-sections in an automated and label-free manner with accuracy of about 85% for DMM subtypes. Even in highly heterogeneous tissue structures, our label-free approach can identify small regions of interest, which can be dissected as homogeneous samples using LCM. Subsequent proteome analysis provides a location specific molecular characterization. Applied to DMM subtypes, we identify 142 differentially expressed proteins, including five protein biomarkers commonly used in DMM immunohistochemistry panels. Thus, FTIR imaging resolves not only morphological alteration within tissue but it resolves even alterations at the level of single proteins in tumour subtypes. Our fully automated workflow FTIR-guided LCM opens new avenues collecting homogeneous samples for precise and predictive biomarkers from omics studies.

Histopathological differentiation between severe urocystitis with reactive urothelial atypia and carcinoma in situ (CIS) can be difficult, particularly after a treatment that deliberately induces an inflammatory reaction, such as intravesical instillation of Bacillus Calmette-Guèrin. However, precise grading in bladder cancer is critical for therapeutic decision making and thus requires reliable immunohistochemical biomarkers. Herein, an exemplary potential biomarker in bladder cancer was identified by the novel approach of Fourier transform infrared imaging for label-free tissue annotation of tissue thin sections. Identified regions of interest are collected by laser microdissection to provide homogeneous samples for liquid chromatographyetandem mass spectrometryebased proteomic analysis. This approach afforded label-free spatial classification with a high accuracy and without interobserver variability, along with the molecular resolution of the proteomic analysis. Cystitis and invasive highgrade urothelial carcinoma samples were analyzed. Three candidate biomarkers were identified and verified by immunohistochemistry in a small cohort, including low-grade urothelial carcinoma samples. The best-performing candidate AHNAK2 was further evaluated in a much larger independent verification cohort that also included CIS samples. Reactive urothelial atypia and CIS were distinguishable on the basis of the expression of this newly identified and verified immunohistochemical biomarker, with a sensitivity of 97% and a specificity of 69%. AHNAK2 can differentiate between reactive urothelial atypia in the setting of an acute or chronic cystitis and nonmuscle invasive-type CIS. (Am J Pathol 2019, 189: 619e631; https://doi.org/10. 1016/j.ajpath.2018.11.018) Bladder cancer is the second most common urogenital malignancy, with approximately 430,000 new cases diagnosed worldwide in 2012. 1,2 Approximately 70% to 75% of patients are newly diagnosed with nonmuscle invasive, mostly low-grade bladder cancer, whereas 25% to 30% of de novo diagnoses are high-grade bladder cancer at the stage of infiltration of the muscularis, with metastases observed in 10% of cases. 3e5 A significant percentage (50% to 70%) of patients diagnosed with nonmuscle invasive cancer will have multiple recurrences, and 10% to 15% will even progress to invasion. 3,6,7 A special case of bladder cancer is carcinoma in situ (CIS), which is a flat, noninvasive tumor with a rate of progression to an invasive state of approximately 54%. 8

Challenging histopathological diagnostics in cancer include microsatellite instability-high (MSI-H) colorectal cancer (CRC), which occurs in 15% of early-stage CRC and is caused by a deficiency in the mismatch repair system. The diagnosis of MSI-H cannot be reliably achieved by visual inspection of a hematoxylin and eosin stained thin section alone, but additionally requires subsequent molecular analysis. Time-and sample-intensive immunohistochemistry with subsequent fragment length analysis is used. The aim of the presented feasibility study is to test the ability of quantum cascade laser (QCL)-based infrared (IR) imaging as an alternative diagnostic tool for MSI-H in CRC. We analyzed samples from 100 patients with sporadic CRC UICC stage II and III. Forty samples were used to develop the random forest classifier and 60 samples to verify the results on an independent blinded dataset. Specifically, 100% sensitivity and 93% specificity were achieved based on the independent 30 MSI-Hand 30 microsatellite stable (MSS)-patient validation cohort. This showed that QCL-based IR imaging is able to distinguish between MSI-H and MSS for sporadic CRC-a question that goes beyond morphological features-based on the use of spatially resolved infrared spectra used as biomolecular fingerprints.

BackgroundIn recent years, hyperspectral microscopy techniques such as infrared or Raman microscopy have been applied successfully for diagnostic purposes. In many of the corresponding studies, it is common practice to measure one and the same sample under different types of microscopes. Any joint analysis of the two image modalities requires to overlay the images, so that identical positions in the sample are located at the same coordinate in both images. This step, commonly referred to as image registration, has typically been performed manually in the lack of established automated computational registration tools.ResultsWe propose a corresponding registration algorithm that addresses this registration problem, and demonstrate the robustness of our approach in different constellations of microscopes. First, we deal with subregion registration of Fourier Transform Infrared (FTIR) microscopic images in whole-slide histopathological staining images. Second, we register FTIR imaged cores of tissue microarrays in their histopathologically stained counterparts, and finally perform registration of Coherent anti-Stokes Raman spectroscopic (CARS) images within histopathological staining images.ConclusionsOur validation involves a large variety of samples obtained from colon, bladder, and lung tissue on three different types of microscopes, and demonstrates that our proposed method works fully automated and highly robust in different constellations of microscopes involving diverse types of tissue samples.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-015-0804-9) contains supplementary material, which is available to authorized users.