Background/Objectives: Performing hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) on the same specimen slide provides advantages that include specimen conservation and the ability to combine the H&E context with biomarker expression at the individual cell level. We previously used invisible deposited chromogens and dual-camera imaging, including monochrome and color cameras, to implement simultaneous H&E and IHC. Using this approach, conventional H&E staining could be simultaneously viewed in color on a computer monitor alongside a monochrome video of the invisible IHC staining, while manually scanning the specimen. Methods: We have now simplified the microscope system to a single camera and increased the IHC multiplexing to four biomarkers using translational assays. The color camera used in this approach also enabled multispectral imaging, similar to monochrome cameras. Results: Application is made to several clinically relevant specimens, including breast cancer (HER2, ER, and PR), prostate cancer (PSMA, P504S, basal cell, and CD8), Hodgkin’s lymphoma (CD15 and CD30), and melanoma (LAG3). Additionally, invisible chromogenic IHC was combined with conventional DAB IHC to present a multiplex IHC assay with unobscured DAB staining, suitable for visual interrogation. Conclusions: Simultaneous staining and detection, as described here, provides the pathologist a means to evaluate complex multiplexed assays, while seated at the microscope, with the added multispectral imaging capability to support digital pathology and artificial intelligence workflows of the future.

Anatomical pathology relies upon visual evaluation of clinical specimens using brightfield microscopy. This necessarily utilizes histological stains and immunohistochemical (IHC) chromogens that absorb visible light, between 430 and 690 nm. To complement the visible stains, we developed chromogens for use in IHC that absorb light outside the visible range, and imaging methods for detecting dyes absorbing from the ultraviolet (UV) through the near infrared (NIR). Invisible chromogens partnered with conventional visible stains on the same slide provides complementary information, enabling pathologists to gain deeper insights from a patient’s tissue. Methods: Invisible IHC was performed using conventional antibodies and antibody-enzyme conjugates utilizing covalently deposited chromogens absorbing in the UV and NIR. This was combined with IHC using visible chromogens or histological stains. Resulting stained specimens were evaluated interactively in real-time using a dual-camera microscope system comprising color and monochrome cameras, or by recording single microscope fields using multispectral imaging and the monochrome camera. Results: Multiplex IHC was performed with different combinations of visible and invisible chromogens, with the highest level of multiplexing - 7 biomarkers + hematoxylin counterstain (5 visible IHC chromogens plus a UV and an NIR chromogen) - demonstrated on a formalin-fixed paraffin-embedded (FFPE) prostate tumor. Images of each stain were recorded using multispectral imaging, and staining patterns were concordant with single stain DAB analysis on serial sections. In addition to increased IHC multiplexing capacity, multiplexing of IHC with histological stain was demonstrated by combining an NIR absorbing chromogen, identifying MART-1 expression, with H&E staining on FFPE melanoma specimens. The dual-camera microscope presented video of the visible H&E staining (color) next to the NIR IHC staining (monochrome) on the computer monitor as the specimen was manually scanned. Archiving was afforded by multispectral imaging which also enabled quantitative analysis. An additional advantage of the NIR chromogen was the suppression of melanin pigment, the absorbance of which was significantly reduced in the NIR. Other conventional stains multiplexed with invisible IHC on colon, lung, pancreas, and tonsil FFPE tissues also showed good separation of conventional and IHC staining. Conclusion: Taking advantage of invisible chromogens, IHC multiplexing capacity can be increased and conventional histological stains can be combined with IHC on a single slide. Clinical benefits include saving precious specimen, enabling differentiation of cell populations by simultaneous expression of multiple biomarkers, and providing biomarker expression within the cellular and morphological context of conventional stains. Citation Format: Larry E. Morrison, Mark R. Lefever, Lauren J. Behman, Monesh J. Kapadia, Daniel R. Bauer. Invisible chromogens expand brightfield multiplexing and enable combined protein expression and morphological analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2040.

Conventional histological stains, such as hematoxylin plus eosin (H&E), and immunohistochemistry (IHC) are mainstays of histology that provide complementary diagnostic information. H&E and IHC currently require separate slides, because the stains would otherwise obscure one another. This consumes small specimen, limiting the total amount of testing. Additionally, performing H&E and IHC on different slides does not permit comparison of staining at the single cell level, since the same cells are not present on each slide, and alignment of tissue features can be problematic due to changes in tissue landscape with sectioning. We have solved these problems by performing conventional staining and IHC on the same slide using invisible IHC chromogens, such that the chromogens are not visible when viewing the conventional stain and the conventional stain is excluded from images of the IHC. Covalently deposited chromogens provided a convenient route to invisible chromogen design and are stable to reagents used in conventional staining. A dual-camera brightfield microscope system was developed that permits simultaneous viewing of both visible conventional stains and invisible IHC chromogens. Simultaneous staining was demonstrated on several formalin-fixed paraffin-embedded tissue specimens using single and duplex IHC, with chromogens that absorb ultraviolet and near infrared light, followed by H&E staining. The concept was extended to other conventional stains, including mucicarmine special stain and Papanicoulou stain, and further extended to cytology specimens. In addition to interactive video review, images were recorded using multispectral imaging and image processing to provide flexible production of color composite images and enable quantitative analysis.

Abstract Introduction. Brightfield microscopy is the current gold standard for tissue-based cancer diagnoses and has long been the choice of pathologists. Accordingly, the clinical use of brightfield-based immunohistochemistry (IHC) far exceeds immunofluorescence (IF), except in multiplexing applications for which IF is predominately used. Brightfield multiplex imaging is currently limited by 1) the broad spectral absorbance of conventional chromogens and 2) color cameras that only have three broad and overlapping color channels. In this work, we combine narrowband covalently deposited chromogens (CDCs; Day et al., Lab Invest 2017 vol.97 p.104) with matched narrow illumination channels and monochrome imaging to demonstrate that high-order multiplexing is possible in brightfield. Material and Methods. Multiplex IHC was performed using 4 or 5 CDCs and hematoxylin (htx) nuclear staining on formalin-fixed paraffin-embedded (FFPE) tissue sections. Spectral widths of 5 CDC absorbance bands ranged from 69 to 85 nm (FWHM), and 150 nm for a 6th broader CDC. Illumination was provided with a tungsten lamp/filter wheel combination (average bandwidth = 28nm) or filtered light emitting diodes (LEDs; average bandwidth = 23nm). Images were recorded on microscopes fitted with either illumination source and monochrome CCD or CMOS cameras. Furthermore, a custom-designed 12 wavelength LED illuminator facilitated high-speed multispectral imaging with short exposure times (average Δt=10ms). On-slide spectra of all chromogens were measured. Spectral unmixing was used to calculate protein abundances, which were pseudo-colored to render a visualization. Results. Multiple assays were tested on FFPE sections of lung, prostate, and colon tumors with up to 5 IHC markers (5 CDCs + htx). Spectral cross-talk between chromogen signals was significantly reduced with spectral unmixing (SNR≈30). Biomarker concentrations were pseudo-colored to produce customizable visualizations of each assay. Brightfield renditions of each assay were nearly identical to a traditional visualization. Multiplex staining results were concordant with single stain DAB IHC of serial sections. Automated control and image acquisition using the 12 LED illuminator permitted acquisition of all 12 wavelengths in 700 ms per 1 mm2 of tissue. Conclusion. Feasibility of brightfield IHC multiplexing was achieved by matching narrowband CDCs with specific light channels to enable detection of up to six analytes (5 CDCs plus nuclear stain), approaching the capabilities of IF. Our 12-color system is expandable to even higher-order multiplexing and boasts imaging speed roughly comparable to that of a clinical brightfield scanner. This system presents an attractive alternative to IF by combining high-order multiplexing with the speed, pathologist familiarity, and broadly established clinical utility of brightfield microscopy. Citation Format: Daniel R. Bauer, Mark Lefever, Torsten Leibold, Lauren Behman, Esteban Roberts, Julia Ashworth-Sharpe, Larry Morrison. Multispectral imaging of brightfield multiplex immunohistochemistry [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4250.

Lung cancer is the most common cancer worldwide and has the highest mortality rate. Carcinomas comprise 95% of all lung malignancies, the vast majority of which are non-small cell lung carcinomas (NSCLC). Increasingly, the diagnosis of lung cancer is established by examination of small tissue specimens obtained by minimally invasive techniques. It is critical to employ these tissues at maximum efficiency in order to render an accurate pathologic diagnosis and to perform theranostic studies, either genomic or by immunohistochemistry, to demonstrate genetic mutations that make patients eligible for molecularly targeted agents. Currently Thyroid Transcription Factor-1 (TTF-1) and Napsin A are the most commonly used immunohistochemical (IHC) stains to identify primary lung adenocarcinoma, and p40 and cytokeratin 5/6 (CK5/6) are used for squamous cell carcinoma. IHC stains for these markers, are performed either individually (IHC brown staining) or in combination as dual immunostains (i.e. TTF-1 + Napsin A and p40 + CK5/6, utilizing brown and red chromogens). Here we present a novel, truly multiplex immunohistochemical approach that combines staining with the above four antibodies on a single tissue section utilizing four different chromogens to accurately diagnose primary lung adenocarcinomas, squamous cell carcinomas, and combined adenosquamous carcinomas of the lung. Each marker is represented by a distinct color that can be read by a pathologist, using standard, bright field microscopy. We evaluated the ability of pathologists to differentiate NSCLCs using the multiplexed assay as compared to standard, single marker per slide diaminobenzidine (DAB)-based IHC. All cases in a cohort of 264 NSCLCs showed concordance of information (including positivity of stain, intensity of stain and coverage) between single IHC stains and the multiplex assay. This new multiplex IHC offers the capability to accurately diagnose and sub-classify primary lung NSCLCs, while conserving precious tissue for additional testing.

Brightfield microscopy is the preferred method of pathologists for diagnosing solid tumors, utilizing common staining techniques such as hematoxylin and eosin staining and immunohistochemistry (IHC). However, as our understanding of the complex tumor microenvironment grows, there is increasing demand for multiplexed biomarker detection. Currently, multiplexed IHC assays are almost exclusively based on immunofluorescence because brightfield techniques are limited by the broad spectral absorption of chromogens and a reliance on conventional 3-channel color cameras. In this work, we overcome these limitations by combining new chromogens possessing narrow absorbance bands with matched illumination channels and monochrome imaging. Multiplex IHC was performed using four or five covalently deposited chromogens and hematoxylin nuclear stain to preserve morphological context and detail. Brightfield illumination was provided with a tungsten lamp/filter wheel combination or filtered light emitting diodes to provide up to 12 illumination wavelengths. In addition, an automated rapid imaging system was developed, using a synchronized 12-LED illuminator, that could capture images at all wavelengths in under 1 s. In one example, a four-biomarker multiplex assay was designed and used to distinguish regions of adenocarcinoma and squamous cell carcinoma in non-small cell lung cancer. The technology was also validated with a five-biomarker assay in prostate cancer. Spectrally unmixed images of each biomarker demonstrated concordant expression patterns with DAB single stain on serial sections, indicating faithful identification of each biomarker. In each assay, all chromogens were well resolved by spectral unmixing to remove spectral crosstalk. While further characterization and refinement of the assay, and improvements in automation and user interface are necessary for pathologist acceptance, this approach to multiplex IHC and multispectral imaging has the potential to accelerate adoption of multiplexing by combining the medical value of high-order multiplexing with the speed, pathologist familiarity, and broadly established clinical utility of brightfield microscopy.

Multiplex brightfield immunohistochemistry (IHC) offers the potential advantage to simultaneously analyze multiple biomarkers in order to, for example, determine T-cell numbers and phenotypes in a patient's immune response to cancer. This paper presents a fully automatic image-analysis framework to utilize multiplex assays to identify and count stained cells of interest; it was validated by comparison with multiple "gold standard" 3,3'-Diaminobenzidine (DAB) singleplex assays. Both multiplex and singleplex assays were digitized using an RGB slide scanner. The proposed image-analysis algorithms consist of 1) a novel color-deconvolution method, 2) cell candidate detection, 3) feature extraction, and 4) cell classification based on supervised machine learning. Fully automated cell counts on the singleplex images were first rigorously verified by comparing to experts' ground truth counts: A total of 72,076 for CD3-, 34,133 for CD8-, and 2,615 for FoxP3-positive T-cells were used in this singleplex algorithm validation. Concordance correlation coefficients (CCC) of the singleplex algorithm-to-observer agreements were 0.945, 0.965, and 0.997, respectively. Then, the singleplex slides were registered to the adjacent multiplex slides and the automated cell counts for each were compared. For this validation of the multiplex assay cell counts, the CCC values were 0.914, 0.943, and 0.877 for 12,828, 2,545, and 1,647 cells, respectively; we observed good slide-to-slide agreement between multiplex and singleplex. We conclude that the proposed fully-automated image analysis can be a useful and reliable tool to assess multiplex IHC assays.

Abstract Introduction Immunohistochemistry (IHC) is a valuable means to examine the distribution of protein expression at the subcellular level throughout human tissues while retaining morphology of the cells and tissue. Given the large and growing number of established protein biomarkers and the often very limited amount of tissue provided by tumor biopsy, the ability to evaluate multiple biomarkers on a single-slide mounted specimen via multiplexed IHC is of growing importance. However, due to the limited number of conventional chromogens, their broad absorbance spectra, and deposition chemistry based on only two enzymes, brightfield multiplexing has been typically limited to only two protein targets. Methods To expand multiplexing capability, our project objective was to reduce the antibody incubation, detection, and enzyme deactivation steps to produce high-level multiplexed chromogenic assays with short run times and minimal exposure to potentially destructive enzyme deactivation reagents. We utilized unmodified and tagged primary antibodies with secondary anti-primary and anti-tag antibodies conjugated to several enzymes employing orthogonal enzymatic detection systems. Peroxidase-catalyzed tyramide signal amplification (TSA) and quinone methide signal amplification (QMSA) chemistry adapted to two different hydrolase enzymes provided several compatible chromogens with relatively narrow absorbance bands and good spectral separation. All assays were fully automated and performed on the Benchmark XT platform. Results Multiplex IHC staining was demonstrated using combinations of TSA and QMSA, with the former catalyzed by horseradish peroxidase (HRP) and the latter established to work successfully with two different hydrolase enzymes: alkaline phosphatase (AP) and neuraminidase. Three- and four-chromogen multiplex assays were demonstrated on a variety of targets including breast cancer (Her2, ER, PR, Ki-67) and immune cell targets (CD3, CD8, cytokeratin, FOXP3, PD-L1) on a variety of tumor and tonsil tissues. Use of both tagged and untagged primary antibodies and up to three enzymes allow reduction of antibody removal and enzyme deactivation steps, significantly reducing assay time. Quantitative comparisons of multiplex staining to ‘gold standard' DAB staining on serial sections verified the multiplex assay performance. Conclusion We have expanded on the current HRP and AP brightfield IHC detection technologies by utilizing TSA- and QMSA-based chromogens and introducing the new neuraminidase detection chemistry. These detection chemistries combined with tagged and untagged primary antibodies provided up to four-chromogen brightfield multiplex IHC with verified assay performance. Citation Format: Esteban Roberts, William Day, Brian D. Kelly, Nathan W. Polaske, Julia Ashworth-Sharpe, Donald Johnson, Mark Lefever, Jerry Kosmeder, Hongjun Zhang, Jian Zhang, Tsu-Shuen Tsao, Mike Farrell, Joerg Bredno, Robert Ochs, Larry Morrison. Expanded multiplexing capability in brightfield immunohistochemistry utilizing multiple chromogen chemistries and detection enzymes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1600.

Background and Aims Preliminary single-institution data suggest that fluorescence in situ hybridization (FISH) may be useful for detecting high-grade dysplasia (HGD) and esophageal adenocarcinoma (EA) in patients with Barrett’s esophagus (BE). This multicenter study aims to validate the measurement of polysomy (gain of at least two loci) by FISH as a way to discriminate degrees of dysplasia in BE specimens. Methods Tissue specimens were collected from four different hospitals and read by both the local pathology department (“Site diagnosis”) and a single central pathologist (“Review diagnosis”) at a separate institution. The specimens then underwent FISH analysis using probes 8q24 ( MYC ), 9p21 ( CDKN2A ), 17q12 ( ERBB2 ), and 20q13 (ZNF217) for comparison. A total of 46 non-BE, 42 non-dysplastic specialized intestinal metaplasia (SIM), 23 indefinite-grade dysplasia (IGD), 10 low-grade dysplasia (LGD), 29 HGD, and 42 EA specimens were analyzed. Results We found that polysomy, as detected by FISH, was the predominant chromosomal abnormality present as dysplasia increased. Polysomy was also the best predictor for the presence of dysplasia or EA when comparing its area under the curve to that of other FISH abnormalities. We observed that if at least 10% of cells had polysomy within a specimen, the FISH probe was able to differentiate between EA/HGD and the remaining pathologies with a sensitivity of 80% and a specificity of 88%. Conclusions This study demonstrates that using FISH to determine the percentage of cells with polysomy can accurately and objectively aid in the diagnosis of HGD/EA in BE specimens.

Multiplexed immunofluorescence imaging of formalin-fixed, paraffin-embedded tissues is a powerful tool for investigating proteomic profiles and diagnosing disease. However, conventional immunofluorescence with organic dyes is limited in the number of colors that can be simultaneously visualized, is made less sensitive by tissue autofluorescence background, and is usually incompatible with commonly used hematoxylin and eosin staining. Herein, we demonstrate the comparative advantages of using time-gated luminescence microscopy in combination with an emissive Tb(III) complex, Lumi4-Tb, for tissue imaging in terms of sensitivity, multiplexing potential, and compatibility with common immunohistochemistry protocols. We show that time-gated detection of millisecond-scale Tb(III) emission increases signal-to-noise ratio relative to conventional steady-state detection of organic dye fluorescence and permits visualization of low-abundance tissue markers such as Bcl-6 or MSH-6. In addition, temporal separation of long- and short-lifetime (∼nanosecond) signals adds a second dimension for multiplexing and also permits detection of intermolecular Tb(III)-to-dye Förster resonance energy transfer. Furthermore, we demonstrate that the Lumi4-Tb complex is compatible with tyramide signal amplification and, unlike conventional organic dyes, can be reliably used on tissue stained with hematoxylin and eosin. Our results indicate that time-gated luminescence microscopy using Tb(III) labels can provide a sensitive and robust method to perform multiplexed immunofluorescence on archived or clinical tissue specimens.

11590 Background: Expression of PD-L1 in tumor cells (TC) and infiltrating immune cells (IC) allows TCs to escape from host’s antitumor immunity by inducing tolerance among tumor-specific T cells. Treatment with the anti-PD-L1 agent atezolizumab has been shown to result in significantly improved outcomes in patients with advanced urothelial bladder cancer (UBC) and non-small cell lung cancer (NSCLC). Higher levels of PD-L1-expression have been associated with a greater efficacy. An ideal assessment of TCs and ICs in patients with anti-PD-L1 treatment requires simultaneous immunohistochemistry (IHC) detection of multiple biomarkers. Methods: A fully automated 5-Plex fluorescence IHC assay was developed on the BenchMark ULTRA automated slide stainer using rabbit monoclonal antibodies PD-L1 (SP142), CD3, CD8, CD68 and FoxP3 by tyramide signal amplification. The stained slides were scanned with Zeiss AXIO Z1, and analyzed with in-house digital image analysis tools. Results: A cohort of archived NSCLC and UBC specimens were evaluated for PD-L1 positive staining in tumor areas or on tumor infiltrating immune cells. The workflow consisted of spectral unmixing to determine marker colocalization, and followed by detection of individual cells within the tumor region and classifying them into tumor or one of the immune phenotypes through a supervised machine learning algorithm trained on expert-provided examples. The spatial relationship of immune and PD-L1-positive tumor cells was quantitatively explained using descriptive statistics of the distances of immune cells to the closest PD-L1 positive tumor and PD-L1 positive immune cells, and density of different immune cell phenotypes within the tumor and peri-tumoral regions. Hot and cold spots of immune cell makeup were also identified in the heat maps of immune cells within the tumor region. Conclusions: The fully automated 5-Plex IHC assay together with its respective digital analysis could serve as a tool for further characterizing tumors and their microenvironment and provide a better understanding of which patients may benefit from immune-therapies.

Multiplexed analysis of multiple biomarkers in a tissue sample requires use of reporter dyes with specific spectral properties that enable discrimination of signals. Conventional chromogens with broad absorbance spectra, widely used in immunohistochemistry (IHC), offer limited utility for multiplexed detection. Many dyes with narrow absorbance spectra, eg rhodamines, fluoresceins, and cyanines, potentially useful for multiplexed detection are well-characterized; however, generation of a chromogenic reagent useful for IHC analysis has not been demonstrated. Studies reported herein demonstrate utility of tyramine-chemistry for synthesis of a wide variety of new chromogenic dye conjugates useful for multiplexed in situ analysis using conventional light microscopes. The dyes, useful individually or in blends to generate new colors, provide signal sensitivity and dynamic range similar to conventional DAB chromogen, while enabling analysis of co-localized biomarkers. It is anticipated that this new paradigm will enable generation of a wide variety of new chromogens, useful for both research and clinical biomarker analysis that will benefit clinicians and patients.

Abstract Cancers may escape immune surveillance and eradication through the expression of programmed death-ligand 1 (PD-L1) on tumor cells and in the tumor microenvironment. PD-L1 expression has been reported in various cell populations within the tumor, and its expression associated with prognosis for various tumors. Further, clinical studies have shown that this pathway is an important target for immunotherapy and PD-L1 expression on tumor cells and in the tumor microenvironment has been associated with enhanced response. Understanding PD-L1's complex biological function not only on tumor cells but also within the tumor microenvironment requires simultaneous interrogation of multiple biomarkers, ranging from cancer immunology checkpoint markers, tumor infiltrating immune cell markers, and tumor specific markers, etc. Multiplex immunohistochemistry (IHC) allows simultaneous detection of multiple markers to explore the potential cellular composition of immune/stromal/cancer cells in tumor microenvironment. Development of a multiplex IHC assay remains challenging due to antibody species similarity and cross reactivity, stability of fluorophores through multiple rounds of processing, balancing high and low signals and measurement of weakly expressed markers. We present here the development of a fully automated multiplex IHC assay (PD-L1, CD3, CD8, CD68 and FoxP3) using rabbit primary antibodies with a heat deactivation process between each antigen staining cycles on the BenchMark ULTRA automated slide stainer. As part of the technology validation, we compared the 5-plex IHC to the respective single-plex chromogenic IHC assays. Using the automated 5-plex fluorescent IHC assay, we tested a cohort of non-small cell lung (NSCLC) and bladder cancer tissue specimens and characterized PD-L1 and immune marker expression in both tumor and infiltrate immune cells. To provide an objective and reliable readout of the assay, image analysis tools are being developed for automated identification and quantification of the labelled biomarkers and their co-expression on a cell-by-cell basis. This automated multiplex PD-L1 5-Plex IHC assay could be utilized as a tool for further characterizing tumors and its microenvironment and gain a better understanding of which patients may benefit from immune-therapies. Citation Format: Wenjun Zhang, Antony Hubbard, Adriana Racolta, Nick Cummins, Mehrnoush Khojasteh, Liping Zhang, Karl Garsha, Joerg Bredno, Dustin Harshman, Srabani Bhaumik, Tobin Jones, Marcin Kowanetz, Sanjeev Mariathasan, Ian McCaffery, Dustin Smith, J Andrew Williams, Lidija Pestic-Dragovich, Larry Morrison, Lei Tang. An automated 5-plex fluorescent immunohistochemistry enabled characterization of PD-L1 expression and tumor infiltrating immune cells in lung and bladder cancer specimens. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5117.

Background: Pathologic complete response (pCR) after neoadjuvant chemotherapy for breast cancer is associated with improved prognosis in aggressive tumor subtypes, including ERBB2-positive tumors. Recent adoption of pCR as a surrogate endpoint for clinical trials in early stage breast cancer in the neoadjuvant setting highlights the need for biomarkers that, alone or in combination, help predict the likelihood of response to treatment.Methods: Biopsy specimens from 29 patients with invasive ductal carcinoma treated with trastuzumab-based therapy prior to definitive resection and pathologic staging were evaluated by dual color bright field in situ hybridization (dual ISH) using probes for MET, TOP2A, PTEN, and PIK3CA genes, each paired with centromeric probes to their respective chromosomes (chromosomes 7, 17, 10, and 3). Ki-67 expression was assessed by immunohistochemistry (IHC). Various parameters describing copy number alterations were evaluated for each gene and centromere probe to identify the optimal parameters for clinical relevance. Combinations of ISH parameters and IHC expression for Ki-67 were also evaluated.Results: Of the four genes and their respective chromosomes evaluated by ISH, two gene copy number parameters provided statistically significant associations with pCR: MET gain or loss relative to chromosome 7 (AUC = 0.791, sensitivity = 92 % and specificity = 67 % at optimal cutoff, p = 0.0032) and gain of PTEN (AUC = 0.674, sensitivity = 38 % and specificity = 100 % at optimal cutoff, p = 0.039). Ki-67 expression was also found to associate significantly with pCR (AUC = 0.726, sensitivity = 100 % and specificity = 42 % at optimal cutoff, p = 0.0098). Combining gain or loss of MET relative to chromosome 7 with Ki-67 expression further improved the association with pCR (AUC = 0.847, sensitivity = 92 % and specificity = 83 % at optimal cutoffs, p = 0.0006).Conclusions: An immunogenotypic signature of low complexity comprising MET relative copy number and Ki-67 expression generated by dual ISH and IHC may help predict pCR in ERBB2-positive breast cancer treated with neoadjuvant chemotherapy and trastuzumab. These findings require validation in additional patient cohorts.

Cytologic examination of serous effusions is of paramount importance because the finding of malignant cells denotes advanced cancer. Immunocytochemical (IHC) methods are valuable tools to ascertain the presence and origin of metastatic tumors in effusions. Ber-EP4 (EP-Cam) is an epithelial marker commonly used to identify epithelial cells of non-mesothelial origin. Calretinin is a marker for mesothelial cells, and …

BACKGROUND:To reduce sampling error associated with cancer detection in prostate needle biopsies, we explored the possibility of using fluorescence in situ hybridisation (FISH) to detect chromosomal abnormalities in the histologically benign prostate tissue from patients with adenocarcinoma of prostate.METHODS:Tumour specimens from 33 radical prostatectomy (RP) cases, histologically benign tissue from 17 of the 33 RP cases, and 26 benign prostatic hyperplasia (BPH) control cases were evaluated with Locus Specific Identifier (LSI) probes MYC (8q24), LPL (8p21.22), and PTEN (10q23), as well as with centromere enumerator probes CEP8, CEP10, and CEP7. A distribution of FISH signals in the tumour and histologically benign adjacent tissue was compared to that in BPH specimens using receiver operating characteristic curve analysis.RESULTS:The combination of MYC gain, CEP8 Abnormal, PTEN loss or chromosome 7 aneusomy was positive in the tumour area of all of the 33 specimens from patients with adenocarcinomas, and in 88% of adjacent histologically benign regions (15 out of 17) but in only 15% (4 out of 26) of the benign prostatic hyperplasia control specimens.CONCLUSIONS:A panel of FISH markers may allow detection of genomic abnormalities that associate with adenocarcinoma in the field adjacent to and surrounding the tumour, and thus could potentially indicate the presence of cancer in the specimen even if the cancer focus itself was missed by biopsy and histology review.