Karl Garsha

Partial plasma cell differentiation as a mechanism of lost major histocompatibility complex class II expression in diffuse large B-cell lymphoma

Loss of major histocompatibility complex class II (MHC II) expression is associated with poor patient outcome in diffuse large B-cell lymphoma (DLBCL). As MHC II molecules are lost with plasmacytic differentiation in normal cells, we asked whether MHC II loss in DLBCL is associated with an altered differentiation state. We used gene expression profiling, quantum dots, and immunohistochemistry to study the relationship between MHC II and plasma cell markers in DLBCL and plasmablastic lymphoma (PBL). Results demonstrate that MHC II(-) DLBCL immunophenotypically overlap with PBL and demonstrate an inverse correlation between MHC II and plasma cell markers MUM1, PRDM1/Blimp1, and XBP1s. In addition, MHC II expression is significantly higher in germinal center-DLBCL than activated B cell-DLBCL. A minor subset of cases with an unusual pattern of mislocalized punctate MHC II staining and intermediate levels of mRNA is also described. Finally, we show that PBL is negative for MHC II. The results imply a spectrum of MHC II expression that is more frequently diminished in tumors derived from B cells at the later stages of differentiation (with complete loss in PBL). Our observations provide a possible unifying concept that may contribute to the poor outcome reported in all MHC II(-) B-cell tumors.

A technique for relative quantitation of cancer biomarkers in formalin‐fixed, paraffin‐embedded (FFPE) tissue using stable‐isotope‐label based mass spectrometry imaging (SILMSI)

We developed a novel technique for the relative quantitation of pairs of cancer biomarkers in formalin-fixed paraffin-embedded (FFPE) tissue. The method utilizes stable isotope labeled (SIL) chromogens deposited during the standard immunohistochemistry (IHC) tissue staining process. The labeled chromogens are precipitated on tissue enzymatically using the standard IHC protocols. The tissue is then imaged with matrix-free laser desorption ionization time-of-flight mass spectrometry, and peak intensities of reporter ions are used to estimate the relative quantitation of protein biomarkers across the tissue. The relative abundance of two breast cancer biomarkers, estrogen receptor (ER) and progesterone receptor (PgR), were quantitated using their ratio of expression in xenograft models, and the ratios were found to be reproducible both within and across serial sections. The relative quantification of multiple biomarkers in situ across a single tissue section adds an additional dimension in cancer histological evaluation by allowing a visual and statistical assessment of tumor heterogeneity. Copyright

Abstract 5117: An automated 5-plex fluorescent immunohistochemistry enabled characterization of PD-L1 expression and tumor infiltrating immune cells in lung and bladder cancer specimens

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-L19s 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.

Multi-modal contrast of tissue anatomy enables correlative biomarker imaging

Optical imaging techniques are being developed that promise to increase the information content related to specific molecular reporters. Such modalities do not produce contrast in the structural context of the surrounding tissue, making it difficult to reconcile molecular information with morphological context. We report a solution that enables visualization of the tissue morphology on formalin-fixed, paraffin embedded sections prepared for analytical biomarker imaging. Our approach combines modes of transmitted darkfield and fluorescence contrast and computer visualization to produce 2-component image data analogous to the classical hematoxylin and eosin histological stain. An interferometric hyperspectral image capture mode enables measurement of multiplexed biomarkers in annotated anatomic regions. The system enables practical correlative analysis of molecular changes within areas of anatomic pathology.

Abstract 2218: Integrated diagnostic methods for detection of multiple gene rearrangements in prostate cancer tissue specimens

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The identification of patients who will benefit from therapy is one of the more difficult questions in prostate cancer disease management. Approximately 60% of men with diagnosed prostate cancer will have an ETS rearranged tumor foci. The prostate cancer gene fusions identified thus far are characterized by the fusion of 5’ genomic regulatory elements (commonly androgen regulated), such as TMPRSS2, with the ETS family of transcription factors which include ERG, ETV1, ETV4, and ETV5. About 40-50% of prostate cancers have the TMPRSS2/ERG gene fusion which leads to the over expression of oncogenic transcription factors. The goal of this study was to develop a novel, integrated diagnostic testing method that accounts for tissue heterogeneity including multiple gene re-arrangements in single transformed nuclei. We selected a method that sequentially reflexes from an initial H&E to ERG immunohistochemistry (IHC), and finally to a quantum dot (Life Technologies) based FISH probes for the detection of multiple gene rearrangements in prostate cancer. To this end, probes specific to the ETS gene rearrangements including, 3’ and 5’ ERG, TMPRSS2, NDRG1, ETV1, and ETV4, were detected with up to four different quantum dot bioconjugates simultaneously in single nuclei. We have shown sensitive and specific detection of gene rearrangements with this testing method in the xenograft models, VCaP, H660, and LNCaP, as well as in samples from prostate needle biopsies and radical prostatectomies. In 6 of 88 cases, the ERG IHC was diagnostic of PIN (prostatic intraepithelial neoplasia) that was missed on initial examination of the H&E stain. Further, the four color quantum dot FISH assay inclusive of ERG gene rearrangements was confirmatory of ERG IHC reactivity. In addition, we also clearly demonstrated instances of multiple gene rearrangements in the 5’ and 3’ ends on TMPRSS2 and NDRG1 in the same cancer nuclei, but not in benign nuclei from the same tissue. The signal patterns associated with various genomic events assessed were: no rearrangement (normal), translocation through insertion, and translocation through deletion. In summary, we propose that the ERG antibody is likely to be a key component in a diagnostic PIN IHC cocktail, that follows the initial H&E, and may be reflexed to a multiplexed quantum dot FISH assay that incorporates the detection of prevalent gene rearrangements. This method may be useful as an aid in detecting clinically relevant diagnostic markers, since multiple gene rearrangements in the same cell may be identified early in prostate cancer progression. Studies are in progress to evaluate this testing strategy for prognostic value in assessing prostate cancer progression in selected prostate cancer and biopsy cohorts. At time of submission, assay is not approved for use in the US. 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 2218. doi:10.1158/1538-7445.AM2011-2218