Multiplexed IHC analysis to enable Hodgkin lymphoma differential diagnosis on a single slide.

e19536 Background: Routine diagnosis of classical Hodgkin lymphoma is performed with a panel of immunohistochemistry markers to evaluate the biomarker expression profile of the relatively rare Hodgkin cells. One of the key challenges of this technique is that serial immunostains are used and hence it can be difficult or impossible to locate the same Hodgkin cell on adjacent slides. Given the rarity of the Hodgkin cells coupled with the number of markers that are needed for a definitive diagnosis, we developed a new technique in which a single patient slide is multiplexed with nine different antibodies . Methods: One FFPE tissue section from 11 cases was probed for the following nine biomarkers: CD30, CD15, CD45, Pax5, CD20, CD79a, OCT2, Bob1, and CD3. An initial 10x whole slide fluorescent image of CD30 was acquired and presented to the pathologist who based on this staining selected regions of interest for higher magnification (40x) imaging of the CD30 and the other antibodies. The fluorescent images acquired were processed for interpretation using an in-house developed viewing tool. The pathologist was able to view each biomarker as a standard grayscale, monochromatic image, an overlay of two or more biomarkers, or as a virtually created molecular DAB image. Results: A correct diagnosis of classical Hodgkin lymphoma vs. other was able to be made using the MultiOmyx platform in all cases. Subjectively, the pathologist noted that the novel methodology allowed for a significantly more confident assessment of marker expression on the Hodgkin cells in the seven cases of classical Hodgkin lymphoma, eliminating many issues of staining ambiguity and allowing recognition of subtle nuances of staining intensity in the Hodgkin cells. The CD30+ cells in the four other cases, three cases of B-cell lymphoma and one case of lymphocyte predominance Hodgkin lymphoma, showed a B-cell profile that was distinguishable from the classical Hodgkin cell phenotype. Conclusions: This new method of fluorescent multiplexing on a single tissue section allows more accurate interpretation of the biomarker expression profile on the same Hodgkin cell. It is likely that this paradigm can be expanded to a greater range of challenging cases in hematopathology.

Non-Genotoxic Targeting of p53-Dependent and p53-Independent Signaling Pathways Efficiently Kills Hodgkin/Reed-Sternberg Cells.

Although the majority of patients with classical Hodgkin lymphoma (cHL) can be cured by conventional chemotherapy, a substantial proportion of patients will finally develop treatment-induced secondary malignancies. Therefore, non-genotoxic targeting of major cellular survival pathways could be an interesting novel treatment strategy for patients with cHL. In this study, we focused on the analysis of p53-dependent and p53-independent signaling pathways in Hodgkin/Reed-Sternberg (HRS) cells. To test whether p53 signaling is functional in cHL and whether activation of the p53 pathway is sufficient to kill HRS cells, we employed a recently developed small-molecule antagonist of MDM2, designated nutlin-3a, that disrupts the p53-MDM2 interaction. Nutlin-3a efficiently increased the level of p53 and induced expression of p53 downstream targets in Hodgkin cell lines with wild-type p53, whereas no effects were observed in Hodgkin cell lines that harbor p53 mutations. Activation of the p53 pathway led to strong induction of apoptosis in p53 wild-type Hodgkin cell lines. Knock-down of p53 by RNA interference protected cells from nutlin-induced apoptosis, demonstrating that nutlin-3a exerts its effects strictly through p53. In addition, MDM2 inhibition strongly sensitized HRS cells to cytotoxic drugs, such as doxorubicin, etoposide, or vincristine. In view of the fact that HRS cells are characterized by high constitutive NF-κB activity, we also analyzed the effects of a second non-genotoxic agent, geldanamycin, which is an inhibitor of the HSP90/NF-κB pathway. Titration experiments showed that the pro-apoptotic effects of geldanamycin correlate with the mutation status of IκB proteins, demonstrating strong induction of apoptosis in cell lines with wild-type IκB. Furthermore, Hodgkin cell lines that contain wild-type IκB but lack functional p53 (through mutation or siRNA knock-down) are resistant to nutlin treatment, but still respond to treatment with geldanamycin. This indicates that inhibitors of HSP90 induce apoptosis in HRS cells in a p53-independent manner. Therefore, combined targeting of p53-dependent and p53-independent pathways could be a promising approach to develop highly effective and less genotoxic treatment strategies for patients with cHL.

The Human Anti-CD30 Antibody 5F11 Activates NF-kB and Sensitizes Lymphoma Cells to Bortezomib Induced Apoptosis.

Purpose: We recently developed a fully human monoclonal antibody (5F11) directed against the CD30 receptor, which is an excellent target for antibody based immunotherapy of malignant lymphoma cells. The antibody is cytotoxic for lymphoma cells in vitro and in vivo, however a partial or even complete resistance of several Hodgkin cell lines has been observed. In this study we analysed, whether the efficacy of 5F11 can be improved by combination with bortezomib. Methods and Results: Bortezomib is an inhibitor of the ubiquitin-proteasomes pathway that is toxic to multiple solid and hemotologic tumor cells. Using XTT viability assays, TUNEL assays and Facs analysis we demonstrate a synergistic toxic effect of 5F11 and bortezomib on the Hodgkin cell lines tested (L540, L1236, L428) and the CD30 expressing ALCL Karpas299. Moreover the growth of subcutaneous L540 derived tumors in SCID mice is inhibited by 5F11 in combination with bortezomib. The synergy depends on the regime of the drugs and is only seen when a pre-incubation with the antibody is followed by exposure to bortezomib. Immunofluorescence analysis demonstrates that the sensitization of the tumor cells correlates with a 5F11 dependent localisation of the survival factor NF-kB into the cell nuclei. A transfected NF-kB responsive reporter gene is 2–3 fold activated in 5F11 treated L428 cells and enhanced NF-kB binding is detected performing EMSA.. We furthermore measured an increased expression of the NF-kB target gene c-flip, that is down regulated after additional incubation of the tumor cells with bortezomib. Conclusion: Our data indicate that initial activation of NF-kB and downstream anti-apoptotic proteins in response to 5F11 via CD30 sensitizes the tumor cells to bortezomib induced cell death. The in vitro and in vivo activity of the combination of 5F11 and bortezomib suggests a therapeutic value for the treatment of HD patients.