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The combination of intravenous Reolysin and gemcitabine induces reovirus replication and endoplasmic reticular stress in a patient with KRAS-activated pancreatic cancer.

Mahalingam D, Patel S, Nuovo G, Gill G, Selvaggi G, Coffey M, Nawrocki ST - BMC Cancer (2015)

Bottom Line: Analysis of a tumor biopsy revealed an activating KRAS mutation (G12D) and the patient was started on first-line treatment with Reolysin in combination with gemcitabine in March 2012.Stable disease was achieved with significant improvement in cancer-related pain.Importantly, co-localization of reoviral protein and active caspase-3 was also observed in the biopsy specimen.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology/Oncology, Cancer Therapy and Research Center at The University of Texas Health Science Center at San Antonio, 7979 Wurzbach Rd, San Antonio, TX, 78229, USA. Mahalingam@uthscsa.edu.

ABSTRACT

Background: Activating mutations in RAS are present in the majority of pancreatic cancer cases and represent an ideal therapeutic target. Reolysin is a proprietary formulation of oncolytic reovirus that is currently being evaluated in multiple clinical trials due to its ability to selectively replicate in cells harboring an activated RAS pathway. Here we report for the first time the presence of reovirus replication and induction of endoplasmic reticular (ER) stress in a primary tumor specimen collected from a pancreatic cancer patient receiving intravenous Reolysin and gemcitabine.

Case presentation: We describe the case of a 54-year old patient diagnosed with pancreatic adenocarcinoma in February 2012. Analysis of a tumor biopsy revealed an activating KRAS mutation (G12D) and the patient was started on first-line treatment with Reolysin in combination with gemcitabine in March 2012. Stable disease was achieved with significant improvement in cancer-related pain. Following 25 cycles of treatment over 23 months, a second biopsy was collected and immunohistochemical analyses revealed the presence of reovirus replication and induction of the ER stress-related gene GRP78/BIP and the pro-apoptotic protein NOXA. Importantly, co-localization of reoviral protein and active caspase-3 was also observed in the biopsy specimen.

Conclusion: This is the first report of reoviral protein detection in primary tumor biopsies taken from a pancreatic cancer patient receiving intravenous Reolysin therapy. The accumulation of reoviral protein was associated with ER stress induction and caspase-3 processing suggesting that Reolysin and gemcitabine treatment exhibited direct pro-apoptotic activity against the tumor.

No MeSH data available.


Related in: MedlinePlus

Co-expression of reoviral protein and caspase-3 is consistent with productive lytic infection in the patient’s pancreatic cancer cells when treated with intravenous Reolysin and gemcitabine. Blue indicates the nucleus of the cancer cell. Fluorescent green is the reoviral protein and fluorescent red is active caspase-3 protein. Yellow represents co-localization of reovirus and active caspase-3 in the same cancer cells after co-expression IHC analysis. The Nuance system converts each signal to a fluorescent-based signal to determine co-expression of the two targets of interest
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Fig3: Co-expression of reoviral protein and caspase-3 is consistent with productive lytic infection in the patient’s pancreatic cancer cells when treated with intravenous Reolysin and gemcitabine. Blue indicates the nucleus of the cancer cell. Fluorescent green is the reoviral protein and fluorescent red is active caspase-3 protein. Yellow represents co-localization of reovirus and active caspase-3 in the same cancer cells after co-expression IHC analysis. The Nuance system converts each signal to a fluorescent-based signal to determine co-expression of the two targets of interest

Mentions: With the patient on treatment, a biopsy of the pancreatic mass was performed after cycle 25 day 8 in February 2014. The biopsy features were consistent with the diagnosis of pancreatic adenocarcinoma, with confirmed KRAS mutation (G12D) and loss of CDKN2A/B. Immunohistochemistry (IHC) was performed on Reolysin-treated or untreated HCT116 colon cancer cells as a positive and negative control for reovirus staining, respectively (Fig. 2a). Viral replication was detected using antibodies against the reovirus protein, as the presence of viral RNA may not necessarily imply infectious virus particles. A polyclonal antibody, raised in goats, was derived from mature reovirus viral capsid proteins [12]. Importantly, IHC analyses of biopsy specimens from a pancreatic cancer patient revealed strong positivity for reoviral protein and activated caspase 3 within the tumor (Fig. 2b). Biopsies from pancreatic cancer patients frequently contain benign fat, which may serve as an excellent internal negative control. Images of the stained fat cells were negative for reovirus and active caspase-3 and were from the same tissues that displayed positive staining for reovirus and active caspase-3 (Fig. 2b). Serial section analysis showed a very high concordance of reoviral protein and activated caspase-3, which is characteristic of a productive reovirus infection. In addition, co-expression analysis demonstrated that the reoviral protein and active caspase-3 were being expressed in many of the same cancer cells (Fig. 3). Our preclinical studies with Reolysin identified induction of ER stress and NOXA to be key determinants for Reolysin-mediated apoptosis [9, 13]. In agreement with the induction of active caspase-3, we also noted a significant increase in the expression of GRP78/BIP, which is commonly induced following ER stress and NOXA in the biopsy sample following Reolysin and gemcitabine treatment (Fig. 4).Fig. 2


The combination of intravenous Reolysin and gemcitabine induces reovirus replication and endoplasmic reticular stress in a patient with KRAS-activated pancreatic cancer.

Mahalingam D, Patel S, Nuovo G, Gill G, Selvaggi G, Coffey M, Nawrocki ST - BMC Cancer (2015)

Co-expression of reoviral protein and caspase-3 is consistent with productive lytic infection in the patient’s pancreatic cancer cells when treated with intravenous Reolysin and gemcitabine. Blue indicates the nucleus of the cancer cell. Fluorescent green is the reoviral protein and fluorescent red is active caspase-3 protein. Yellow represents co-localization of reovirus and active caspase-3 in the same cancer cells after co-expression IHC analysis. The Nuance system converts each signal to a fluorescent-based signal to determine co-expression of the two targets of interest
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4496814&req=5

Fig3: Co-expression of reoviral protein and caspase-3 is consistent with productive lytic infection in the patient’s pancreatic cancer cells when treated with intravenous Reolysin and gemcitabine. Blue indicates the nucleus of the cancer cell. Fluorescent green is the reoviral protein and fluorescent red is active caspase-3 protein. Yellow represents co-localization of reovirus and active caspase-3 in the same cancer cells after co-expression IHC analysis. The Nuance system converts each signal to a fluorescent-based signal to determine co-expression of the two targets of interest
Mentions: With the patient on treatment, a biopsy of the pancreatic mass was performed after cycle 25 day 8 in February 2014. The biopsy features were consistent with the diagnosis of pancreatic adenocarcinoma, with confirmed KRAS mutation (G12D) and loss of CDKN2A/B. Immunohistochemistry (IHC) was performed on Reolysin-treated or untreated HCT116 colon cancer cells as a positive and negative control for reovirus staining, respectively (Fig. 2a). Viral replication was detected using antibodies against the reovirus protein, as the presence of viral RNA may not necessarily imply infectious virus particles. A polyclonal antibody, raised in goats, was derived from mature reovirus viral capsid proteins [12]. Importantly, IHC analyses of biopsy specimens from a pancreatic cancer patient revealed strong positivity for reoviral protein and activated caspase 3 within the tumor (Fig. 2b). Biopsies from pancreatic cancer patients frequently contain benign fat, which may serve as an excellent internal negative control. Images of the stained fat cells were negative for reovirus and active caspase-3 and were from the same tissues that displayed positive staining for reovirus and active caspase-3 (Fig. 2b). Serial section analysis showed a very high concordance of reoviral protein and activated caspase-3, which is characteristic of a productive reovirus infection. In addition, co-expression analysis demonstrated that the reoviral protein and active caspase-3 were being expressed in many of the same cancer cells (Fig. 3). Our preclinical studies with Reolysin identified induction of ER stress and NOXA to be key determinants for Reolysin-mediated apoptosis [9, 13]. In agreement with the induction of active caspase-3, we also noted a significant increase in the expression of GRP78/BIP, which is commonly induced following ER stress and NOXA in the biopsy sample following Reolysin and gemcitabine treatment (Fig. 4).Fig. 2

Bottom Line: Analysis of a tumor biopsy revealed an activating KRAS mutation (G12D) and the patient was started on first-line treatment with Reolysin in combination with gemcitabine in March 2012.Stable disease was achieved with significant improvement in cancer-related pain.Importantly, co-localization of reoviral protein and active caspase-3 was also observed in the biopsy specimen.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology/Oncology, Cancer Therapy and Research Center at The University of Texas Health Science Center at San Antonio, 7979 Wurzbach Rd, San Antonio, TX, 78229, USA. Mahalingam@uthscsa.edu.

ABSTRACT

Background: Activating mutations in RAS are present in the majority of pancreatic cancer cases and represent an ideal therapeutic target. Reolysin is a proprietary formulation of oncolytic reovirus that is currently being evaluated in multiple clinical trials due to its ability to selectively replicate in cells harboring an activated RAS pathway. Here we report for the first time the presence of reovirus replication and induction of endoplasmic reticular (ER) stress in a primary tumor specimen collected from a pancreatic cancer patient receiving intravenous Reolysin and gemcitabine.

Case presentation: We describe the case of a 54-year old patient diagnosed with pancreatic adenocarcinoma in February 2012. Analysis of a tumor biopsy revealed an activating KRAS mutation (G12D) and the patient was started on first-line treatment with Reolysin in combination with gemcitabine in March 2012. Stable disease was achieved with significant improvement in cancer-related pain. Following 25 cycles of treatment over 23 months, a second biopsy was collected and immunohistochemical analyses revealed the presence of reovirus replication and induction of the ER stress-related gene GRP78/BIP and the pro-apoptotic protein NOXA. Importantly, co-localization of reoviral protein and active caspase-3 was also observed in the biopsy specimen.

Conclusion: This is the first report of reoviral protein detection in primary tumor biopsies taken from a pancreatic cancer patient receiving intravenous Reolysin therapy. The accumulation of reoviral protein was associated with ER stress induction and caspase-3 processing suggesting that Reolysin and gemcitabine treatment exhibited direct pro-apoptotic activity against the tumor.

No MeSH data available.


Related in: MedlinePlus