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Clinical Immunobiology Correlative Studies Laboratory

Overview
The Clinical Immunobiology Correlative Studies laboratory (CICSL) was founded in 2004, based on an identified need for a central laboratory dedicated to the development and implementation of cutting–edge immune correlative studies to support clinical trial protocols in the Cancer Center.
The CICSL has developed and has available a variety of assays that are based on molecular, flow Cytometry, molecular, functional, and biochemical platforms; the laboratory operates under principles of Good Laboratory Practice (GLP).
The laboratory also houses as the Quality Control (QC) unit for the Cell Therapeutics Program within HCT, developing and performing characterization and release tests for therapeutic cell products.
 
Objective
The research objective of the Clinical Immunobiology Correlative Studies Laboratory (CICSL) is to support Cancer Center investigators through the development, validation, and implementation of highly quantitative immune correlative studies that will be applied to evaluate the efficacy of immunotherapy-based and other clinical trials at the City of Hope. CICSL assay platforms are also available to support translational projects that utilize in-vitro and model systems.
 
Importance
  • The ability to quantitatively evaluate what has happened immunologically and molecularly in patients after receiving treatment is critical in order to obtain insight on why a particular regimen did or did not work, to guide the rational design of ultimately more effective immunotherapy strategies
  • Correlative studies that are well designed and performed are increasingly being viewed as critical components to clinical trials, and standards of quality such as assay qualification/validation, statistical significance of results, and GLP lab environment will be required for results to be published in top-tier peer reviewed publications
  • Since a comprehensive understanding of the effect of treatment on patients is in most circumstances lacking, there is a strong rationale to develop and apply a broad series of platforms and assays to evaluate samples and specimens obtained from patients enrolled in clinical protocols
 
Diagram illustrating the need for broad and comprehensive
correlative assays
 
The laboratory has developed a wide variety of platforms and assays.
 
Specifically:
Flow Cytometry:
Characterization of immune cell activation/differentiation/homing status
Absolute count determination and quantification of immune cell subsets
Analysis of immune cell effector phenotypes (ICC, CD107)
MHC tetramer analysis
Intracellular cytokine staining.

Molecular:
Q-RT-PCR-based quantification of immune cell subsets (including Treg cells)
Q-RT-PCR-based quantification of mRNA levels for immune effector genes
Q-PCR-based quantification of adoptively transferred cells
Vb spectratyping

Biochemistry:
Simultaneous quantification of multiple cytokine/chemokine/growth factors in serum, tissue samples, tissue culture using Luminex bead array
Simultaneous quantification of multiple phosphoproteins in tissue culture and tissue samples using Luminex bead array
 
Cell-based:
CD8 and NK cytolysis bioassays
Cell proliferation assays
Blood processing, PBMC/serum/plasma isolation, freezing, and storage
In-vitro CD8 and CD4 T cell culture
 
Last updated: 06/30/2008
 

Abstract for Grants

The City of Hope Clinical Immunobiology Correlative Studies laboratory (CICSL) is a Shared Resource for the City of Hope Comprehensive Cancer Center. The laboratory occupies approximately 1000 square feet of laboratory space in the Shapiro building (rooms 1002, 1044, and 1045). This space includes a 275 square foot tissue culture area, a 275 square foot area dedicated to qPCR and ELISA, a 250 square foot room dedicated to liquid nitrogen, -80°, and -20° C storage, and a 200 square foot room housing dedicated to Luminex equipment. CICSL laboratory operations are guided by GLP principles of operation, with all possible equipment 21 CFR part 11 compliant.
 
The CICSL includes state-of-the-art equipment for monitoring immune responses to cancer therapy. Specific equipment includes a fully automated microplate based multi-detection reader capable of reading fluorescence intensity, time-resolved fluorescence, high-performance luminescence (flash and glow), and absorbance, a Luminex FLEXMAP 3D bead array reader, a Luminex BioPlex™-HTX bead array reader, two MJR Chromo 4™ Q-PCR machine, and a Guava PCA-96 analytical cell counter and analyzer. The CICSL additionally makes use of access to instruments within the City of Hope Analytical Flow Cytometry Core, including a Beckman Coulter Gallios three-laser flow cytometer capable of evaluating fluorescence in ten distinct colors, and analytical cell sorters.
 
Current assays within the capabilities of the laboratory include:
 
  • Flow Cytometry: Characterization of immune cell activation/differentiation/homing status; analysis of immune cell effector phenotypes and functionality (ICC, CD107); absolute count determination and quantification of immune cell subsets.
 
  • Molecular: quantitative real-time PCR-based quantification of immune cell subsets (including Treg cells) and mRNA levels for immune effector genes; qPCR-based quantification of adoptively transferred cells; Vβ spectratyping.
 
  • Biochemical: Quantification of multiple biomarkers and analytes in serum, plasma, tissue samples and cell culture medium using Luminex bead array and ELISA platforms. These analytes include cytokines and chemokines, growth factors, endocrine markers and hormones, cardiovascular markers, bone metabolism markers, cell signaling molecules, and cancer markers.
 
  • Cell-based: Blood processing; PBMC/serum/plasma isolation; freezing, and storage; CD8 and NK cytolysis bioassays.
 

Assays

In general, the CICSL develops assay platforms that can be applied to multiple clinical studies. Please contact the CICSL director if you are interested in having the laboratory develop an assay not listed below.
 
Current Available Assays and Procedures
 
Flow Cytometry
Characterization of immune cell activation/differentiation/homing status
Analysis of immune cell effector phenotypes (ICS, CD107 degranulation)
Absolute count determination and quantification of immune cell subsets
MHC:tetramer analyses
Proliferation assays (CFSE)
 
Molecular
Nucleic acid (DNA, RNA) isolation
cDNA synthesis
Q-RT-PCR-based quantification of immune cell subsets
Q-RT-PCR-based quantification of mRNA levels for immune and other factors
Q-PCR-based quantification of adoptively transferred cells
Vβ spectratyping
 
Biochemical
 
i. Luminex X-MAP bead array-based
Quantification of multiple cytokine/chemokine/growth factors in serum, tissue samples, tissue culture
ii. ELISA-based
Quantification of cytokine/chemokine/growth factors in serum, culture, tissue samples
iii. Quantification of phosphorylation status of intracellular signal transduction molecules, transcription factors by Luminex X-MAP bead array
 
Cell-based
Blood processing, PBMC/serum/plasma isolatio
 

Equipment

The CICSL is equipped with state-of-the-art equipment to allow for the evaluation and monitoring of immune responses in patients post-immunotherapy. Specific equipment available in the laboratory include:

 

Luminex FLEXMAP 3D System (Luminex multi-bead array)

View Luminex FlexMAP3D System website


The new FLEXMAP 3D® system takes Luminex xMAP® technology to three dimensions. The FLEXMAP 3D system combines differentially dyed fluorescent microsphere sets with an innovative instrument design to allow multiplexing of up to 500 unique assays within a single sample, both rapidly, and precisely. The FLEXMAP 3D accommodates 96 and 384-well formats for enhanced sample volume flexibility and increased throughput. Sample plate heating control also allows the user to run thermally sensitive applications such as nucleic acids.The FLEXMAP 3D system works in the same basic manner as the Luminex® 100TM analyzer. Mixtures of internally dyed microspheres are used in an assay, each having been prepared to bind to a specific analyte of interest. After the assay has been performed the mixture of microspheres is aspirated into the system and then each individual microsphere is individually excited by a red and green laser. Each microsphere is impregnated with different amounts of three internal dyes. All dyes excite at the same red laser wavelength, but fluoresce at three separate wavelengths. Monitoring the relative intensity of the three signals allows the system to discriminate up to 500 different microsphere sets. The green laser is then used to excite the reporter molecules, normally in the form of phycoerythrin, and the resulting fluorescence is measured to determine the amount of analyte present.

 

 

Bio-Rad Bio-Plex HTF System (Luminex multi-bead array)
View Bio-Rad Bio-Plex System website

The Bio-Rad Bio-Plex 100 allows for the simultaneous assay of up to 100 distinct proteins in a single well of a microtiter plate, using very small sample volumes. The system delivers fast and cost-effective bioassay results on many assay formats including cytokines, immunoassays and enzymatic assays, nucleic acid assays, receptor-ligand assays, and phosphorylation assays. The Bio-Plex utilizes Luminex xMAP® technology which is based on the use of 100 distinctly color-coded microspheres. Each microsphere set can be coated with a reagent specific to a particular bioassay, for example individual anti-cytokine antibodies, allowing the capture and specific detection of multiple distinct cytokines from a sample in a single microplate well. Within the Luminex 100 compact analyzer, lasers excite the internal dyes that identify each microsphere particle, and also any reporter dye captured during the assay.
 
MJR Chromo 4™ Thermal Cycler
The MJR Chromo 4 is a Q-PCR instrument that allows for the quantitation of specific nucleic acid sequences in samples. The instrument is capable of excitation and detection in four different channels allowing for PCR multiplex analyses, has a linear dynamic range of up to ten orders of magnitude, and can detect a single copy of initial template in samples.
 
BMG FLUOstar OPTIMA
View BMG FLUOstar OPTIMA website
The FLUOstar OPTIMA is a fully automated microplate based multi-detection reader which incorporates five different measurement principles: fluorescence intensity, time-resolved fluorescence, high-performance luminescence (flash and glow), and absorbance. It is designed for the widest possible range of non-radioactive labeled applications, including immunoassays (ELISA), Ca2+ Measurements (e.g. Ca2+ Flux with Fura-2 or Indo-1), enzyme activity, cell toxicity, proliferation and viability, cytotoxicity and ATP quantification. The instrument covers a wavelength range from 240 to 740 nm and can read all plate formats from six up to 384-well plates in all four measurement modes. The versatile optical system allows instant switching from top to bottom reading. Temperature-regulated heating plates above and below the entire plate movement area provide uniform incubation up to 60°C. The instrument is equipped with a single syringe pumps. Injection speed and timing can be adjusted as is appropriate for particular assays. Delivery volume is adjustable between 3-350 µL in 1µL increments and is individually adjustable for each well allowing control of dilution schemes, concentration ranges, etc.
 
Tissue Culture/LN2 Storage
The CICSL is equipped with class II type A2 bio-safety cabinets and CO2 incubators to allow the culture and analysis of immune cell subsets, as well as a controlled rate-cell freezer and liquid N2 freezers for long term storage of biological samples.
 

Using the Facility

Scheduling
Please contact the core director to schedule an initial consultation regarding the correlative studies you are proposing for your clinical trial. A research plan will then be assembled by the director and submitted to the principal investigator for approval. A lead time of three to six months is generally required for protocol development.
 
Note: Equipment operation is restricted to qualified CICSL personnel.
 
Quality Control
Protocol development, instrumentation, reagent, and technical quality control in the CICSL will be guided by GLP principles of operation. To the extent possible, all CICSL equipment is compliant with 25 CFR part 11 guidelines.
 

Immunobiology Correlative Studies Team

Clinical Immunobiology Correlative Studies Laboratory

Clinical Immunobiology Correlative Studies Laboratory

Overview
The Clinical Immunobiology Correlative Studies laboratory (CICSL) was founded in 2004, based on an identified need for a central laboratory dedicated to the development and implementation of cutting–edge immune correlative studies to support clinical trial protocols in the Cancer Center.
The CICSL has developed and has available a variety of assays that are based on molecular, flow Cytometry, molecular, functional, and biochemical platforms; the laboratory operates under principles of Good Laboratory Practice (GLP).
The laboratory also houses as the Quality Control (QC) unit for the Cell Therapeutics Program within HCT, developing and performing characterization and release tests for therapeutic cell products.
 
Objective
The research objective of the Clinical Immunobiology Correlative Studies Laboratory (CICSL) is to support Cancer Center investigators through the development, validation, and implementation of highly quantitative immune correlative studies that will be applied to evaluate the efficacy of immunotherapy-based and other clinical trials at the City of Hope. CICSL assay platforms are also available to support translational projects that utilize in-vitro and model systems.
 
Importance
  • The ability to quantitatively evaluate what has happened immunologically and molecularly in patients after receiving treatment is critical in order to obtain insight on why a particular regimen did or did not work, to guide the rational design of ultimately more effective immunotherapy strategies
  • Correlative studies that are well designed and performed are increasingly being viewed as critical components to clinical trials, and standards of quality such as assay qualification/validation, statistical significance of results, and GLP lab environment will be required for results to be published in top-tier peer reviewed publications
  • Since a comprehensive understanding of the effect of treatment on patients is in most circumstances lacking, there is a strong rationale to develop and apply a broad series of platforms and assays to evaluate samples and specimens obtained from patients enrolled in clinical protocols
 
Diagram illustrating the need for broad and comprehensive
correlative assays
 
The laboratory has developed a wide variety of platforms and assays.
 
Specifically:
Flow Cytometry:
Characterization of immune cell activation/differentiation/homing status
Absolute count determination and quantification of immune cell subsets
Analysis of immune cell effector phenotypes (ICC, CD107)
MHC tetramer analysis
Intracellular cytokine staining.

Molecular:
Q-RT-PCR-based quantification of immune cell subsets (including Treg cells)
Q-RT-PCR-based quantification of mRNA levels for immune effector genes
Q-PCR-based quantification of adoptively transferred cells
Vb spectratyping

Biochemistry:
Simultaneous quantification of multiple cytokine/chemokine/growth factors in serum, tissue samples, tissue culture using Luminex bead array
Simultaneous quantification of multiple phosphoproteins in tissue culture and tissue samples using Luminex bead array
 
Cell-based:
CD8 and NK cytolysis bioassays
Cell proliferation assays
Blood processing, PBMC/serum/plasma isolation, freezing, and storage
In-vitro CD8 and CD4 T cell culture
 
Last updated: 06/30/2008
 

Abstract for Grants

Abstract for Grants

The City of Hope Clinical Immunobiology Correlative Studies laboratory (CICSL) is a Shared Resource for the City of Hope Comprehensive Cancer Center. The laboratory occupies approximately 1000 square feet of laboratory space in the Shapiro building (rooms 1002, 1044, and 1045). This space includes a 275 square foot tissue culture area, a 275 square foot area dedicated to qPCR and ELISA, a 250 square foot room dedicated to liquid nitrogen, -80°, and -20° C storage, and a 200 square foot room housing dedicated to Luminex equipment. CICSL laboratory operations are guided by GLP principles of operation, with all possible equipment 21 CFR part 11 compliant.
 
The CICSL includes state-of-the-art equipment for monitoring immune responses to cancer therapy. Specific equipment includes a fully automated microplate based multi-detection reader capable of reading fluorescence intensity, time-resolved fluorescence, high-performance luminescence (flash and glow), and absorbance, a Luminex FLEXMAP 3D bead array reader, a Luminex BioPlex™-HTX bead array reader, two MJR Chromo 4™ Q-PCR machine, and a Guava PCA-96 analytical cell counter and analyzer. The CICSL additionally makes use of access to instruments within the City of Hope Analytical Flow Cytometry Core, including a Beckman Coulter Gallios three-laser flow cytometer capable of evaluating fluorescence in ten distinct colors, and analytical cell sorters.
 
Current assays within the capabilities of the laboratory include:
 
  • Flow Cytometry: Characterization of immune cell activation/differentiation/homing status; analysis of immune cell effector phenotypes and functionality (ICC, CD107); absolute count determination and quantification of immune cell subsets.
 
  • Molecular: quantitative real-time PCR-based quantification of immune cell subsets (including Treg cells) and mRNA levels for immune effector genes; qPCR-based quantification of adoptively transferred cells; Vβ spectratyping.
 
  • Biochemical: Quantification of multiple biomarkers and analytes in serum, plasma, tissue samples and cell culture medium using Luminex bead array and ELISA platforms. These analytes include cytokines and chemokines, growth factors, endocrine markers and hormones, cardiovascular markers, bone metabolism markers, cell signaling molecules, and cancer markers.
 
  • Cell-based: Blood processing; PBMC/serum/plasma isolation; freezing, and storage; CD8 and NK cytolysis bioassays.
 

Assays

Assays

In general, the CICSL develops assay platforms that can be applied to multiple clinical studies. Please contact the CICSL director if you are interested in having the laboratory develop an assay not listed below.
 
Current Available Assays and Procedures
 
Flow Cytometry
Characterization of immune cell activation/differentiation/homing status
Analysis of immune cell effector phenotypes (ICS, CD107 degranulation)
Absolute count determination and quantification of immune cell subsets
MHC:tetramer analyses
Proliferation assays (CFSE)
 
Molecular
Nucleic acid (DNA, RNA) isolation
cDNA synthesis
Q-RT-PCR-based quantification of immune cell subsets
Q-RT-PCR-based quantification of mRNA levels for immune and other factors
Q-PCR-based quantification of adoptively transferred cells
Vβ spectratyping
 
Biochemical
 
i. Luminex X-MAP bead array-based
Quantification of multiple cytokine/chemokine/growth factors in serum, tissue samples, tissue culture
ii. ELISA-based
Quantification of cytokine/chemokine/growth factors in serum, culture, tissue samples
iii. Quantification of phosphorylation status of intracellular signal transduction molecules, transcription factors by Luminex X-MAP bead array
 
Cell-based
Blood processing, PBMC/serum/plasma isolatio
 

Equipment

Equipment

The CICSL is equipped with state-of-the-art equipment to allow for the evaluation and monitoring of immune responses in patients post-immunotherapy. Specific equipment available in the laboratory include:

 

Luminex FLEXMAP 3D System (Luminex multi-bead array)

View Luminex FlexMAP3D System website


The new FLEXMAP 3D® system takes Luminex xMAP® technology to three dimensions. The FLEXMAP 3D system combines differentially dyed fluorescent microsphere sets with an innovative instrument design to allow multiplexing of up to 500 unique assays within a single sample, both rapidly, and precisely. The FLEXMAP 3D accommodates 96 and 384-well formats for enhanced sample volume flexibility and increased throughput. Sample plate heating control also allows the user to run thermally sensitive applications such as nucleic acids.The FLEXMAP 3D system works in the same basic manner as the Luminex® 100TM analyzer. Mixtures of internally dyed microspheres are used in an assay, each having been prepared to bind to a specific analyte of interest. After the assay has been performed the mixture of microspheres is aspirated into the system and then each individual microsphere is individually excited by a red and green laser. Each microsphere is impregnated with different amounts of three internal dyes. All dyes excite at the same red laser wavelength, but fluoresce at three separate wavelengths. Monitoring the relative intensity of the three signals allows the system to discriminate up to 500 different microsphere sets. The green laser is then used to excite the reporter molecules, normally in the form of phycoerythrin, and the resulting fluorescence is measured to determine the amount of analyte present.

 

 

Bio-Rad Bio-Plex HTF System (Luminex multi-bead array)
View Bio-Rad Bio-Plex System website

The Bio-Rad Bio-Plex 100 allows for the simultaneous assay of up to 100 distinct proteins in a single well of a microtiter plate, using very small sample volumes. The system delivers fast and cost-effective bioassay results on many assay formats including cytokines, immunoassays and enzymatic assays, nucleic acid assays, receptor-ligand assays, and phosphorylation assays. The Bio-Plex utilizes Luminex xMAP® technology which is based on the use of 100 distinctly color-coded microspheres. Each microsphere set can be coated with a reagent specific to a particular bioassay, for example individual anti-cytokine antibodies, allowing the capture and specific detection of multiple distinct cytokines from a sample in a single microplate well. Within the Luminex 100 compact analyzer, lasers excite the internal dyes that identify each microsphere particle, and also any reporter dye captured during the assay.
 
MJR Chromo 4™ Thermal Cycler
The MJR Chromo 4 is a Q-PCR instrument that allows for the quantitation of specific nucleic acid sequences in samples. The instrument is capable of excitation and detection in four different channels allowing for PCR multiplex analyses, has a linear dynamic range of up to ten orders of magnitude, and can detect a single copy of initial template in samples.
 
BMG FLUOstar OPTIMA
View BMG FLUOstar OPTIMA website
The FLUOstar OPTIMA is a fully automated microplate based multi-detection reader which incorporates five different measurement principles: fluorescence intensity, time-resolved fluorescence, high-performance luminescence (flash and glow), and absorbance. It is designed for the widest possible range of non-radioactive labeled applications, including immunoassays (ELISA), Ca2+ Measurements (e.g. Ca2+ Flux with Fura-2 or Indo-1), enzyme activity, cell toxicity, proliferation and viability, cytotoxicity and ATP quantification. The instrument covers a wavelength range from 240 to 740 nm and can read all plate formats from six up to 384-well plates in all four measurement modes. The versatile optical system allows instant switching from top to bottom reading. Temperature-regulated heating plates above and below the entire plate movement area provide uniform incubation up to 60°C. The instrument is equipped with a single syringe pumps. Injection speed and timing can be adjusted as is appropriate for particular assays. Delivery volume is adjustable between 3-350 µL in 1µL increments and is individually adjustable for each well allowing control of dilution schemes, concentration ranges, etc.
 
Tissue Culture/LN2 Storage
The CICSL is equipped with class II type A2 bio-safety cabinets and CO2 incubators to allow the culture and analysis of immune cell subsets, as well as a controlled rate-cell freezer and liquid N2 freezers for long term storage of biological samples.
 

Using the Facility

Using the Facility

Scheduling
Please contact the core director to schedule an initial consultation regarding the correlative studies you are proposing for your clinical trial. A research plan will then be assembled by the director and submitted to the principal investigator for approval. A lead time of three to six months is generally required for protocol development.
 
Note: Equipment operation is restricted to qualified CICSL personnel.
 
Quality Control
Protocol development, instrumentation, reagent, and technical quality control in the CICSL will be guided by GLP principles of operation. To the extent possible, all CICSL equipment is compliant with 25 CFR part 11 guidelines.
 

Immunobiology Correlative Studies Team

Immunobiology Correlative Studies Team

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City of Hope embodies the spirit of scientific collaboration by sharing services and core facilities with colleagues here and around the world.
 

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