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Biomarkers, Prevention and Interventions for HIV-associated Malignancies and NCDs Core

The Biomarkers, Prevention and Interventions for HIV-associated Malignancies and NCDs (BPIN) Core provides assays and data instruments to enable studies of HIV-associated malignancies.

Cancer has become the leading cause of death among persons with HIV infection worldwide. Kaposi sarcoma, non-Hodgkin lymphoma, and cervical cancer were recognized as AIDS-Defining Malignancies (ADM) early in the epidemic. In the setting of broadly available ART, cancer now impacts persons living with HIV both at the time of diagnosis and as a consequence of long-term HIV infection. In addition to ADM, people living with HIV are at increased risk of a range of cancers, including lung cancer, anal cancer, Hodgkin lymphoma, liver cancer, and head and neck cancer. Importantly, comorbid HIV infection is associated with more than a two-fold increased risk of death among persons with cancer compared with persons of the same age, gender, and stage of cancer without HIV infection. Despite more than three decades of recognizing an association between HIV and cancer, significant disparities remain in our knowledge about the etiology, natural history and treatment of HIV-associated malignancies (HIVAM). Immunotherapeutic approaches to treating cancer in the setting of HIV are emerging as important options. To address unanswered question in the field of HIV and cancer, we are proactively continuing to foster and strengthen research in our local and global HIV communities through these specific aims:

  1. Support design and implementation of studies addressing the changing epidemiology of HIVAM across the globe
  2. Foster development and evaluation of new technologies for the treatment and prevention of HIVAM through rigorous pre-clinical, translational and early-clinical studies
  3. Promote state-of-the-art technology for the pathologic and molecular diagnosis and monitoring of HIVAM
HIVAM Laboratory and Biorepositories (HCRI-Uganda)
The Uganda Cancer Institute Fred Hutchinson Cancer Research Center Facility has an 8,000
ft 2 laboratory with facilities that can support IRB approved HIVAM research. The laboratory has
specimen processing and storage, histopathology, BSL2/3 studies, and a steadily expanding
capability for molecular analysis that includes standard real-time PCR and a 16-channel
GeneXpert as well as high throughput sequencing on the Illumina MiSeq platform. The
laboratories are supported by 6 full-time lab technicians and data staff and are overseen by an
Acting Laboratory Director.

The laboratory also has a dedicated 337 sq. foot climate and access controlled biospecimen
repository that supports HIVAM research. The repository is setup to accommodate up to six -80-
degree Fahrenheit freezers, one -20-degree Fahrenheit freezer, four ambient temperature
specimen storage cabinets, and one Liquid Nitrogen (LN2) freezer with two LN2 supply tanks.
All freezers are setup with a real-time temperature monitoring system that logs all temperature
data and sends SMS to identified individuals in cases of temperatures occurring outside of
acceptable ranges.

Storage of specimens is also available via refrigerator and freezer storage and is provided in
separate, secured room in the laboratory; all refrigerators and freezers are supported by a back-
up generator and uninterruptible power supplies. Laboratory results and specimen storage data
are managed by a secure database (LIMS) system. The Laboratory staff have shipping
protocols for the efficient transfer of specimens to North America on either dry ice or liquid
nitrogen and hold annual import permits for the US CDC, the National Drug Authority of Uganda
and additional international permits as needed. Office space for the lab supervisor and auxiliary
staff contains six computer stations, printers, fax, and copy machines.

Sample processing services include standard plasma, serum, and buffy coat separation, PBMC
isolation by Ficoll separation, granulocyte isolation, solid tissue formalin fixing, snap-freezing of
tissue and cells, and cryopreservation capabilities. The laboratory equipment is maintained in
accordance with the standards set by Good Laboratory Practice (GLP) and CAP.

Please contact Manoj Menon for more information.

HIVAM Research Consultation
The HIVAM Core provides consultation services for researchers performing clinical or population science HIVAM research. Please contact us if you are planning assay development. To request a consultation, please email tuldrick@fredhutch.org.

Please contact Manoj Menon for more information.

Human Papillomavirus Genotyping (HCRI-Uganda)
Through 2020, we have employed the Roche Linear Array HPV Genotyping Test for HPV-
associated malignancy research. This commercial assay is based on four major processes:
specimen preparation/DNA Extraction using the Qiagen MinElute Extraction Kit and Vacuum
System; PCR Amplification of the HPV polymorphic L1 region using pool of HPV Primers;
hybridization of the amplified products to oligonucleotide probes; and detection of the probe-
bound amplified products by colorimetric determination. This assay has the ability to detect
HPV DNA of 37 anogenital HPV types.

These HPV genotypes included: 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 55,
56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73 (MM9), 81, 82 (MM4), 83 (MM7), 84 (MM8),
IS39, and CP6108.

The ideal sample type is a cervical, genital, or rectal brush or swab collected and stored in a
PCR Buffer such as the Roche PCR Cell Collection Media. As of 2020, this assay is being discontinued by Roche diagnostics no longer available. The HIVAM Core is pursuing a comparable assay to replace this legacy assay at our site for 2021.

Please contact Manoj Menon for more information.

Kaposi Sarcoma Herpesvirus (KSHV) LANA Immunohistochemistry (HCRI-Uganda)
Immunohistochemistry (IHC) for KSHV latency associated nuclear protein (LANA) helps
visualize the localization and distribution of KSHV infected cells in tissue sections based on the
antigen-antibody binding reaction. The laboratory follows manual processes of IHC staining of
LANA utilizing the Novocastra liquid mouse monoclonal antibody from Leica, NCL-L-
HHV-8-LNA (Clone13B10, class IgG1).

Please contact Manoj Menon for more information.

Real-Time Quantitative Epstein Barr Virus (EBV) PCR (HCRI-Uganda)
EBV is a necessary etiology agent of endemic Burkitt’s lymphoma, as well as a range of
aggressive B-cell lymphomas that are strongly associated with HIV, including plasmablastic
lymphoma, AIDS-associated primary CNS lymphoma and a proportion of cases of classical
Hodgkin lymphoma, diffuse large B-cell lymphoma and primary effusion lymphoma.

EBV RT-PCR assay can be performed on clinical samples including saliva, genital secretion,
CSF, plasma, serum, body fluids and tissues. Viral DNA is extracted and quantified using PCR
techniques. DNA extraction is performed using Qiagen chemistry and single column procedure.
PCR is performed on a high-throughput ABI 7900 thermocycler, in addition to two new 7500
systems. To date, more than 75,000 PCR tests have been resulted in this laboratory, which
undergoes regular Quality Control audits by the Seattle-based molecular diagnostics lab.
The Real-Time Taqman PCR assay utilizes fluorescent labeled probes to amplify, detect and
quantify EBV. Primers and probes specific to the BALF5 region of the EBV genome. The beta-
Globin region of the human genome may be used to quantify cellular content of the specimen.
These assays were designed and tested for sensitivity and specificity at the University of
Washington Molecular Diagnostic Lab in Seattle, Washington, USA. An internal control is
spiked into each PCR reaction to ensure that no non-specific inhibition of the reaction has taken
place. A negative result is accepted only if the internal control amplification is detected.
Inhibited samples are re-extracted or chelexed and the analysis repeated.

Please contact Manoj Menon for more information.

Real-Time Quantitative Kaposi Sarcoma Herpesvirus (KSHV) PCR (HCRI-Uganda)
KSHV is the necessary but insufficient infectious etiologic agent of Kaposi sarcoma (KS), a form
of multicentric Castleman disease (KSHV-MCD) and primary effusion lymphoma (PEL). KSHV
prevalence is greater than 60% in the general population in Uganda. KSHV PCR is available for
research on these important HIVAM.

KSHV RT-PCR assay can be performed on clinical samples including saliva, genital secretion,
CSF, plasma, serum, body fluids and tissues. Viral DNA is extracted and quantified using PCR
techniques. DNA extraction is performed using Qiagen chemistry and single column procedure.
PCR is performed on a high-throughput ABI 7900 thermocycler, in addition to two new 7500
systems. To date, more than 75,000 PCR tests have been resulted in this laboratory, which
undergoes regular Quality Control audits by the Seattle-based molecular diagnostics lab.

The Real-Time Taqman PCR assay utilizes fluorescent labeled probes to amplify, detect and
quantify KSHV. Primers and probes specific to the ORF73 and T.07-K12 regions of the KSHV
genome. The beta-Globin region of the human genome may be used to quantify cellular content
of the specimen. These assays were designed and tested for sensitivity and specificity at the
University of Washington Molecular Diagnostic Lab in Seattle, Washington, USA. An internal
control is spiked into each PCR reaction to ensure that no non-specific inhibition of the reaction
has taken place. A negative result is accepted only if the internal control amplification is
detected. Inhibited samples are re-extracted or chelexed and the analysis repeated.

Please contact Manoj Menon for more information.

The BPIN Core continues to cultivate research in the Seattle biomedical community through the provision of assistance with lab assay development, consultation, and data tools. Additionally, the number of researchers within this field is growing with many of our junior investigators and next generation leaders initiating independent research careers in the field of HIVAM with the support of UW/Fred Hutch CFAR. Further, the NIH funding base for research in HIVAM has grown to over $15 million, and several grants, including K23s, R21s, R01s, P01s, and U54s have been awarded to HIVAM members, furthering CFAR’s mission of adding value to the local research community.
Co-Director
Corey Casper, MD, MPH
Corey.Casper@idri.org
 
Co-Director
Manoj Menon, MD, MPH
mmenon@fredhutch.org
 
Associate Director
Warren Phipps, MD, MPH
wtphipps@fredhutch.org
 
Core Manager
TBD