False-positive HIV nucleic acid amplification testing during CAR T-cell therapy (2024)

Abstract

Advancements in immunotherapy have opened a new era in oncology, to include genetic modification of human T-cells to express a chimeric antigen receptor (CAR) that enables targeted tumor recognition (Kochenderfer et al., 2015; Lee et al., 2015; Maus and Levine 2016; Rosenberg et al., 2008). Herein, we report a false-positive HIV testing in a patient who had undergone CAR T-cell therapy created with a lentiviral vector.

Keywords: HIV, CAR T-cell therapy, False-positive, Immunotherapy

1. Case description

A 54-year-old female patient diagnosed with recurrent progressive follicular lymphoma was enrolled in a clinical trial of anti-CD19 CAR T-cell therapy. Her treatment course was complicated with cytokine release syndrome (CRS). The patient’s re-staging positron emission tomography–computed tomography performed 2 months after the completion of CAR T-cell infusion demonstrated a substantial decrease in lymphoma burden with a formal staging result of partial response. In order to consolidate therapy, the patient was evaluated for hematopoietic stem cell transplantation (HSCT). As part of the routine pre-transplant workup, NAAT for HIV was performed, which results were positive. As the patient had no prior HIV history, and to rule out laboratory error, HIV testing was repeated 1 week later on a fresh specimen, which again was positive. In parallel, specimens were sent to a reference laboratory for a 4th generation HIV screening assay and HIV viral load quantification. The HIV viral load testing was positive, at 74 copies/mL. However, the 4th generation HIV screening assay result was negative. The CD4 count at this time was normal at 837 cells/mL. The patient denied having any risk factors for HIV; no intravenous drug use, tattoos, incarceration, high risk sexual behavior, and had been married for over 20 years”. Furthermore, she denied any symptoms of acute HIV, including fever, malaise, or lymphadenopathy.

At this point, as a laboratory error was ruled out, we concentrated our efforts on evaluating the differential diagnosis for positive HIV NAAT and negative HIV antibody test results. Acute HIV infection during the window period also was unlikely, owing to the patient’s clinical and epidemiological history. Furthermore, her viral load had remained low on confirmatory testing, and it would have been expected to rise in a case of acute HIV infection. After further investigation, we learned that the CAR T-cells the patient had received were created using a lentiviral vector (Yang et al., 2012; Zhou et al., 2003). A recently published case report described false-positive HIV polymerase chain reaction (PCR) testing following an ex-vivo lentiviral-based gene transfer treatment for X-linked immunodeficiency (De Ravin et al., 2014). The challenge in our case was to confirm, with certainty, that the positive HIV viral load was due to the presence of lentiviral-derived CAR T-cells in our patient, thus allowing her to safely undergo HSCT.

Lentiviral vectors contain portions of the HIV viral genome, which are integrated into the genomes of cells transfected by these vectors. Commercially available HIV NAATs detect HIV by amplifying the genomic regions of 1 or 2 viral genes specific to HIV (Izopet, 2014; Wittek et al., 2007). To determine whether the HIV NAATs used in our case could discriminate between cells infected by wild-type HIV versus cells transfected by a lentiviral vector, we contacted the manufacturers of each assay. The initial test was performed using the Procleix Ultrio assay (Hologic), which amplifies 2 regions of the HIV genome: the pol gene and the long terminal repeat (LTR) sequence. The reference laboratory performed the HIV viral load assay on the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test (Roche Molecular Systems), which detects the LTR sequence and gag gene. The research protocol for generating the patient’s CAR T-cells used a lentiviral vector that contained the HIV LTR and gag gene sequences, which could explain why these 2 assays yielded HIV-positive results (Yang et al., 2012). Of interest, the reference laboratory had a second commercial platform, the RealTime HIV-1 assay (Abbott Molecular), a qualitative assay that detects the only the pol gene, which was not part of the lentiviral vector (Table 1). Our patient had HIV-negative findings on the RealTime HIV-1 platform, making infection with wild-type HIV unlikely. Furthermore, because the cells transfected by the lentiviral vector are incapable of producing HIV proteins, including p24 antigen (Yang et al., 2012), the 4th generation serological testing remained negative, even after six months follow-up, providing further evidence against acute HIV infection.

Two very important teaching points arise from this case of a false-positive HIV result in a patient who received CAR T-cell therapy for a hematological malignancy. First, positive HIV screening results on NAAT warrant critical interpretation when the patient has a low risk of the disease. Second, treatment with lentiviral-derived CAR T-cells may lead to false-positive results on HIV RNA testing.

In clinical practice, HIV-positive results on NAAT and viral load testing with negative results for HIV antibody testing can result from: false-positive test results due to rare laboratory errors (Feucht et al., 2012; Rich et al., 1999), acute HIV infection (Busch and Satten, 1997; Wittek et al., 2007), administration of an HIV-1 vaccine (Schwartz et al., 1997), or cross-reactivity with gene transfer therapy (De Ravin et al., 2014). Screening for acute HIV infection with plasma viral load testing is recommended only for patients who have a high pretest probability of a positive result (Centers for Disease Control and Prevention and Association of Public Health Laboratories; Facente et al., 2011; Pilcher et al., 2005). In the evaluation of transplant donors and recipients, some centers, including ours, use serology and NAAT for screening HIV, hepatitis B and C viruses (Ison et al., 2013; Tomblyn et al., 2009). This approach is useful in deceased solid organ transplant donors and in potential living donors at high risk for one or more of the viruses; however, it may lead to potential false-positive results in donors with no identified risk factors (Humar et al., 2010).

Our case corresponds to HIV cross-reactivity resulting from gene transfer therapy, which utilizes lentiviruses to engineer CAR T-cells. These vectors are used to transduce T-cells of patient’s ex-vivo, and then are infused into patients (Maus and Levine, 2016). These T-cells are not capable of producing new lentivirus, as they are replication incompetent. However, components of the lentivirus become a permanent part of the infused CAR –cell product (Yang et al., 2012; Zhou et al., 2003). The genomes of these lentiviruses, but no those that rely on gamma retrovirus (Liechtenstein et al., 2013), share a similar organization with other retroviruses, including HIV (De Ravin et al., 2014; Escors and Breckpot, 2010; Yang et al., 2012). This similarity explains the cross-reactivity that occurs with some molecular HIV PCR tests (Table 1). False-positive HIV-1 testing (Roche COBAS AmpliPrep) has also been described in two patients with X-linked severe combined immunodeficiency, whom received lentiviral gene-transfer therapy (De Ravin et al., 2014). Other clinical applications of lentivirus vectors where we could possibly observe the same effect include patients with: sickle cell disease (Ribeil et al., 2017), X-linked adrenoleukodystrophy (Cartier et al., 2009), and transfusion-dependent β-thalassemia (Thompson et al., 2016).

As more patients receive immunotherapy for diverse malignancies (Rosenberg et al., 2008), clinicians need to be aware of potential false-positive HIV test findings resulting from CAR T-cell therapy that use lentiviral vectors (Kochenderfer et al., 2015; Lee et al., 2015), and recommendations for HIV testing need to be developed to avoid delays on therapy and unwanted stress for the patients and their families.

Acknowledgments

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