Molecular pathology selected abstracts

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Editors: Donna E. Hansel, MD, PhD, chair of pathology, Oregon Health and Science University, Portland; Richard D. Press, MD, PhD, professor and director of molecular pathology, OHSU; James Solomon, MD, PhD, assistant professor, Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York; Sounak Gupta, MBBS, PhD, senior associate consultant, Mayo Clinic, Rochester, Minn.; Tauangtham Anekpuritanang, MD, molecular pathology fellow, Department of Pathology, OHSU; Hassan Ghani, MD, molecular genetic pathology fellow, Department of Pathology, OHSU; and Fei Yang, MD, assistant professor, Department of Pathology, OHSU. 

Circulating tumor DNA as a clinical test in resected pancreatic cancer

October 2019—Pancreatic ductal adenocarcinomas are associated with high rates of mortality due, in part, to a lack of effective screening strategies and advanced disease at diagnosis. Residual occult disease is thought to contribute to disease recurrence in up to 80 percent of patients treated surgically for localized disease. These findings highlight the critical need for biomarkers for detecting disease early and monitoring tumor dynamics. Current strategies involve a combination of serum markers (carbohydrate antigen [CA] 19–9) and imaging modalities, both of which have limitations, particularly for detecting early disease recurrence postoperatively. While circulating tumor DNA (ctDNA) as a clinical biomarker of tumor recurrence is gaining popularity, one of the major challenges to broad clinical implementation of ctDNA detection assays involves the vast diversity of molecular alterations that may be present. Pancreatic ductal adenocarcinomas are unique in that more than 90 percent of cases harbor alterations of KRAS at select hotspots—G12D, G12V, G12R, and Q61H. The authors of this study exploited this unique molecular signature by developing KRAS hotspot-specific digital droplet polymerase chain-reaction (ddPCR) assays that were used in a CLIA-certified setting in a prospective manner. They evaluated primary tumors from 59 patients using next-generation sequencing to determine baseline KRAS status. Four ddPCR assays—for G12D, G12V, G12R, and Q61H—were validated for their ability to detect KRAS hotspot alterations in ctDNA. Because ddPCR has high sensitivity, thresholds to stratify specimens into negative, borderline, and positive categories were determined after evaluating normal plasma samples from healthy donors to establish acceptable levels of background noise. Key findings of this study included higher levels of preoperative ctDNA in patients that were chemotherapy naïve compared with those that had received prior neoadjuvant chemotherapy, which were associated with worse outcomes, including overall survival. Therefore, these data may help guide clinical decision-making about the use of neoadjuvant chemotherapy. Furthermore, higher rates of recurrence and poor outcomes, including overall survival, were associated with patients in whom ctDNA persisted postoperatively, while the majority of patients who were recurrence free had undetectable ctDNA levels in follow-up samples. Consequently, patients with persistent postoperative ctDNA may represent a high-risk group that could benefit from intervention with systemic therapy. More importantly, ctDNA detection provided a lead time of more than 80 days compared to imaging, thereby providing an opportunity to switch patients with refractory disease to second- or even third-line therapeutic regimens in a timely manner. Limitations of this study include its exclusion of patients that lack KRAS alterations and the small sample size of the cohort. However, prospectively assessing ctDNA alterations in a CLIA-certified setting has the potential to alter the standard of care for patients with pancreatic ductal adenocarcinomas.

Groot VP, Mosier S, Javed AA, et al. Circulating tumor DNA as a clinical test in resected pancreatic cancer. Clin Cancer Res. 2019;25(16):4973–4984.

Correspondence: Dr. James R. Eshleman at jeshlem@jhmi.edu

Re-engineering anti-HIV CAR-T cells to overcome limitations: a humanized mouse model

The number of people living with AIDS/HIV worldwide was estimated to be approximately 36.9 million in 2017. HIV treatment strategies involve sustained viral suppression using antiretroviral agents. Alternative cellular immunotherapeutic approaches using chimeric antigen receptor-modified T cells (CAR-T) have had limited success in treating HIV infection, despite their proven efficacy for refractory B-cell malignancies. Limitations in the development of first-generation anti-HIV CAR-T cells included the high rate of mutation of HIV envelope-derived epitopes leading to immune escape and CAR-T cells’ susceptibility to HIV infection. The authors conducted a study in which they re-engineered anti-HIV CAR-T cells to overcome these limitations. In the initial step in HIV infection of susceptible T cells, gp120 viral envelope proteins bind to CD4 T-cell receptors. This interaction leads to folding changes in gp120 envelope proteins. These conformational changes were exploited in the design of the anti-HIV CAR-T cells. One of the domains in the re-engineered CAR-T cells targeted conserved portions of the CD4 receptor on T cells, while a second domain bound a CD4-induced gp120 viral envelope protein. Additional domains could inhibit HIV in vitro and in vivo. For instance, the C46 peptide is functionally similar to the FDA-approved HIV fusion inhibitor enfuviritide. The authors evaluated three groups of anti-HIV CAR-T cells: those that were re-engineered with a single domain (monospecific) or multiple domains (bispecific or trispecific). In a series of in vitro experiments, they showed that these proteins were expressed on the surface of T cells and could neutralize HIV-infected cells without causing unwanted or off-target effects on nonHIV-infected T cells. In addition, the proteins demonstrated T helper cell activation based on the expression of downstream markers and cytokines such as interferon-γ. More importantly, the investigators overcame a limitation of first-generation anti-HIV CAR-T cells by showing that the newer multispecific agents were protected from HIV infection. The persistence of these CAR-T clones in humanized mouse models infected with HIV for up to 30 days suggests that these agents have the potential to continuously suppress HIV infection without unwanted or off-target effects on T cells. A limitation of this study is that these constructs were not evaluated in in vivo models of established HIV infection. In this context, future directions could involve combining anti-HIV CAR-T cells with agents that can reverse HIV latency in an attempt to eradicate latent HIV reservoirs. This study provides proof of principle for future clinical trials of CAR T-cells that have the potential to outperform antiretroviral drugs by aiming to eradicate HIV.