Uso de ctDNA para el Manejo de

Inmunoterapia en Cancer

Eduardo M. Sotomayor, MDThe Dr. Cyrus Katzen Family Director of the

George Washington University Cancer Center

Professor of Medicine,

George Washington University School of Medicine

-Response rates for the currently approved checkpoint inhibitors typically range from ~10-30% for solid tumors (Brahmer et al., 2010; Brahmer et al., 2012; Hodi et al., 2010; Le et al., 2015; Topalian et al., 2015)

- Complete and durable responses to CAR T-cells (three different platforms) are seen in approximately 30-40% patients (ASH 2019)

• To move the field forward towards precision immunotherapy:

• Identification of biomarkers of response (…or resistance)

• Tumor cells and their microenvironment

• Lymphoid organs

• Bone marrow

• Peripheral blood

Despite growing success, many patients do not

respond to Immunotherapy

“Classical biopsies”

“Liquid biopsy”

• Given the complexity of the immune responses and tumor biology it is unlikely that a single “classical” biopsy will “tell us the whole story” of what it is a very dynamic interaction between immune cells and tumors and as such, help us to predict clinical outcomes in response to immunotherapy….

• Integration of multiple tumor and immune response parameters (ie. protein expression, genomics, transcriptomics) from a classical biopsy may be necessary for prediction of clinical benefit…..

Predicting responses to immunotherapy

Liquid biopsySerial liquid biopsies would allow us to better “visualize” dynamics of antitumor

immune responses before (pre-existing), during, and after treatment with

checkpoint blockade or CAR T-cells.

Do circulating immune cells and/or tumor cells or their products (ie, ctDNA)

provide a good assessment of the antitumor immune responses elicited by

Immunotherapy?

• ctDNA

• Tumor-antigen specific T-cell responses

Emerging Biomarkers of Response to

Immunotherapy in Liquid Biopsies

Anagnostou, V. et al. Dynamics of tumor and immune responses during immunotherapy with checkpoint blockade. Cancer Research 79(6), March 2019

Current Clinical Utility of ctDNA Analysis

Wu, T-H. et al. Cancer Treatment Reviews 78: 31-41, 2019

• Assessment of therapeutic responses to checkpoint blockade using radiographic imaging has been challenging due to tumor immune infiltration. RECIST do not consistently capture the unique patterns and timing of antitumor immune responses (pseudo-progression).

• Tumors displaying high TMB or with dMMR/MSI respond better to immune checkpoint blockade. However, more cost-effective tests for routine clinical practice are needed

Clinical Utility of ctDNA Analysis in Immune

checkpoint blockade

• ctDNA-based liquid biopsy has the potential for:

• Identifying high responders to checkpoint blockade:

• Detection of bTMB

• Detection of dMMR …..“Matching” the “mistmach”

• Assessment of response to immune checkpoint blockade

• Differentiate pseudo-progression from true progression

• Due to advances in technology ctDNA analysis can be carried out in large scales and integrated into clinical practice….

• ctDNA analysis may be predictive of outcomes in melanoma patients treated with anti-CTLA4 (Lipson, EJ et al. J Immunother Cancer, 2014)

• ctDNA changes have been associated with therapeutic outcomes during immune checkpoint blockade in NSCLC (Goldberg, SB et al. Clin. Cancer Res 2018)

• …however, low sensitivity of these approaches have limited their broader application…… new ctDNA approaches

• Target error sequencing (TEC-Seq) ctDNA a custom capture and sequencing approach for sensitive and specific detection of low abundance sequence alterations using NGS (Anagnostou, V. et al. Cancer Research 2019)

• Cancer Personalized Profiling by Deep Sequencing (CAPP-seq) ctDNA analysis, a NGS-based method that tracks multiple mutations per patients with lower limits of detection ~0.002% (Chaudhuri, A et al. Cancer Discovery, 2017)

Predicting efficacy of Immune checkpoint

blockade with ctDNA

TEC-seq ctDNA and response to anti-PD1 in

NSCLC

Anagnostou, V. et al.. Cancer Research 79(6), March 2019

Responder Non-responder

Early ctDNA clearance predicts PFS and OS in

response to anti-PD1 in NSCLC

Anagnostou, V. et al. Cancer Research 79(6), March 2019

CAPP-seq ctDNA: Early detection of Molecular

Residual Disease in NSCLC

Chaudhuri, A et al. Cancer Discovery, 2017

• ctDNA

• Tumor-antigen specific T-cell responses

Emerging Biomarkers of Response to

Immunotherapy in Liquid Biopsies

• Only few autologous T-cell clones seems to be needed for a clinically effective antitumor response

• “Holy Grail”: Identification of antigen-specific T-cells responsible for a clinically effective antitumor effect in response to checkpoint blockade….

• Given their low frequency, novel immune technologies, bio-informatics and computational biology tools are needed for identification of those T-cells in liquid biopsies

Pre-existent tumor-antigen specific

T-cell responses

• Conventional Assays:

• Multimer reagents including tetramers permit identification of antigen-specific T cells by direct binding of T cells to reagent.

• ELISPOT, intracellular cytokine staining (ICS) and proliferation assays, enumerate antigen-specific T cells based on detection of activation following stimulation of T cells with antigen.

• Novel Assays:

• Next generation sequencing (NGS) of T cell receptor (TCR) repertoires: identification and quantitation of all rearranged T cell antigen receptors, or clonotypes within a sample

• However, TCR sequence alone does not permit identification of the antigen that a T-cell recognizes

• MANAFEST : Mutation Associated Neo-Antigens + Functional Expansion of Specific T-cells

Identification and enumeration of antigen specific T-cells in liquid biopsies

• ctDNA

• NGS of TCR repertoires

“Visualizing” antitumor immune responses to

checkpoint blockade in Liquid Biopsies

Anagnostou, V. et al. Dynamics of tumor and immune responses during immunotherapy with checkpoint blockade. Cancer Research 79(6), March 2019

“Visualizing” an effective antitumor immune

responses to checkpoint blockade in Liquid Biopsies

Anagnostou, V. et al. Dynamics of tumor and immune responses during immunotherapy with checkpoint blockade. Cancer Research 79(6), March 2019

“Visualizing” lack of antitumor immune responses

to checkpoint blockade in Liquid Biopsies

Anagnostou, V. et al. Dynamics of tumor and immune responses during immunotherapy with checkpoint blockade. Cancer Research 79(6), March 2019

MANAFEST : Mutation Associated Neo-Antigens + Functional Expansion of Specific T-cells

-Whole Exome Sequencing (WES) in pre-treatment tumors and matched normal

tissue: tumor-specific alterations are analyzed using a neo-antigen prediction

pipeline to identify candidate MANAs specific to the patient's HLA haplotype

(Bioinformatics and computational science)

-T cells are isolated from patients 4 weeks after initiation of anti-PD1 and cultured

for 10 days with putative MANAs

Resulting expanded CD8b T cells are isolated for TCR Vb CDR3 sequencing and MANAFEST analysis

MANAFEST: Visualizing mutation-associated, neo-antigen specific T-cells in patients treated with checkpoint blockade

CONCLUSIONS

• Yes, circulating T-cells and tumor cells (or their products, ie ctDNA) in

serial liquid biopsies can provide a good assessment of the dynamics of

antitumor immune responses elicited by checkpoint blockade

• Although these initial studies are promising and encouraging, more data

in larger number of patients is needed, before these analysis can

become part of our routine clinical practice

• We are “just learning” how to identify in liquid biopsies, those T-cell

clones responsible for an effective antitumor immune response and the

tumor antigens they recognize…..

• Potential to engineer a “strong army” of highly specific autologous T-

cells, with hopefully, less toxicity than CAR T-cells

THANK YOU

Esotomayor@ mfa.gwu.edu

Identification and tracking of mutation-associated neo-antigen specific T-cells in patients treated with anti-PD1

- Patients with untreated, surgically resectable stage I, II, or IIIA NSCLC.

- Two preoperative doses of Nivolumab (at a dose of 3 mg/ kg, IV every 2

weeks). Surgery planned approximately 4 weeks after the first dose.

- Primary end point: Safety and feasibility.

- Secondary endpoints: Tumor pathological response, expression of PD-

L1, mutational burden, and mutation-associated, neoantigen-specific T-

cell responses.

Neoadjuvant PD-1 blockade in resectable Lung

cancer