Benjamin Holmes, DVM

During the 10th Annual Meeting of the Society of Hematologic Oncology, 

, will explore the emergence of small molecule–inhibitor targeted therapies. His presentation, “

Workup of CLL in the Era of Small Molecules,

Wierda is a professor; section chief, chronic lymphocytic leukemia (CLL); and center medical director, Department of Leukemia, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, where he is also executive medical director. He will address the role of Bruton tyrosine kinase (BTK) inhibitors, PI3K inhibitors, and inhibitors of BCL2 during the session.

CLL is the most common leukemia in adults in Western countries; estimates indicate more than 20,000 individuals in the United States will receive CLL diagnoses and approximately 4410 will die of CLL in 2022.

 Improved knowledge of the B-cell receptor (BCR) signaling pathway and its relationship to the behavior and pathophysiology of CLL has resulted in substantial advances in therapeutic options.

 The BCR is responsible for an antigen-independent survival signal involving PI3K and an antigen-dependent signal involving intracellular molecules such as LYN, SYK, BTK, and PI3K leading to proliferation and activation of the cells.

 CLL B cells are “stuck” in a later stage of development characterized by the inability to further differentiate, impaired immune function, and prolonged life span due to overexpression of antiapoptotic proteins (primarily BCL2).

 Downstream components of the BCR signaling pathway continue to be expressed in CLL B cells, propagating signaling.

 Increased understanding of the role of BCR in CLL pathophysiology led to the development of small molecule inhibitor–targeted therapies with tyrosine kinase inhibitors against BTK and PI3K and inhibitors of BCL2.

BTK inhibitors block the adenosine triphosphate–binding site by binding irreversibly via covalent bonds with the sulfhydryl group of Cys481 or reversibly with noncovalent binding affinity, disrupting the ability of the CLL cells to interact with the microenvironment, resulting in death of the malignant cells.

 Current BTK inhibitors approved by the FDA for treatment of CLL are irreversible and include acalabrutinib (Calquence) and ibrutinib (Imbruvica).

Results from the phase 3 ELEVATE CLL TN trial (NCT02475681) showed acalabrutinib plus or minus obinutuzumab (Gazyva) produced improved progression-free survival (PFS) compared with chlorambucil (Leukeran) plus obinutuzumab in patients with treatment-naïve CLL.

 The combination of acalabrutinib and obinutuzumab also produced a longer PFS than acalabrutinib alone at 48 months (87% vs 78%), but the study was not powered to compare the PFS between the acalabrutinib arms.

The phase 3 RESONATE-2 trial (NCT01722487) in CLL patients without del(17p) who were 65 years or older resulted in approval of ibrutinib as first-line therapy for all patients.

 In 2 separate studies (ECOG-ACRIN Cancer Research Group (E1912) study [NCT02048813] and FLAIR study [ISRCTN01844152]) ibrutinib with rituximab (Rituxan) proved more effective than fludarabine/cyclophosphamide/rituximab in patients lacking del(17p)/

 Results also indicated ibrutinib is a good option for younger patients with unmutated 

 Ibrutinib plus rituximab or obinutuzumab proved more effective than chemoimmunotherapy for treatment-naïve CLL without del(17p) or 

 mutations across several randomized phase 3 trials; however, the clinical outcomes were not improved with the addition of rituximab compared to ibrutinib monotherapy.

 Therefore, ibrutinib is recommended as monotherapy in first-line or relapsed/refractory CLL.

Zanubrutinib (Brukinsa) is another irreversible BTK inhibitor, but is highly selective/specific and has FDA approval for Waldenström macroglobulinemia and relapsed/refractory mantle cell lymphoma.

 The phase 3 SEQUOIA trial (NCT03336333) evaluated zanubrutinib vs bendamustine plus rituximab and found a greater overall response rate (95% vs 85%) with zanubrutinib in patients with untreated CLL without del(17p)/

 mutation along with significantly improved PFS (HR, 0.42; 

 did not experience a significant improvement in PFS (HR, 0.67; 

PI3K activation results in greater B-lymphocyte proliferation, apoptosis, adhesion, and migration.

 Approved PI3K inhibitors for treatment of CLL include idelalisib (Zydelig) and duvelisib (Copiktra).

 Idelalisib received FDA approval in 2014 for R/R CLL and duvelisib was approved in 2018 for the treatment of R/R CLL.

 Due to poorer toxicity profiles including autoimmune complications such as colitis, pneumonitis, and increased susceptibility of infections, BTK inhibitors are preferred to PI3K inhibitors.

 Additionally, acalabrutinib monotherapy proved more effective with fewer adverse events than idelalisib plus rituximab in the phase 3 randomized ASCEND trial (NCT02970318).

The BCL2 family of proteins are important regulators of apoptosis, containing proteins that support apoptosis and others that support survival.

 Cancer cells avoid apoptosis by shifting toward more proteins in support of survival.

Venetoclax (Venclexta) is the only FDA-approved BCL2 inhibitor for the treatment of CLL and is a fixed-duration treatment option

 Venetoclax plus obinutuzumab significantly improved PFS compared with chlorambucil plus obinutuzumab in patients 65 years or older or patients younger than 65 years with comorbidities in the CLL14 study (NCT02242942).

 Venetoclax also resulted in a greater rate of undetectable minimal residual disease (74% vs 34%; 

 < .0001) along with a lower rate of conversion to MRD-positive status 1 year after treatment.

 Recently presented results from the phase 3 CLL13 trial (NCT02950051) indicate superior PFS with venetoclax plus obinutuzumab plus ibrutinib (HR, 0.32; 97.5% CI, 0.19-0.5; 

 < .0001) and venetoclax plus obinutuzumab (HR, 0.42; 97.5% CI, 0.26-0.68; 

Given the advantages of specific therapies in patients with particular gene mutations and characteristics, the National Comprehensive Cancer Network (NCCN) recommends certain treatments depending upon the findings during work-up.2 For patients with CLL without del(17p) or 

 mutations, the NCCN guidelines give the strongest recommendation for acalabrutinib plus or minus obinutuzumab followed by zanubrutinib.

 Venetoclax and obinutuzumab is a recommended option for patients 65 years or older or younger patients with significant comorbidities and is included as a lesser recommended option for patients younger than 65 years without comorbidities due to a lack of current available evidence.

 mutations there are limited data for recommendations and the NCCN recommends enrolling these patients in an appropriate clinical trial.

 However, currently available data suggest acalabrutinib plus Obinutuzumab; ibrutinib; venetoclax plus Obinutuzumab; and zanubrutinib are preferred treatment options for first-line therapy.

1.Tambaro FP, De Novellis D, Wierda WG. The role of BTK inhibition in the treatment of chronic lymphocytic leukemia: a clinical view. 

. 2021;13:923-935. doi:10.2147/JEP.S265284

2.Wierda WG, Brown J, Abramson JS, et al. NCCN Guidelines Insights: chronic lymphocytic leukemia/small lymphocytic lymphoma, version 3.2022. J

. 2022;20(6):622-634. doi:10.6004/jnccn.2022.0031

3.Hus I, Pula B, Robak T. PI3K inhibitors for the treatment of chronic lymphocytic leukemia: current status and future perspectives. 

 (Basel). 2022;14(6):1571. doi:10.3390/cancers14061571

4.Hallek M, Al-Sawaf O. Chronic lymphocytic leukemia: 2022 update on diagnostic and therapeutic procedures. 

. 2021;96(12):1679-1705. doi:10.1002/ajh.26367

5.Eichhorst B, Niemann C, Kater A, et al. Time-limited venetoclax-obinutuzumab +/- ibrutinib is superior to chemoimmunotherapy in frontline chronic lymphocytic leukemia (CLL): PFS co-primary end point of the randomized phase 3 GAIA/CLL13 trial. Presented at: European Hematology Association 2022 Congress; June 9-12, 2022; Vienna, Austria. https://library.ehaweb.org/eha/2022/eha2022-congress/366209/barbara.eichhorst.time-limited.venetoclax-obinutuzumab.2B.-.ibrutinib.is.html

Articles

William Wierda, MD, PhD, will explore the emergence of small molecule–inhibitor targeted therapies.

Greater Understanding of CLL Pathophysiology Spurs Small Molecules Development

The discovery of antitumor activities of pro-inflammatory cytokines in animal models led to subsequent approvals of recombinant interferon alpha (INF-α) and IL-2.

 Challenges of targeted cancer treatment with cytokines include the short half-life of cytokines in the circulation, significant toxicities at therapeutic doses, and initial lack of efficacy.

 Subsequent immunotherapies such as immune checkpoint inhibitors (ICIs) and chimeric antigen receptor T cells were found to be more effective, and their use superseded and overshadowed that of cytokine-based therapies.

Advances in genomics, molecular engineering, and molecular analysis have helped renew interest in the antitumor properties of cytokines and their use as single agents or in combination with other immunotherapies such as ICIs.

 Current- generation cytokines are being engineered to extend their half-life and improve tumor targeting using polyethylene glycol conjugation, fusion to tumor-targeting antibodies, alterations of cytokine/cell receptor–binding affinity, and other strategies.

 A significant number of recent studies have evaluated the clinical use of these cancer therapeutics based on numerous cytokines that include, but are not limited to, IFN-α, IL-2, IL-15, IL-21, IL-12, and IL-10.

Svetomir N. Markovic, MD, PhD, a professor of medicine and oncology and consultant in the Division of Medical Oncology at Mayo Clinic in Rochester, Minnesota, discussed cytokine-based cancer treatments in an interview with 

™. Markovic noted that initial antitumor cytokines were used like most chemotherapeutic and cytotoxic drugs.

Cytokines are hormones of the immune system that were developed the way we develop most drugs in cancer, by giving people the maximally tolerated dose.” He pointed out that cytokine therapies, in contrast, should likely follow the “Goldilocks principle.” “There are optimal doses for hormones.

Think of diabetes and insulin; there can be too much, or too little, or just right.” This is likely the reason behind the initial poor performance of recombinant IFN-α and IL-2. “The IL-2 doses gave us approximately 30 seconds of benefit and then the body started fighting us at every additional treatment, trying to shut down all of the toxicity we were generating.”

Here we discuss some of the most recent trial results and upcoming trials designed to evaluate new cytokine-based cancer therapies.

SOT101 is a fusion protein consisting of IL-15 linked to the Sushi domain of IL-15 receptor α that displayed a favorable safety profile and promising preliminary efficacy signals in a phase 1 study enrolling patients with advanced solid tumors (NCT04234113).4 This dose-escalation study is evaluating subcutaneous injections of SOT101 as monotherapy and in combination with pembrolizumab (Keytruda) administered in 3-week cycles.

A total of 51 patients have been treated: 30 with monotherapy and 21 with combination therapy.

 Pyrexia, chills, lymphopenia, anemia, transaminase elevation, and vomiting were the most common treatment-emergent adverse events (TEAEs), and all were transient.

 The majority of TEAEs were mild (grade 2 or less), and no treatment-related deaths were observed. A recommended phase 2 dose of 12 μg/kg for SOT101 has been determined.

The observed clinical benefit rate of SOT101 monotherapy was 38% among the 13 patients who received doses of 6 μg/kg to 12 μg/kg. Dose ranges for the monotherapy group were 0.25 μg/ kg to 15 μg/kg and 1.5 μg/kg to 12 μg/kg in the combination therapy group.4 A single patient with ICI-refractory squamous cell carcinoma of the skin experienced a partial response (PR; duration, 46 days), and 4 ICI-pretreated patients had stable disease (SD) ranging from 33 to 183 days.

 The median duration of clinical benefit was 190 days, with a median duration on treatment of 84 days.

The observed clinical benefit rate in patients receiving the pembrolizumab combination was 63% across all SOT101 doses.

 A single ICInaïve patient with mesothelioma experienced a complete response (CR) that was ongoing in cycle 5 as of January 2022.

 Three patients (2 ICI-pretreated) experienced PR (range, 51-232 days; 2 ongoing), and 5 patients (3 ICI pretreated) experienced SD (range, 92-340 days; 2 ongoing).

 The preliminary median duration of clinical benefit for combination treatment was 131 days, with a median treatment duration of 113 days (range, 7-429).

Intravesical administration of N-803 (Anktiva), a mutant IL-15–based immunostimulatory fusion protein complex (IL15RαFc), in combination with BCG in BCG-naïve patients with non–muscle-invasive bladder cancer (NMIBC) resulted in CRs in all patients for the study duration of 24 months in a recent phase 1b trial.5 This open-label, 3-cohort, multicenter trial (QUILT-3.032; NCT03022825) evaluating intravesical injection of N-803 with BCG in BCG-unresponsive high-grade NMIBC resulted in significant response rates and clinical outcomes.

Primary end points included CR at any time for patients with carcinoma in situ (CIS) and disease-free survival (DFS) rate at 12 months for patients with papillary NMIBC.

 A total of 160 patients 83 CIS, 77 papillary) have been enrolled to date, with a median of 12 prior BCG doses and a mean of 4 prior transurethral resections of bladder tumors.5 Patients with CIS had a CR rate of 71% (59/83), and the median duration of CR was 24.1 months; 91% avoided cystectomy and 96% achieved 24-month bladder cancer–specific progression-free survival (PFS; progression was defined as progression to MIBC).

 Patients with papillary disease had a 57% 12-month DFS rate and a 48% 24-month DFS rate, and 95% avoided cystectomy. 5 For responders requiring cystectomy (n = 4), median time to procedure was 12.9 months vs 7.8 months for nonresponders requiring cystectomy (n = 8).

No grade 4 or 5 treatment-related AEs (TRAEs) were observed, and the most common grade 1 or 2 TRAEs were dysuria (22%), pollakiuria (19%), hematuria (18%), fatigue (16%), and urgency (12%).5 Bladder cancer–specific overall survival (OS) at 2 years was 99% in patients with BCG-unresponsive NMIBC, and the activity of N-803 was confined to the bladder, with no measurable systemic levels.5 The developer of N-803, ImmunityBio, Inc, submitted a biologics license application to the FDA on May 23, 2022, for the use of N-803 with BCG to treat BCG-unresponsive NMIBC CIS with or without Ta or T1 disease.

SRF388 is a fully human IgG1 blocking antibody that targets IL-27, thereby preventing  it from upregulating inhibitory immune checkpoint receptors and downregulating proinflammatory cytokines.

 A phase 1 dose-escalation study recruited patients with advanced treatment-refractory solid tumors to determine preliminary safety and recommended phase 2 doses of SRF388 as monotherapy and in combination with pembrolizumab (NCT04374877).

A total of 29 patients were enrolled to receive SRF388 monotherapy doses of 0.003 to 20 mg/kg; 62% were female, 80% had prior PD-L1 blockade, and 48% had 4 or more prior therapies.

 The median age of patients was 64 years, with a median time on study of 9 weeks (range, 0-59).

 TRAEs were observed in 21% of patients, with only treatment-related fatigue reported in more than 10% (n = 3)  and all AEs were considered low grade.

A confirmed PR was observed in a patient with highly treatment-refractory NSCLC at 8 weeks; the PR was durable for 20 weeks.

 SD was observed in 9 patients (31%) at 8 weeks, with 6 of 9 maintaining durable disease control at 6 months.

 Durable disease control for more than 20 weeks was observed in 43% (n = 3/7) of patients with clear cell renal cell carcinoma (ccRCC). The pembrolizumab safety cohort (n = 10) and stage 1 of the ccRCC monotherapy expansion (n = 17) have fully enrolled.

 A phase 2 dose of 10 mg/kg was selected based on study results, with dosing every 3 to 4 weeks.

TransCon IL-2 β/γ is a novel, longacting prodrug with sustained release of an IL-2Rβ/γ–selective IL-2 analogue designed to address the shortcomings of current IL-2 cancer immunotherapies such as aldesleukin (a recombinant form of IL-2; Proleukin). The severe AEs associated with these agents, such as cytokine release syndrome and vascular leak syndrome, likely result from the frequent high doses necessitated by their short half-life and high peak serum concentration (C

In a preclinical study, cynomolgus monkeys received 2 to 3 doses of TransCon IL-2 β/γ intravenously every 2 weeks.8 TransCon IL-2 β/γ produced a robust increase in absolute lymphocyte count (ALC) driven by potent activation and expansion of CD8-positive (+) T cells, natural killer (NK) cells, and γδ T cells relative to CD4+ T cells and T-regulatory cells (Tregs).

 After administration, investigators observed the following increases compared with predose levels: greater than 20-fold in ALC, 19-foldin CD8+ T cells, 50-fold in effector memory CD8+ T cells, 18-fold in NK cells, and 400-fold in γδ T cells.

 TransCon IL-2 β/γ had a long effective half-life (> 30 hours) and a low Cmax. Levels of IL-5, IL-6, IFN-β, and tumor necrosis factor-α remained low, whereas an induction of soluble CD25 and the chemokine MCP-1 was seen.

 No signs of vascular damage, tissue necrosis, pulmonary edema, or eosinophilia were observed on histopathology.

These results suggest the potential for substantial improvement in the therapeutic index beyond that of current recombinant IL-2 therapies. TransCon IL-2 β/γ is being evaluated in the phase 1/2 IL Believe trial (NCT05081609) as monotherapy and in combination with pembrolizumab (see page 35).

Markovic explained the concept behind this new IL-2–based therapy. “One potential explanation for the failure of the original IL-2 treatment [aldesleukin] was that it engaged the high-affinity [α/β/γ] receptor, thereby preferentially stimulating [immunosuppressive Tregs]. If we remove the [part of IL-2 that] binds to the high-affinity receptor and [bind it to] the low-affinity [β/γ] receptor [instead], there is potential for greater benefit.”

However, Markovic has his doubts about this approach: “I think one of the reasons high-dose IL-2 failed is not just because it triggered Tregs but because it triggered such an explosion of immune activation that the entire immunoregulatory system [was stimulated] to shut it down.”

Modakafusp alfa (TAK-573) is a unique immune-targeting cytokine that is also

undergoing evaluation. The agent targets CD38-expressing cells with attenuated  IFNα2b moieties.9 Increased target selection is achieved by the specificity for CD38 and reduced IFN receptor–binding affinity of this agent’s 2 attenuated IFNα2b molecules, which are genetically fused to the Fc portion of a humanized, anti-CD38, IgG4 monoclonal antibody.

 Although modakafusp alfa has shown strong clinical responses and immune activation in refractory/relapsed multiple myeloma, a phase 1b/2 study (NCT04157517) was conducted to investigate its safety and preliminary efficacy in metastatic solid tumors.

A total of 21 patients with a median age of 63 years and 3 median prior lines of therapy, 71.4% of whom had gastrointestinal malignancies, received a dose of modakafusp alfa 0.1 to 1.5 mg/kg every 3 weeks.

 TRAEs occurred in 81% of patients and included infusion-related reactions (52.4%), chills (47.6%), and nausea (33.3%); grade 3 or greater TRAEs were reported in 42.9% of patients and included neutropenia (14.3%) and hypertension (9.5%).

A greater than dose-proportional exposure increase was observed across the dosing range, with no exposure accumulation associated with dosing every 3 weeks. All patients (100%) developed posttreatment antidrug antibodies; the investigators are currently characterizing this response and its impact.

 A best response of SD was observed in 7 of the 14 response-evaluable patients, including 1 patient with cutaneous melanoma who experienced 21% target lesion reduction.

 These results provided proof of mechanism and a recommended phase 2 dosage of 1.0 mg/kg every 3 weeks, which will be tested in combination with an ICI in selected tumor types.

Phase 2a of the ongoing KEYNOTE A60 trial (NCT04332653) is evaluating NT-I7 (efineptakin alfa), recombinant human IL-7 protein fused to a hybrid Fc region of a human antibody, in combination with pembrolizumab. Patients with ICI-naïve relapsed/ refractory microsatellite-stable colorectal cancer (MSS-CRC) or pancreatic ductal adenocarcinoma (PDAC) are eligible, as are those with ICI-treated triple-negative breast cancer (TNBC), non–small cell lung cancer (NSCLC), and SCLC.10 The primary end point is objective response rate, with secondary end points of duration of response, disease control rate, PFS, and OS.

Ninety-two patients with metastatic or locally advanced cancer who had received a median of 3 prior treatments were enrolled as of January 2022 (PDAC, n = 32; MSS-CRC, n = 28; NSCLC, n = 22; TNBC, n = 6; SCLC, n = 4).10 Results from the 71 evaluable patients are shown in the 

A sustained peripheral lymphocyte increase of approximately 3 times the baseline level was observed in all arms.10 NT-I7– related AEs occurred in 67 (72.8%) of the 92 treated patients, including 56.5% grade 1 or 2, 14.1% grade 3, and 2.2% grade 4.10 These results suggest the addition of NT-I7 to ICI therapy can help overcome primary resistance to ICIs in ICI-naïve MSS-CRC and PDAC, and acquired ICI resistance in ICI-treated TNBC, NSLC, and SCLC.

AU-007 is a computationally designed monoclonal antibody that binds to IL-2 on its CD25-binding epitope, resulting in redirection of IL-2 (either endogenously produced or exogenously administered, eg, as aldesleukin) toward activation of immune-stimulating T effector and NK cells while diminishing Treg activation and expansion.

AU-007 also converts a Treg-mediated autoinhibitory loop into an immune-stimulating loop by binding and redirecting newly secreted endogenous IL-2.

 A recently initiated phase 1/2 study (CP-AU-007-01; NCT05267626) is evaluating the safety, tolerability, and initial efficacy of AU-007 alone or combined with aldesleukin in patients with advanced solid tumors, including melanoma, RCC, and 17 others.

 Phase 1 consists of 3 escalation arms. In arm A, patients receive escalating doses of AU-007 monotherapy every 2 weeks; in arm B, a single dose of aldesleukin is received with the initial AU-007 dose; and in arm C, patients receive combination therapy with both drugs every 2 weeks.

 Matching expansion cohorts of up to 20 patients each are planned for phase 2.

IL Believe is a multicenter, first-inhuman, phase 1/2 study in 3 parts evaluating

TransCon IL-2 β/γ in adults with locally advanced or metastatic solid tumors.

 Patients who are included need 1 or more measurable lesions per RECIST 1.1 and an ECOG score of 2 or less.

Evaluating safety and tolerability and defining the maximum tolerated dose along with the recommended phase 2 dose are primary objectives.12 Parts 1 and 2 (phase 1; n = 15 patients each) involve dose escalation of TransCon IL-2 β/γ alone or in combination with pembrolizumab.12 Part 3 is a phase 2 evaluation of the preliminary clinical efficacy of TransCon IL-2 β/γ combined with standard-of-care chemotherapy.

RTX-224 is an off-the-shelf allogeneic therapy consisting of red blood cells engineered to express the costimulatory 4-1BB ligand and IL-12.13 RTX-224 distribution is restricted to the vasculature and spleen, which should limit toxicities previously observed with agonist 4-1BB monoclonal antibodies and recombinant IL-12, which are distributed systemically.

RTX-224-01 (NCT05219578) is a phase 1/2, first-in-human, multicenter, doseescalation and -expansion study of RTX-224 in patients with relapsed/refractory urothelial cancer, squamous cell carcinoma of the head and neck, NSCLC, TNBC, and cutaneous melanoma.

Approximately 28 patients will be enrolled to evaluate safety, tolerability, pharmacokinetics, pharmacodynamics, and antitumor activity.

XTX202 is a masked, tumor-selective recombinant human IL-2 protein designed to be pharmacologically active (unmasked by matrix metalloproteases) only in tumor tissue and inactive in the systemic circulation.

The agent was designed with the goal of producing a localized antitumor immune response and limiting exposure of the active form in nontumor tissue.

 XTX202-01 (NCT05052268) is a first-in-human phase 1/2 study of XTX202. Phase 1 will determine the recommended phase 2 dose, and phase 2 will evaluate the efficacy of XTX202 monotherapy in patients with metastatic RCC and unresectable or metastatic melanoma.

Markovic compared the immune system to an orchestra, with 800 to 1000 different regulators working in concert. The interplay between these regulators driving acute or chronic inflammation remains to be elucidated. “I think cytokines have a role, but I also sense chemokines have a bigger role, though they are much more difficult to target,” he stated concerning ICIs and cytokine-based therapies.

He added that the first iteration of cytokine-based therapies taught investigators that increasing doses until toxicity occurs is not the answer but focusing on biological measurements is. Herein lies another issue for Markovic and colleagues: “We measure many parameters, but finding those most relevant remains a challenge.” He again equated this to diabetes and insulin; without assessing blood glucose levels, you cannot determine an adequate dose of insulin. Therefore, what should be measured to determine the adequate dose of cytokines?

Markovic remains proud of the advances made in immunotherapy cancer treatments and has significant hope for the future. “The more we understand and unravel the complexities of multicytokine regulation in blood—what happens in the lymph node downstream of cancer, what is happening in the tumor itself, why the immune system is allowing the tumor to grow instead of eliminating it—[the closer we will get to highly efficacious and safe cytokine-based cancer therapies]. We’ve learned more in the past decade than in the previous 5 decades,” summarized Markovic.

“I think working through those conceptual frames with novel agents will get us there, and science will win out.”

1. Berraondo P, Sanmamed MF, Ochoa MC, et al. Cytokines in clinical cancer immunotherapy. Br J Cancer. 2019;120(1):6-15. doi:10.1038/s41416-018-0328-y

2. Xue D, Hsu E, Fu YX, Peng H. Next-generation cytokines for cancer immunotherapy. Antib Ther. 2021;4(2):123-133. doi:10.1093/abt/tbab014

3. Rallis KS, Corrigan AE, Dadah H, et al. Cytokine-based cancer immunotherapy: challenges and opportunities for IL-10. Anticancer Res. 2021;41(7):3247-3252. doi:10.21873/anticanres.15110

4. Garralda E, Naing A, Galvao V, et al. Interim safety and efficacy results from AURELIO-03: a phase 1 dose escalation study of the IL-2/IL-15 receptor superagonist SOT101 as a single agent and in combination with pembrolizumab in patients with advanced solid tumors. J Clin Oncol. 2022;40(suppl 16):2502-2502. doi:10.1200/JCO.2022.40.16_suppl.2502

5. Chamie K, Chang SS, Gonzalgo M, et al. Final clinical results of pivotal trial of IL-15RαFc superagonist N-803 with BCG in BCG-unresponsive CIS and papillary nonmuscle-invasive bladder cancer (NMIBC). J Clin Oncol. 2022;40(suppl 16):4508-4508. doi: 10.1200/JCO.2022.40.16_suppl.4508

6. ImmunityBio submits biologics license application for N-803 Plus BCG for patients with BCG-unresponsive non-muscle invasive bladder cancer carcinoma in situ. ImmunityBio; May 23, 2022, 2022. Accessed July 11, 2022. https://bit.ly/3P2m3CZ

7. Naing A, Mantia C, Morgensztern D, et al. First-in-human study of SRF388, a first-in-class IL-27 targeting antibody, as monotherapy and in combination with pembrolizumab in patients with advanced solid tumors. J Clin Oncol. 2022;40(suppl 16):-2501. doi:10.1200/JCO.2022.40.16_suppl.2501

8. Rosen D, M.nsson Kvarnhammar A, Olling JD, et al. TransCon IL-2 β/γ, a novel long-acting prodrug with sustained release of an IL-2Rβ/γ-selective IL-2 analog, demonstrates improved pharmacokinetics and profound expansion of cytotoxic immune cells in non-human primates. J Clin Oncol. 2022;40(suppl 16l):2563-2563. doi:10.1200/JCO.2022.40.16_suppl.2563

9. Johnson ML, Abdul-Karim R, Sommerhalder D, et al. Dose escalation of a phase 1b/2 study of modakafusp alfa, an immune-targeting attenuated cytokine, in patients (pts) with metastatic solid tumors. J Clin Oncol. 2022;40(suppl 16):2503-2503. doi:10.1200/JCO.2022.40.16_suppl.2503

10. Naing A, Mamdani H, Barve MA, et al. Efficacy and safety of NT-I7, long-acting interleukin-7, plus pembrolizumab in patients with advanced solid tumors: Results from the phase 2a study. J Clin Oncol. 2022;40(suppl 16):2514-2514. doi:10.1200/JCO.2022.40.16_suppl.2514

11. Vasselli JR, Frentzas S, Weickhardt AJ, et al. Trial in progress: A phase 1-2, first-in-human, open label, dose escalation and expansion study of AU-007, a monoclonal antibody that binds to IL-2 and inhibits IL-2Rα binding, in patients with advanced solid tumors. J Clin Oncol. 2022;40(suppl 16):TPS2671-TPS2671. doi:10.1200/JCO.2022.40.16_suppl.TPS2671

12. Starodub A, Gabrail NY, Zhang Y, et al. IL believe: A phase 1/2, open-label, dose escalation and dose expansion study of TransCon IL-2 β/γ alone or in combination with pembrolizumab or standard-of-care chemotherapy in patients with locally advanced or metastatic solid tumors. J Clin Oncol. 2022;40(suppl 16):TPS2695-TPS2695. doi:10.1200/JCO.2022.40.16_suppl.

13. Spira AI, Johnson ML, Ren A, et al. A phase 1/2 study of RTX-224, an engineered red blood cell expressing 4-1BB ligand and membrane- bound IL-12, for the treatment of patients with select advanced solid tumors. J Clin Oncol. 2022;40(16_suppl):TPS2680-TPS2680. doi:10.1200/JCO.2022.40.16_suppl.TPS2680

14. McKean M, Powderly JD, Duncan M, et al. A first-in-human, multicenter, phase 1/2, open-label study of XTX202, a masked and tumor-selective recombinant human interleukin-2 (IL-2) protein, in patients with advanced solid tumors. J Clin Oncol. 2022;40(suppl 16):TPS2697-TPS2697. doi:10.1200/JCO.2022.40.16_suppl.TPS2697

Current- generation cytokines are being engineered to extend their half-life and improve tumor targeting using polyethylene glycol conjugation, fusion to tumor-targeting antibodies, alterations of cytokine/cell receptor–binding affinity, and other strategies.

Interleukin-Based Treatments Find Footing in Ongoing Research 

Circulating tumor (ctDNA) technology has the potential to improve diagnosis, treatment, and monitoring of patients with colorectal cancer (CRC).

Research is ongoing to explore all potential uses for ctDNA to help reduce the global burden of CRC.

Although CRC is preventable and curable when diagnosed early, most patients receive diagnoses at advanced stages.

 Early and accurate detection of CRC tumors is critical for success, and biopsy with tissue pathology remains the gold standard for diagnosis despite the associated costs, invasive and uncomfortable nature, technical limitations based on tumor location, and ineffi ciency in targeting tumor subpopulations. Fragmented DNA in bodily fluids originating from a primary or metastatic tumor, known as ctDNA, has the potential to provide a simple, cost-effective, non-invasive (or limited) approach to diagnosing and characterizing CRC and is being evaluated and incorporated in numerous clinical trials. 

Current assays for the detection of ctDNA are either tumor informed or tumor uninformed.

 Newer tumor-uninformed liquid biopsies are cancer specific, require a simple blood sample, and pair an epigenomic signature with the ctDNA component to identify neoplasm-specific genetic and epigenetic abnormalities.

 Tumor-informed assays are patient specific, requiring a matched sample of the tumor to generate a custom genetic panel for identifying ctDNA within the patient.

 Emerging ctDNA technologies provide opportunities for minimal residual disease (MRD) detection in patients with early-stage disease, enhanced risk stratifi cation, identification of tumor heterogeneity and genomic evolution, and improved ctDNA-guided use of neoadjuvant and adjuvant therapies.

Although much of the research for ctDNA in CRC to date has been focused on MRD and the metastatic setting, there are also potential applications in the adjuvant setting.

 The detection of ctDNA after surgery correlates with a high risk of recurrence. After curative resection, 10% to 15% of stage II patients will have detectable ctDNA compared with almost 50% of stage IV patients.

In addition, ctDNA is proving a valuable prognostic indicator. One meta-analysis of patients with CRC determined a strong association between ctDNA and recurrence-free survival (RFS; HR, 2.78; 95% CI, 2.08-3.72) and overall survival (OS; HR, 3.03; 95% CI, 2.51-3.66), suggesting the presence of ctDNA as a negative prognostic indicator for RFS and OS.

Two experts discussed the latest research and opinions regarding ctDNA within CRC in interviews with 

. Pashtoon M. Kasi, MD, MS, is director for colon cancer research and precision medicine and director of liquid biopsy research at the Sandra and Edward Meyer Cancer Center at Weill Cornell Medicine Englander Institute of Precision Medicine, New York, New York. Tanios S. Bekaii-Saab, MD, is medical director of the Cancer Clinical Research Offi ce and vice chair and section chief for medical oncology in the Department of Internal Medicine at Mayo Clinic in Phoenix, Arizona. 

The CIRCULATE-Japan collection of trials aims to refine precision medicine for patients with CRC by improving adjuvant therapy choice through the utilization of ctDNA technology.

 The study involves the GALAXY, VEGA, and ALTAIR trials. 

GALAXY is a prospective large-scale registry trial designed to monitor ctDNA for patients with stage II to IV or recurrent CRC who are candidates for complete resection.

The VEGA trial is a randomized phase 3 trial open to patients from GALAXY with high-risk stage II or low-risk stage III colon cancer who are ctDNA negative 4 weeks postoperatively, and aims to determine whether surgery alone is noninferior to standard post-operative therapy with capecitabine plus oxaliplatin for 3 months.

 The ALTAIR trial (NCT04457297) is a double-blind phase 3 trial open to ctDNA positive patients from the GALAXY trial and will evaluate the use of trifl uridine/tipiracil (TAS-102, Lonsurf) compared with placebo. Collectively, the CIRCULATE-Japan trials involve both de-escalation and escalation trials for ctDNA-negative and ctDNA-positive patients to help determine the postoperative prognostic and/or predictive value of ctDNA. 

A total of 1365 patients with CRC were enrolled in the GALAXY study, comprising 116 with stage I disease, 478 with stage II, 503 with stage III, and 268 with oligometastatic resectable stage IV disease.

 Six-month disease-free survival (6M-DFS) rate analysis excluded patients enrolled in VEGA or ALTAIR. The ctDNA dynamics (positive or negative) were evaluated from week 4 to week 12 to assess changes from baseline. 

At a median follow-up of 11.4 months, a significant difference was observed between the “positive to negative” and “positive to positive” groups with an HR of 15.8 (95% CI, 5.7-44.2; 

< .001), and compared with the “negative to positive” group with (HR, 9.2; 95% CI, 3.0-28.4;

Ninety-six of 183 evaluable patients who were positive at 4 weeks and had an available 12-week status were treated with standardof-care (SOC) adjuvant chemotherapy (ACT).

Across most subgroups, the ctDNA clearance rate was higher for those who received ACT vs those without ACT. However, in patients with stage IV disease (n = 68), the clearance rate was 23% in patients who received ACT vs 53% in those who did not receive ACT (

 < .001), whereas the rates were 68% and 29% (

< .001), respectively, among patients with stage III disease (n = 90). The cumulative incidence of ctDNA clearance by 24 weeks was significantly higher in the ACT group vs the non-ACT group (68% vs 10%; HR, 9.3; 95% CI, 4.6-18.9; 

< .001) and the 6M-DFS rate among patients who were ctDNA-positive at 4 weeks was significantly higher in the ACT vs non-ACT group across disease stages. 

GALAXY is the largest MRD study to date and demonstrates a relationship between ctDNA dynamics and improved clinical outcomes, suggesting ctDNA can help identify patients more likely to benefit from ACT therapy. VEGA and ALTAIR are ongoing and will help further establish ctDNA-guided ACT strategies. 

“ctDNA is a dynamic variable,” stated Kasi, “and with GALAXY we were able to see patients who were positive convert to negative with ‘mop-up’ chemotherapy. The RFS was the same for those who remained negative and those who were positive but converted to negative.” 

Kasi and Bekaii-Saab were interested in the data regarding ACT. “The one group that is very intriguing is the ctDNA-negative cohort,” Bekaii-Saab commented, “where there wasn’t much difference between patients receiving ACT and those who did not. [Although] this study wasn’t randomized, it indicates a possible predictive value for this test.” 

Kasi added, “The GALAXY results are very provocative and give rationale for future de-escalation evaluation and support ongoing studies, such as CIRCULATE-US.” ctDNA 

A retrospective, single-center cohort study evaluated a ctDNA assay (Signatera; Natera) compared with standard radiographic imaging and carcinoembryonic antigen (CEA) level measurements as evaluation of recurrence in patients with resected CRC.

 Of the 48 patients included in the study, 15 had disease recurrence. The sensitivity of the ctDNA assay (53.3%; 95% CI, 27.4%-77.7%) was numerically less than imaging alone (60.0%; 95% CI, 32.9%-82.5%) and the combination of imaging with CEA measurement (73.3%; 95% CI, 44.8%-91.1%; 

= .55). Additionally, there was no significant differenced noted in the median time to detection of recurrence between ctDNA (14.3 months), imaging (15.0 months), or the combination of imaging with CEA (15.0 months). Although these results are retrospective, taken from a single-center, and comprise a relatively small patient population, they suggest that ctDNA assays may not improve surveillance compared with imaging and CEA levels. However, this study did find a low correlation between low-burden lung recurrence and negative ctDNA results that should be further investigated in larger prospective studies. 

However, Bekaii-Saab and Kasi were equally surprised by the amount of attention this study received, given bias, vague end points, and lack of blindness. Bekaii-Saab commented, “I personally put it in the interesting observation category with no real practice application. It isn’t just how early you detect cancer; it’s [whether] a difference can be made in survival outcomes.”

A small study consisting of 14 patients with metastatic CRC who were treated using chemotherapy alone or in combination with a targeted agent indicates ctDNA analysis of plasma samples obtained within 4 to 10 weeks after treatment initiation can support molecular response assessments.

A consistent reduction in mean variant allele fraction (mVAF) as assessed by the GuardantOMNI 500-gene liquid biopsy assay was observed in 11 of 13 evaluable patients. One outlier had an initial increase in mVAF followed by consistent decrease thereafter, whereas the second outlier had elements of KRAS alterations that were not previously detected by tissue biopsy. These results suggest patient-centric liquid biopsy has an advantage in detecting potential resistance mutations prior to therapy over genetic testing of tumor samples. 

Disease Monitoring During and After Treatment 

A recent study evaluating ctDNA via ultradeep sequencing found ctDNA measurements correlated with the stage of disease, with lower levels of ctDNA concentration in stage I patients vs stage IV patients.

Additionally, ctDNA testing identified 112 alterations in 14 genes in 69% of patients studied (69/100). The most frequent mutations were in APC (29%),

 family mutations were identified in 37 patients; 89% of those identified by tissue sampling were identified via ctDNA testing. Overall, ctDNA was found to be a greater performance predictor than CEA measurement, and ctDNA mutation levels reflected 5 patients’ clonal evolution and clinical outcomes. Therefore, monitoring disease progression and clonal evolution via ctDNA testing has the potential to improve patient management. 

ctDNA Inadequate in Determining Recurrence 

A recent retrospective study evaluated data from 595 patients from 3 earlier CRC clinical trials that included information on high-risk factors and postsurgical ctDNA status.

 RFS in patients with high-risk stage II CRC who were ctDNA negative did not differ between patients who did or did not receive ACT (

 = .1758) despite high-risk patients having significantly reduced RFS compared with low-risk patients (HR, 3.283; 95% CI, 1.398-14.18; 

 = .0116). These results suggest ctDNA alone is not enough to suggest which patients will experience recurrence and post-operative ctDNA status, and risk factor combinations may need to be assessed for more accurate therapeutic decisions. 

“Currently, negative predictive value can be problematic and varies,” stated Bekaii-Saab. “We would want to rely on these tests, because of the potential to prevent unnecessary chemotherapy exposure if negative. However, if the negative predictive value isn’t as accurate then it becomes more challenging for decision-making and conversations with the patient.” 

Kasi added, “For the negative patient, you can add 2 additional points for prolonged tumor assessment, increasing the sensitivity and specificity.” 

The BESPOKE CRC trial (NCT04264702) is a prospective, multicenter trial evaluating the clinical impact of serial testing with Signatera in patients with resectable CRC, including as a potential guide for adjuvant therapy use.

 Eligible patients have undergone surgery for stage I to IV CRC and have formalin-fixed paraffin-embedded tissue available. Also, ctDNA will be evaluated using the Signatera test, and patients may be recommended for post-operative systemic therapy or observation with a follow-up of up to 2 years. A control arm will consist of matched stage I to IV CRC cases with a minimum of 2 years’ follow-up data available. Currently, the study has over 100 participating locations and 1000 patients enrolled of the 2000 enrollment target with an interim analysis planned for later this year. 

Prospective Dutch Colorectal Cancer Cohort Study 

The Prospective Dutch Colorectal Cancer cohort study (NCT02070146) will analyze patients with CRC in nearly all hospitals within the Netherlands to further research in the field; the trial includes subcohorts for varying stages of disease.

Under this trial umbrella, the MEDOCC-CrEATE study aims to evaluate how many patients with stage II colon cancer who are ctDNA-positive postoperatively will accept ACT, and whether ACT reduces the rate of recurrence.

The study will enroll 1320 patients without an indication for ACT who will be randomized 1:1 into the experimental and control arms. In the experimental arm, ctDNA is identifi ed using the PGDx elio platform, and patients with detectable ctDNA are offered 8 cycles of adjuvant capecitabine and oxaliplatin. The control arm and patients without detectable ctDNA will receive standard follow-up care. The proportion of patients accepting ACT when ctDNA is detected is the primary end point with secondary end points of 2-year and 5-year response rates. 

The PROVENC3 study aims to determine the prognostic value of post-operative ctDNA in stage III colon cancer in patients treated with ACT.

 The observational study consists of 267 patients with stage III colon cancer who will be evaluated by PGDx elio plasma resolve assay to determine the association between postoperative ctDNA and progressionfree survival and OS. A ctDNA-guided interventional trial will be designed based on this study’s results. 

The CIRCULATE-US study (NCT05174169) will evaluate approximately 2000 patients with stage II or III colon cancer post resection.

Research is ongoing to explore all potential uses for circulating tumor DNA to help reduce the global burden of colorectal cancer.

Benjamin Holmes, DVM | Authors

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