Cancer Associated Macrophage-Like Cells Research Articles

Abstract 3297: Expression of pd-l1 on circulating stromal cells predicts immunotherapy response in unresectable non-small cell lung carcinoma after definitive chemoradiotherapy

Abstract Background: Circulating stromal cells (CStCs) have been found to be common in the peripheral blood of cancer patients and hypothesized to be a blood based biomarker for monitoring cancer treatment. It has previously been described that the dynamic changes of PD-L1 expression during chemoradiotherapy (CRT) could be tracked using circulating stromal cells. However, how these changes relate to PD-L1/PD-1 immunotherapy (IMT) response is unstudied. We prospectively monitored PD-L1 expression in 2 cell types found in circulation (Circulating Tumor Cells [CTCs] and Cancer Associated Macrophage-like Cells [CAMLs]) in locally advanced non-small cell lung cancer (NSCLC) patients (pts) treated with Atezolizumab (Atezo) after definitive CRT (n=39) or in pts with CRT alone (n=40). Methods: A 2 year single blind prospective study was undertaken in pts with locally advanced NSCLC in 40 patients treated with CRT alone, or from a phase II DETERRED trial (NCT02525757) where Atezo was added for 1 year after completing CRT (n=10), or concurrently and after CRT (n=30). Samples from 39 of 40 pts from the DETERRED study were available for analysis. Baseline blood samples (7.5 ml) were drawn prior to start of CRT (T0), and a second sample was drawn ~1 month after completing CRT (T1), but prior to induction of Atezo. Blood was processed by CellSieve™ microfilters; stained for cytokeratin/PDL1/CD45 to identify CTCs and CAMLs. PD-L1 intensity was measured and grouped by 4 scores: 0-negative, 1-low, 2-medium, & 3-high. PD-L1 levels from circulating cells were used to evaluate PFS and OS. Significance was assessed by log-rank testing. Results: At least one CTC and/or CAML was found in 91% of available T0 samples and 96% of available T1 samples. In the 40 patients that received CRT alone, 35 pts had measurable cells at T0 and PD-L1 expression in CStCs was low (0-1) in 20 pts and high (2-3) in 15 pts. Further, at T1 PD-L1 expression in CStCs was low in 12 pts and high in 28 pts, with no relationship to PFS (T0 HR=1.1, 95%CI 0.5-2.8, p=0.96 and T1 HR=0.8, 95%CI 0.3-2.0, p=0.82) or OS (T0 HR=0.8, 95%CI 0.2-2.8, p=0.99 and T1 HR=1.1, 95%CI 0.3-3.5, p=0.84). In the patient arm that received Atezo at T0, PD-L1 expression in CStCs was low in 21 pts and high in 17 pts, with no relationship to PFS (HR=0.5, 95%CI 0.2-1.7, p=0.18) or OS (HR=1.6, 95%CI 0.4-6.0, p=0.75). However at T1, pts with high PD-L1 had significantly improved PFS response to Atezo (HR 5.4, 95%CI 1.7-17.0, p=0.009), and improved OS (HR 21.5, 95%CI 4.5-91.9, p<0.001). Conclusions: PD-L1 expression in tissue is often not used in IMT treatment decisions due to limited correlation with clinical responses. However, it has been suggested that sequential monitoring of PD-L1 expression in circulating stromal cells in blood may predict for patients who will respond to IMT. These data suggests that CRT altered PD-L1 expression, and monitoring dynamic changes of PD-L1 in CStCs may predict immunotherapy effectiveness in NSCLC after CRT. Citation Format: Daniel L. Adams, Jianzhong He, Yawei Qiao, Kirby P. Gardner, John V. Haymach, Anne S. Tsao, Ashvathi Raghavakaimal, Cha-Mei Tang, Alexander Augustyn, Steven H. Lin. Expression of pd-l1 on circulating stromal cells predicts immunotherapy response in unresectable non-small cell lung carcinoma after definitive chemoradiotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3297.

Abstract CT233: Phase Ib/II study of leronlimab (PRO 140) combined with carboplatin in CCR5+ mTNBC patients

Abstract Introduction: Triple Negative Breast Cancer (TNBC) represents the most deadly form of invasive disease. It is associated with clinical and pathological features of highly proliferative and rapidly spreading cancer demonstrating higher incidence in younger women, particularly of African-American ethnicity and accounting for a disproportionately high percentage of early development of metastatic disease and breast-cancer-related death. While chemotherapy remains the main treatment option for both primary and metastatic TNBC (mTNBC) there is no optimized therapy for its management due to tumor heterogeneity, leaving the condition with an unmet medical need. Recent preclinical research demonstrated an important role for chemokine receptor type 5 (CCR5) in modulating cell migration and immune microenvironment suggesting a potential new therapeutic target in mTNBC [Jiao X et al, Can Res 2018;78:1657-71]. Leronlimab (PRO 140) is a humanized IgG4,к monoclonal antibody (mAb) to the C-C chemokine receptor type 5 (CCR5). Leronlimab has been administered and generally well tolerated in more than 750 healthy and HIV-1 infected individuals in Phase I/II/III studies. An ongoing Phase Ib/II study is being conducted to assess the safety and clinical outcomes of leronlimab combined with carboplatin in patients with untreated CCR5+ metastatic Triple Negative Breast Cancer (mTNBC). Methods: Eligible patients are required to have confirmed HER2 negative, ER <1%, PR<1% and demonstrate CCR5+ by immunohistochemistry assay (IHC) (>10% of primary or metastatic cancer cells shows membranous staining and/or high predominance of CCR5+ tumor-infiltrating leukocytes (see representative IHC staining images below) Phase Ib of the CD07_TNBC study is a multicenter, single arm, dose escalation phase with 3 dose levels of leronlimab administered in combination with a fixed dose of carboplatin at AUC 5. The starting dose is 350 mg with escalation to 525 mg and 700 mg in the absence of dose-limiting toxicities. Leronlimab is administered as subcutaneous injection in the abdomen weekly and can be self-administered by subjects at home after proper training by a healthcare professional. Carboplatin is administered at AUC 5 every 3 weeks. The maximum tolerated dose (MTD) of leronlimab determined during the Phase Ib portion will be administered to 30 patients during the Phase II portion of the study. Correlative Data: Blood samples are collected at Day 1 of each treatment cycle (every 21 days) to assess changes in circulating tumor cells (CTCs) and cancer associated macrophage-like cells (CAMLs) after treatment and to perform correlative analysis with CCR5 expression. Conclusions: Leronlimab (PRO 140), a CCR5 antagonist mAb, in combination with carboplatin is currently enrolling newly diagnosed mTNBC. The preliminary analysis shows safety and tolerability of the combination and continue to enroll patients with initial promising clinical activity, potentially suggesting the future availability of a new effective agent in the management of this serious condition. Citation Format: Massimo Cristofanilli, Milana Dolezal, Jay Lalezari, Hallgeir Rui, Bruce Patterson, Cha-Mei Tang, Daniel Adams, Qiang Zhang, Kazem Kazempour, Nader Pourhassan, Natalie Rabb, Kush Dhody. Phase Ib/II study of leronlimab (PRO 140) combined with carboplatin in CCR5+ mTNBC patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT233.

Giant Circulating Cancer-Associated Macrophage-Like Cells Are Associated With Disease Recurrence and Survival in Non–Small-Cell Lung Cancer Treated With Chemoradiation and Atezolizumab

BackgroundCancer-associated macrophage-like cells (CAMLs) are a potential peripheral blood biomarker for disease progression. This study used data from a phase 2 clinical trial to evaluate prognostic utility of CAMLs for locally advanced non–small-cell lung cancer treated with definitive chemoradiotherapy (CRT) and atezolizumab (DETERRED; ClinicalTrials.gov NCT02525757). Patients and MethodsSample collection occurred at baseline (T0), during CRT (T1), at end of CRT (T2), and at first follow-up (T3). CAMLs were captured and quantified by the CellSieve system using multiplex immunostaining. Giant CAMLs were defined as characteristic CAMLs ≥ 50 μm. Kaplan-Meier methodology estimated progression-free survival, distant failure-free survival, relapse-free survival, and overall survival at 30 months. ResultsThirty-nine patients were evaluated between December 2015 and March 2018. Median follow-up was 27 months. Most disease was stage III (85%) and comprised squamous-cell carcinoma (38%) or adenocarcinoma (59%). In total, 267 blood samples were analyzed. Giant CAMLs were identified in 57%, 60%, 64%, and 63% of patients at T0, T1, T2, and T3, respectively. Patients with giant CAMLs at T3, occurring at a median of 30 days after completion of CRT, had significantly worse distant failure-free survival (hazard ratio [HR] 4.9, P = .015), progression-free survival (HR 2.5, P = .025), recurrence-free survival (HR 2.4, P = .036), and overall survival (HR 3.5, P = .034) compared to patients with small or no CAMLs. ConclusionsPresence of giant CAMLs after CRT completion was associated with development of metastatic disease and poorer survival despite the use of maintenance immunotherapy. Monitoring CAMLs may help risk-stratify patients for adaptive treatment strategies.

Sequential monitoring of circulating stromal cells from blood is predictive of progression in NSCLC patients undergoing anti-PD-L1 therapy after definitive chemoradiation therapy.

3051 Background: Cancer Associated Macrophage-Like cells (CAMLs) are a recently described circulating stromal cell common in the peripheral blood of patients with solid tumors. In non-small cell lung carcinoma (NSCLC), patients with CAMLs ≥50µm after completion of chemoradiation therapy (CRT) have been shown to have worse progression free survival (PFS). However, with the recent addition of anti-PD-L1 therapies in conjunction with CRT as standard of care, it has never yet to be evaluated whether CAMLs remain predictive for monitoring progression in NSCLC patients post anti-PD-L1 therapy. Methods: A 2 year single blind prospective study was undertaken to test the relationship of ≥50µm CAMLs to PFS based on imaging in lung patients before and after induction of CRT and PD-L1. We recruited 104 patients with pathologically confirmed unresectable NSCLC Stage II (n = 14), Stage III (n = 83), Stage IV (n = 3), and locally recurrent disease (n = 4). Baseline (BL) blood samples were taken prior to start of therapy. A second time point blood sample (T1) was taken at the end of radiotherapy (~40 days). A third time blood sample (T2) was taken after induction of anti-PD-L1 therapy (e.g. Imfinizi, Keytruda, etc.). Blood was filtered by CellSieve filtration and CAMLs were quantified. Analysis by CAML size of < 49 µm or ≥50 µm was used to evaluate PFS hazard ratios (HRs) by censored univariate & multivariate analysis. Results: CAMLs were found in 87% of samples averaging 2.9 CAMLs/7.5mL sample. At BL, CAMLs ≥50 µm had similar PFS to patients with < 50 µm CAMLs (HR = 1.1 95%CI 0.6-1.95 p = 0.8661). However, after CRT (T1), patients with CAML size ≥50 µm had worse PFS (HR = 3.2, 95%CI 1.8-5.8 p = 0.0002). After induction of anti-PD-L1 therapy (T2), patients with ≥50 µm CAMLs also had worse PFS (HR = 2.8 95%CI 1.5-5.4 p = 0.0037). CAML size at BL was not accurate at predicting progression within 24 months; however ≥50 µm CAMLs after CRT or after 1 cycle of anti-PDL1 therapy was 71% accurate at predicting progression of disease. Conclusions: Our data suggests that in NSCLC, ≥50 µm CAMLs after completion of CRT or appearing after induction of anti-PD-L1 therapy appears to predict progressive disease. If validated, additional studies are needed to determine if CAMLs can serve as a significantly prognostic blood based marker for predicting survival in NSCLC patients early in the treatment regime.

Circulating stromal cells as a potential blood-based biomarker for screening invasive solid tumors.

3535 Background: Peripheral blood allows for a simple non-invasive method for isolating various cancer associated circulating stromal cells (CStCs) which may predict for cancer presence. Cancer Associated Macrophage-Like cells (CAMLs), a specific CStC, are phagocytic myeloid cells that derive from an immunological response to cancer and emanate from primary tumors. Using a filtration platform we screened the peripheral blood of untreated newly diagnosed cancer patients (n = 308) for CAMLs. In parallel, we screened patients with newly diagnosed non-malignant diseases, i.e. lupus, benign cysts, etc. (n = 39), and healthy control samples (n = 76). We found that CAMLs are highly prevalent (87%) in the blood of cancer patients, but uncommon in non-malignant conditions (20%) & absent in healthy individuals (0%). Methods: Anonymized peripheral blood were taken from 308 cancer patients after confirmation of invasive malignancy [stage I (n = 76), stage II (n = 73), stage III (n = 72), stage IV (n = 65) and unstaged non-metastatic (n = 22)] with pathologically confirmed lung (n = 65), pancreas (n = 53), breast (n = 52), prostate (n = 40), esophageal (n = 30), renal cell (n = 18), hepatocellular (n = 15), neuroblastoma (n = 10), melanoma (n = 8), and other (n = 17). Further, anonymized blood was taken from patients with untreated non-malignant conditions including benign breast masses (n = 19), lupus (n = 11), liver cirrhosis (n = 5), benign prostatic hyperplasia (BPH) (n = 3), and viral infection (n = 1); or from healthy control volunteers (n = 76). CAMLs were isolated from whole peripheral blood by the CellSieve™ microfiltration technique and defined as enlarged, multinuclear cells with cytokeratin and/or CD45/CD14 positive. Results: CAMLs were found in 87% of all cancer patients regardless of stage, ~5.4 CAMLs/7.5mL blood. Specifically, CAMLs were found in 80% of Stage I, 90% Stage II, 89% Stage III, and 97% Stage IV patients. No CAMLs were found in any healthy controls, but were found in 26% of benign breast masses, 18% of lupus, 0% of BPH and 0% of cirrhosis. In total, CAML sensitivity in cancer vs healthy was 87% (CI95% 82-90%), specificity = 100% (CI95% 95-100%), PPV = 100% (CI95% 100%), NPV = 67% (CI95% 58-71%). CAML sensitivity in cancer vs benign was 87% (CI95% 82-90%), specificity = 80% (CI95% 64-91%), PPV = 97% (CI95% 95-98%), NPV = 43% (CI95% 35-51%). Conclusions: CAMLs, a Circulating Stromal Cell subtype, is a sensitive blood based biomarker found in all stages of cancer that is rare in non-malignant conditions and absent in healthy individuals.

P2.01-93 Detection of Giant Cancer-Associated Macrophage-Like Cells After Concurrent Chemoimmunoradiation Is Associated with Poor Survival in NSCLC

Circulating cancer-associated macrophage-like cells (CAMLs) are a recently described stromal cell found in the peripheral blood of cancer patients that have been shown to be associated with disease progression. The presence of giant CAMLs (≥50 μm) was previously reported to be predictive of disease progression in multiple tumor types. In this phase II DETERRED trial of patients with unresectable locally advanced non-small cell lung cancer (NSCLC) treated with atezolizumab (atezo) combined with concurrent chemoradiation, we explored the utility of CAMLs in predicting progression based on blood samples collected throughout treatment and follow up. Patients were enrolled between February 2016 and April 2018. Patients were treated with carboplatin/paclitaxel (CP) and conventionally fractionated radiation therapy (60 – 66 Gy) with atezo, followed by CP-atezo, followed by maintenance atezo. Median follow up after the completion of concurrent chemoimmunoradiation (cCIRT) was 13.5 months. CAMLs were collected by obtaining peripheral blood from patients at baseline at the beginning, during, at the end, at first follow up, and final follow up after cCIRT. Blood was filtered using CellSieveTM filtration and CAMLs quantified. CAML size ≤49 μm or ≥50 μm was quantified with the observer blinded to clinical information. Relapse free survival (RFS), distant metastasis free survival (DMFS), progression free survival (PFS), and overall survival (OS) were analyzed at each time point. We evaluated 40 patients with unresectable locally advanced NSCLC and obtained a total of 375 samples. CAMLs were identified in 80.5% of samples, averaging 2.5 CAMLs per 7.5 mL sample. Patients with giant CAMLs (≥50 μm) compared to those with smaller CAMLs (≤49 μm) exhibited no difference in RFS, PFS, DMFS, or OS at baseline, during, or immediately after completion of cCIRT. Patients with detectable giant CAMLs at the first follow up (median time 29 days from completion of cCIRT) demonstrated significantly worse RFS (HR=11.79, 95% CI 4.27-32.56, p=0.0021), DMFS (HR=6.48, 95% CI 2.15-19.54, p=0.0009), and PFS (HR=12.47, 95% CI 4.66-33.37, p=0.0014) while OS trended towards statistical significance (HR=5.39 95% CI 1.33-21.81, p=0.071). Long term evaluation of patients with CAML ≥50 μm at first follow up (N=16) revealed 3 patients who converted to CAML<49 μm at last follow up. Patients who converted did not experience any relapses, while all 13 patients who continued to have CAML ≥50 μm experienced progression or death. Giant CAMLs at first follow up after completion of concurrent chemoimmunoradiation is predictive of disease progression and death. This may represent an immediate surrogate marker for poor response at the completion of definitive therapy. Long term follow up with maintenance immunotherapy indicates that a subset of patients convert from giant CAMLs to smaller CAMLs, with better outcomes than those that do not, suggesting that these patients may have derived benefit from maintenance immunotherapy. Continued prospective validation of CAMLs as a peripheral blood-based biomarker is needed to validate these findings.

Presence of Giant Circulating Cancer-Associated Macrophage-like Cells after Definitive Chemoradiation Predicts for Progression in Locally Advanced Non-Small Cell Lung Cancer Patients

Presence of Giant Circulating Cancer-Associated Macrophage-like Cells after Definitive Chemoradiation Predicts for Progression in Locally Advanced Non-Small Cell Lung Cancer Patients

Abstract 437: Combining circulating stromal cells with cell free DNA for increased sensitivity in profiling oncogenic mutations and indicates highly aggressive non small cell lung cancer

Abstract Background: Circulating cell free DNA (cfDNA) in the plasma of cancer patients may provide oncogenic mutation status for NSCLC. However, the low sensitivity of current cfDNA has clinical utility limited to late stage multifocal or some recurrent diseases. Recently, specific phagocytic stromal cells found in blood, i.e. cancer associated macrophage-like cells (CAMLs), were shown to contain large quantities of tumor DNA. We hypothesized that a single blood sample can provide both cfDNA and parallel CAML DNA, providing more sensitive tumor mutation screening in a broader array of patients. We screened untreated NSCLC patients with a range of stages (stage I=3, stage II=5, stage IIIa=10, stage IIIb=7, &amp; stage IV=5), that had available primary tissue for NGS. Blood was drawn prior to induction of radiotherapy, and plasma was sequenced using a 50 gene oncopanel. Separately, CAMLs were isolated from the same blood sample, lysed and sequenced using the same panel. Our data suggests that CAMLs contain clinically relevant oncogenic variants that correspond to both the primary tumor and the matched plasma. Methods: Whole peripheral blood was taken from 30 newly diagnosed NSCLC patients with confirmed stage I-IV disease in a preservative collection tube. Plasma was separated and frozen at -80C. CAMLs were purified from the cell pellet using a standard CellSieveTM Assay, which isolates CAMLs based on size separation. Purified CAMLs were enumerated, then removed from the filter and lysed in 150 µL of buffer. The primary tumor biopsies, plasma and lysed CAMLs were blinded and anonymized for sequencing against a standard 50 oncogene NGS panel. Total mutations in tumor/plasma/CAMLs were used to evaluate progression free survival (PFS) by censored univariate analysis. Results: Forty three mutations were identified from primary tumor DNA (averaging 1.4 variants in 87% of patients) vs 28 mutations from cfDNA (0.9 variants in 47% of patients) with no exon variant matches, although a nonmatching variant was found on the TP53 loci. CAML lysate had 2.8X more mutations than cfDNA, with 78 variants averaging 2.6 variants in 80% of patients. Further, 2 CAML variants exactly matched the primary tumor and 3 more matched at the gene level. cfDNA and CAMLs had the best concordance, with 6 matching exon variants and 6 more matching on the same gene. Notably, patients with cfDNA or CAML mutations had lower PFS, with ≥1 variants in cfDNA (HR=4.0, 95%CI=1.4-11.3, p=0.021) and ≥4 variants in CAMLs (HR=4.3, 95%CI=1.4-12.9, p=0.020). Conclusions: Our data suggests that a single blood sample to screen for oncogenic tumor mutations from cfDNA and CAMLs provides increased sensitivity and specificity than cfDNA alone. Further, while preliminary, we find that high mutation burden in cfDNA or CAMLs may be indicative of aggressive NSCLC that is more likely to progress within 2 years. Citation Format: Daniel L. Adams, Steven H. Lin, Ashvathi Raghavakaimal, Glenn Weiss, Andrew Ford, Charmaine Brown, Chen-Hsiung Yeh. Combining circulating stromal cells with cell free DNA for increased sensitivity in profiling oncogenic mutations and indicates highly aggressive non small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 437.

Abstract 2915: A circulating stromal cell subpopulation that accurately predicts resistance and progression in treatment naïve lung cancer patients receiving definitive radiotherapy

Abstract Background: Cancer Associated Macrophage-Like cells (CAMLs) are recently described circulating stromal cells common in the peripheral blood of cancer patients hypothesized to be a mechanism in cancer pathogenesis. We previously described that treatment naive patients with circulating CAMLs ≥50µm is a significant independent prognostic indicator of shorter progression free survival (PFS) in a variety of cancers. However, the clinical value of CAMLs in specific diseased cohorts for predicting response to treatment has not been evaluated. We present the results of a prospective study on treatment naive lung cancer patients before induction and directly after completion of definitive radiotherapy to determine if CAMLs are predictive of treatment response and cancer progression. Methods: A 2 year single blind prospective study was undertaken, testing the relationship of enlarged CAMLs (≥50µm) to PFS of lung cancer patients before &amp; after induction of definitive radiation therapy. To achieve a 2-tailed 95% power (α=0.05) we recruited a training set of 55 patients, all with pathologically confirmed lung cancer: Stage I (n=13), Stage II (n=7), Stage IIIa (n=10), Stage IIIb (n=18) &amp; Stage IV (n=7). Baseline (BL) blood samples were taken prior to start of therapy. If possible, a second blood sample (T1) was taken after completion of radiotherapy (~60 days), n=46 patients. Blood was filtered by CellSieveTM filtration and CAMLs quantified. Analysis by CAML size of &amp;lt;50 µm or ≥50 µm was used to evaluate PFS hazard ratios (HRs) by censored univariate &amp; multivariate analyses. Results: CAMLs were found in 93% of BL samples averaging 3.2 CAMLs/7.5mL. Patients with at least one CAML ≥50 µm had reduced PFS (HR=2.9, 95%CI 1.4-6.0, p=0.010). 46 patients agreed to a follow up blood draw. 13 of the 46 patients had an increase of CAML size to ≥50 µm, but 3 patients had a decrease to &amp;lt;50 µm, resulting in an increase of PFS (HR=7.7, 95%CI 2.6-12.5, p&amp;lt;0.001). Combined, 90% of patients with a ≥50 µm CAML at BL progressed within 2 years vs 46% of patients with &amp;lt;50 µm CAMLs. Enlarged CAMLs at T1 was more predictive of progression, with 92% of patients with a ≥50 µm CAML progressing vs 21% of patients with &amp;lt;50 µm CAMLs. Notably, 100% of patients that had a ≥50 µm CAML at both BL and T1 progressed. By comparison, only 11% of patients with &amp;lt;50 µm CAMLs at both BL and T1 progressed. In a multivariate analysis, CAML size was the most significant indicator of PFS and OS, independent of all other clinical variables, including stage. Conclusions: Our data suggests that for lung cancer patients undergoing definitive radiation therapy, the presence of enlarged CAMLs appears to predict patients that are resistant to treatment. Furthermore, these circulating stromal cells appear useful for sequential monitoring of patients that may prognosticate who are likely to progress after treatment. Citation Format: Daniel L. Adams, Jianzhong He, Yawei Qiao, Hui Gao, James Reuben, Ignacio I. Wistuba, Cha-Mei Tang, Steven H. Lin. A circulating stromal cell subpopulation that accurately predicts resistance and progression in treatment naïve lung cancer patients receiving definitive radiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2915.

Real-time monitoring of circulating stromal cells in the blood to predict responsiveness of new-line therapies in metastatic breast cancer.

e14048 Background: Cancer associated macrophage like cells (CAMLs), a circulating stromal cell subtype, has been shown as an independent prognostic indicator of survival in late stage metastatic breast cancer when cells enlarge to ≥50µm. However, no study has evaluated the clinical relationship between changes in CAML size and their ability to predict treatment response. While CAMLs are prognostic for progression, we hypothesized that monitoring changes in CAMLs after initiation of therapy may be predictive for responsiveness of new treatment regimens. Methods: A prospective 12 months blind multi-institutional pilot study was undertaken to evaluate CAMLs before, and after, induction of a new line of investigational therapy based on CT scans. Patients with progressive metastatic breast cancer (n = 29) who had failed at least 2 prior therapies were recruited. A baseline (BL) blood sample was taken prior to induction of a new therapy and a 2nd sample (T1) taken after initiation (~30 days). Blood was filtered by CellSieve filtration.The quantities and sizes of CAMLs were analyzed based on PFS hazard ratios (HRs) by censored univariate analysis. Results: CAMLs were found in 97% of BL samples and 93% of T1 samples. At first CT scan, after the assigned dose of investigational treatment, 17 of 29 patients had clinical progression with 14/17 (82%) patients having an increased CAML size and 3/17 (18%) having a decreased CAML size. The remaining 12 of 29 patients saw clinically stable, or regression, of disease with 10/12 (83%) having decreased CAML size and 2/12 (17%) having increased CAML size. Overall CAML size change after therapy induction was 83% accurate at predicting response or progression based on CT scans. Further, patients with increasing CAML size at T1 had a 4 month mPFS vs 10 month mPFS for decreasing CAMLs, with a lower 12 month PFS HR = 3.7 (95%CI = 1.5-10.1, p = 0.020). Conclusions: Our data suggests that in metastatic breast cancer, monitoring CAMLs changes over the first 30 days of treatment accurately predicts responsiveness of disease to new treatments. Further, we suggests using blood sampling may increase the clinical value of blood based diagnostics by rapidly predicting the benefit of subsequent therapies.

Training and validation study for sequential monitoring of CAMLs in circulation to predict ongoing progression in lung cancer patients undergoing definitive radiotherapy.

3053 Background: Cancer Associated Macrophage-Like cells (CAMLs) are a recently described circulating stromal cell common in the peripheral blood of cancer patients that are prognostic for progressive disease. Further, it has been shown that changes in CAML size (i.e. enlargement above 50µm) can predict progression free survival (PFS) in thoracic cancers (e.g. lung). We enrolled 104 unresectable non-small cell lung cancer (NSCLC) patients, with an initial training set review of 54 patients, to determine if change in CAML size after radiation therapy was predictive PFS. Methods: A 2 year single blind prospective study was undertaken to test the relationship of ≥50µm CAMLs to PFS based on imaging in lung patients before and after induction of chemo radiation, or radiation therapy. To achieve a 2-tailed 90% power (α = 0.05) we recruited a training set of 54 patients and validation set of 50 patients all with pathologically confirmed unresectable NSCLC: Stage I (n = 14), Stage II (n = 16), Stage III (n = 61) &amp; Stage IV (n = 13). Baseline (BL) blood samples were taken prior to start of therapy &amp; a 2nd blood sample (T1) was taken after completion of radiotherapy (~30 days). Blood was filtered by CellSieve filtration and CAMLs quantified. Analysis by CAML size of &lt; 49 µm or ≥50 µm was used to evaluate PFS hazard ratios (HRs) by censored univariate &amp; multivariate analysis. Results: CAMLs were found in 95% of samples averaging 2.7 CAMLs/7.5mL sample at BL, with CAMLs ≥50 µm having reduced PFS (HR = 2.2, 95%CI1.3-3.8, p = 0.003). At T1, 18 patients had increased CAML size ≥50 µm with PFS (HR = 4.6, 95%CI2.5-8.3, p &lt; 0.001). In total, ≥50 µm CAMLs at BL was 76% accurate at predicting progression within 24 months while ≥50 µm CAMLs at T1 was 83% accurate at predicting progression. Conclusions: In unresectable NSCLC patients, enlargement of CAMLs during treatment is an indicator active progression. We identify that a single ≥50 µm CAML after induction of radiotherapy, in our training set and confirmed in our validation set, is an indicator of poor prognosis. We suggest that changes in CAML size during therapy may indicate the efficacy of therapy and could potentially help shape subsequent therapeutic decisions.

Safety and efficacy of a phase I/IIa trial (NCT03066947) of a modified whole tumor cell targeted immunotherapy in patients with advanced breast cancer.

e14026 Background: SV-BR-1-GM is a GM-CSF transfected breast cancer cell line which expresses HLA class I &amp; II antigens and has functional antigen-presenting cell activity. Prior studies suggest that partial matching of the HLA type of the patient with SV-BR-1-GM may be predictive of tumor regression. Methods: Subjects received low-dose cyclophosphamide 2-3d prior to ID injection of irradiated SV-BR-1-GM (20 million cells divided into 4 sites) and interferon-α into the inoculation sites ~2 &amp; 4 days subsequently. Cycles were q2 weeks x 3 then q mo. Results: A total of 30 patients were screened and 23 inoculated (Table). The patients were heavily pretreated with a median of 4 prior chemo/biological therapy regimens. There were no serious or unexpected adverse events. Local injection-site irritation was the most common toxicity. Objective tumor regression was seen in 3 patients, all of whom matched SV-BR-1-GM at least at one HLA locus: one patient with regression or clearing of 20 lung metastases; one with reduction in cutaneous involvement of the breast from 80% to 30% and one with regression of a breast lesion. Another 3 patients had decreases in circulating cancer-associated macrophage-like cells (CAMLs), which has been shown to correlate with tumor stage. They also all matched at least at one HLA allele. Circulating tumor cells and circulating epithelial cells were present in low numbers and tended to parallel trends in CAMLs which were present in larger numbers. CAMLs in 21/23 patients stained positive for PD-L1. Patients with tumor regression had robust DTH responses to SV-BR-1-GM. Conclusions: SV-BR-1-GM in this regimen appears to be safe and well-tolerated and is associated with objective regression of metastatic breast cancer and/or with decreases in circulating cancer-associated cells in 6/23 (26%) or patients. HLA matching may be a predictor of response. Clinical trial information: NCT03066947. [Table: see text]

Monocytes and cancer: promising role as a diagnostic marker and application in therapy

Interrelationship between a malignant tumor and the immunity are provided by the involvement of both adaptive and innate immune systems. Monocytes are major participants in nonspecific immune response and mediate their key function through refilling the pool of tumor-associated macrophages, dendritic cells and myeloid suppressor cells. All these populations regulate the relationship of tumor-infiltrating immunocompetent cells with tumor cells and with other components of the microenvironment, as well as tumor cell proliferation, angiogenesis, and dissemination. Monocytes, being direct participants of the chronic persistent inflammation, are involved in the inflammation impact on both tumor origin and progression. The study of the molecular mechanisms of monocyte recruitment and differentiation in malignant neoplasms seems to be a promising direction, both for a diagnostic purpose and as a search for targeting molecules for the control of macrophages and dendritic cells in the tumor microenvironment. In this review, the characteristics of peripheral blood monocytes are given, taking into account the heterogeneity of their population. Tie2+ cells and macrophage-polarized CD163+ and CD204+ -monocytes, as well as cancer-associated macrophage-like cells (CAMLs), are described as contributors to cancer disease progression and outcome. The involvement of monocyte subpopulations in the pathogenesis of oncological diseases of different localizations at the stages of the formation of monocyte precursors in the bone marrow, circulation in peripheral blood and differentiation in tumor tissue is shown.

Abstract A012: SV-BR-1-GM, a whole-cell targeted immunotherapy for advanced breast cancer: Pharmacodynamic markers of response

Abstract Background: SV-BR-1-GM is a GM-CSF-engineered breast cancer cell line employed – after irradiation – as a targeted immunotherapy for advanced breast cancer. Tumor regressions at metastatic sites have been observed, most notably in patients with HLA allele matches to the cell line. We are assessing SV-BR-1-GM in the phase IIa portion of a phase I/IIa clinical trial in metastatic and locally recurrent breast cancer (ClinicalTrials.gov identifier NCT03066947). Additionally, we are co-developing a companion diagnostic (BriaDX™) to identify patients likely to respond to SV-BR-1-GM. Currently, BriaDX™ consists of HLA typing; however, we have begun assessing biomarkers in sera, lymphocyte characteristics, circulating tumor cells (CTCs), and cancer-associated macrophage-like cells (CAMLs) from patients collected at baseline and after inoculation of SV-BR-1-GM to layer in additional components to improve accuracy, with the number/subtyping of CTCs and CAMLs being prognostic indicators. Methods: Subjects are pretreated with low-dose cyclophosphamide to reduce immune suppression. SV-BR-1-GM is inoculated intradermally with follow-up local injections of IFNα2. Cycles are every 2 weeks x 3, then monthly. HLA typing was conducted via LabType R-SSO Kits (One Lambda). Cytokines were measured via single- or multiplex assays. Anti-SV-BR-1 antibodies were determined by incubation of SV-BR-1 cells with diluted patient sera followed by staining with a fluorescently-labeled anti-IgG antibody and detection by flow cytometry. CTCs and CAMLs were evaluated by CellSieve™ at Creatv MicroTech. Results: To date, 16 clinical trial subjects have been inoculated with the SV-BR-1-GM regimen as rescue immunotherapy. All were treatment refractory and had received a median of 4.5 prior chemo/biologic therapy regimens (range 1-13). Two of the 16 patients remained on study for ≥3 months (5 cycles) with 4 patients currently on study not having reached the 3-month evaluation time point. Objective regression of tumor was seen in 2 subjects. One subject had virtually complete regression of 20 of 20 lung metastases noted at 3 and 6 months (but with progressive bone and liver metastases). Another subject had improvement of chest wall metastases and quality of life but expired due to nontreatment-related causes. Response appeared to correlate with HLA allele-matching to SV-BR-1-GM. Anti-SV-BR-1 antibody titers increased in several patients. Among the cytokines assessed, interleukin (IL)-8 levels increased in HLA-DRB3 allele-matched subjects after SV-BR-1-GM inoculation. Of 15 patients evaluated, CTCs were present in 6 patients at baseline while CAMLs were present in all 15. Five of 5 patients evaluated for PD-L1 expression had mostly low-to-medium expression of PD-L1 on their CTCs/CAMLs. In the patient who had regression of lung metastases but progression of liver metastases, PD-L1 expression and maximum CAML size increased, but the number of CAMLs decreased during treatment. CAML number also decreased in a patient who reached the 3-month evaluation visit without progression and in a patient with inflammatory breast cancer who dropped out due to worsening inflammation. Conclusions: In addition to the patients’ HLA types, several pharmacodynamic parameters correlated with tumor regression and/or HLA matching status. CTCs or CAMLs are frequently detectable in this population, and PD-L1 expression appears common on these cells. Both CAML number and size appear to correlate with response, though larger studies are needed. Future steps include evaluation of SV-BR-1-GM with checkpoint inhibitors. Citation Format: Markus D. Lacher, Sanne Graeve, Vivekananda (Vivek) Sunkari, Daniel L. Adams, Cha-Mei Tang, Pete Amstutz, Charles L. Wiseman, George E. Peoples, William V. Williams. SV-BR-1-GM, a whole-cell targeted immunotherapy for advanced breast cancer: Pharmacodynamic markers of response [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A012.

Circulating Hybrid Cells Join the Fray of Circulating Cellular Biomarkers.

Gastrointestinal cancers account for more cancer-related deaths than any other organ system, owing in part to difficulties in early detection, treatment response assessment, and post-treatment surveillance. Circulating biomarkers hold the promise for noninvasive liquid biopsy platforms to overcome these obstacles. Although tumors shed detectable levels of degraded genetic material and cellular debris into peripheral blood, identifying reproducible and clinically relevant information from these analytes (eg, cell-free nucleotides, exosomes, proteins) has proven difficult. Cell-based circulating biomarkers also present challenges, but have multiple advantages including allowing for a more comprehensive tumor analysis, and communicating the risk of metastatic spread. Circulating tumor cells have dominated the cancer cell biomarker field with robust evidence in extraintestinal cancers; however, establishing their clinical utility beyond that of prognostication in colorectal and pancreatic cancers has remained elusive. Recently identified novel populations of tumor-derived cells bring renewed potential to this area of investigation. Cancer-associated macrophage-like cells, immune cells with phagocytosed tumor material, also show utility in prognostication and assessing treatment responsiveness. In addition, circulating hybrid cells are the result of tumor–macrophage fusion, with mounting evidence for a role in the metastatic cascade. Because of their relative abundance in circulation, circulating hybrid cells have great potential as a liquid biomarker for early detection, prognostication, and surveillance. In all, the power of the cell reaches beyond enumeration by providing a cellular source of tumor DNA, RNA, and protein, which can be harnessed to impact overall survival.

Detection of Circulating Giant Cancer Associated Macrophage like Cells During and after Radiation Therapy Is Associated with Disease Progression in Thoracic Cancers

Personalizing therapy in cancer patients requires individualized determination of the likelihood of cancer progression after start of therapy. We assessed the predictive value of circulating giant cancer associated stromal cell type called Cancer Associated Macrophage-Like cells (CAMLs) in predicting disease progression or death during and after definitive radiotherapy in non-small cell lung cancer (NSCLC) and esophageal cancer (EC). This is a 2-year prospective, single blinded study to assess the role of CAMLs in determining progression free survival (PFS) in patients after completing definitive radiotherapy or chemoradiotherapy. We have previously described that CAMLs ≥50 μm found in pretreatment samples, which we call giant CAMLs, were strongly prognostic. This has led us to hypothesize that giant CAMLs that are detected throughout therapy will also be predictive of disease progression. For this, we analyzed 52 NSCLC (stage I, n=7, stage II, n=7, stage III, n=29, and stage IV, n=9) and 21 esophageal cancer (stage III n=20 and Stage IV (n=1) patients. Baseline (BL) blood sample was obtained prior to start of radiation, a 2nd blood sample (T1) was taken midway during therapy and a 3rd sample (T2) was taken at the end of radiotherapy. Peripheral blood was processed using CellSieve® microfiltration and CAML size was determined. CAMLs <49 or ≥50 μm sizes were quantified but the observer was blinded to the disease type or clinical information. This measure was used to evaluate PFS hazard ratios (HRs) by censored univariate & multivariate analysis at each time point. CAMLs were found in 97% of all BL samples. On average 2.9 CAMLs/7.5 mL of blood were found. Giant CAMLs at BL had significantly reduced PFS for NSCLC (HR=2.9, 95%CI 1.3-6.2, p=0.015) and marginally for EC (HR=3.0, 95%CI 0.9-9.9, p=0.14). At T1, patients with detectable giant CAMLs further reduced PFS after treatment (NSCLC, HR=5.0, 95%CI 2.3-10.9, p<0.001; EC, HR=4.0, 95%CI 1.2-13.2, p=0.05), and even more significantly at T2 (NSCLC, HR=7.1, 95%CI 3.4-14.8, p<0.001; EC, HR=5.6, 95%CI 1.6-18.8, p=0.01). Combining the two disease data sets, giant CAMLs at BL was 70% accurate at predicting eventual progression within 24 months while giant CAMLs at T2 was 84% accurate at predicting progression. On multivariable analysis, giant CAMLs was the most significant independent prognostic factor, regardless the site of disease. Our data suggests monitoring the presence of giant CAMLs throughout the course of radiotherapy could be predictive of cancer progression or death. Prospective validation of giant CAMLs as a blood-based biomarker to stratify patient risk is needed to individualize cancer therapies in disease subtypes.

Cancer-associated circulating atypical cells with both epithelial and macrophage-specific markers

In 2014, Adams et al . (1) reported on atypical circulating macrophages, that they named circulating cancer-associated macrophage-like cells (CAMLs), detected in the blood of breast and pancreatic cancer patients following enrichment by blood filtration.

Abstract 1560: Real-time monitoring of solid tumor progression by circulating stromal cells from early- to late-stage disease

Abstract Background: Blood-based biomarkers (PSA, CEA, CA125) are used to track real-time progression of disease in parallel with imaging. However, while numerous blood biomarkers exist, they are specific to cancer type (i.e., PSA to prostate and CEA to colon) and may not appear in all diseased individuals. Recently, cancer-associated macrophage-like cells (CAMLs), a circulating stromal cell subtype, were identified in various solid cancer types, which were observed increasing in size and in hyperploidy during progressive disease. To assess whether CAML enlargement is a biomarker of progression/response, we tracked CAML growth/shrinkage in a pilot study of patients (n=34). Blood was drawn from patients with lung, prostate, or breast cancers over a 3-month period, baseline through 2 treatment cycles, followed by continued monitoring for 2 years. These data suggest that morphologic assessment of CAMLs (growth/hyperploidy) appear to parallel cancer progression, or response to treatment, in multiple solid tumors. Methods: A prospective multi-institutional study used anonymized peripheral blood samples from 34 cancer patients undergoing therapy [stage I (n=2), II (n=3), III (n=8) &amp; IV (n=21)] with breast (n=10), lung (n=16), &amp; prostate (n=8). Samples were taken prior to therapy (BL), at 1 month (FU1) follow-up and a 3-month (FU2) follow-up, after induction of therapy. Blood was processed by the CellSieve™ microfiltration technique at 4 institutions and stained for cytokeratin 8, 18 and 19, CD14 and CD45. After identification and quantification, CAMLs were measured based on their hyperploidy and cell size. Results: CAMLs were found in 97% of cancer patients at BL, 97% at FU1 and 91% of FU2. Over the 2-year follow-up, 7 patients showed no signs of clinical disease progression, while 27 patients had observable progression. Of the 7 patients who did not progress, only two had CAMLs of ≥50µm at BL and at FU1, but whose CAMLs shrunk to &amp;lt;50 µm by FU2. Of the 27 patients who progressed, 22 patients had ≥50µm CAMLs at all time points, while 5 patients had small &amp;lt;50µm CAMLs at BL. Interestingly, CAMLs in these 5 patients had enlarged to ≥50µm by the FU2 time point. Conclusions: We show that increased polynucleation and CAML enlargement indicate shorter progression-free survival in a number of cancer types after baseline. By monitoring CAML changes over time for the 34 individual patients, we demonstrated correlation of ongoing progression, or response, in tumors to the enlargement or shrinkage in CAMLs at follow-up time points from treatment induction. This pilot study suggests that CAMLs have the potential to monitor the progression/regression of malignancy in real time and suggests the need for larger validation studies. Citation Format: Daniel L. Adams, Raymond Bergan, Martin J. Edelman, Stuart S. Martin, Rena Lapidus, Saranya Chumsri, Cha-Mei Tang, Steven H. Lin. Real-time monitoring of solid tumor progression by circulating stromal cells from early- to late-stage disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1560.

Sequential monitoring of CAMLs in circulation as predictive of progression in lung cancer patients undergoing definitive radiotherapy.

e21062Background: Cancer Associated Macrophage-Like cells (CAMLs) are a recently described circulating stromal cell common in the peripheral blood of patients with solid tumors. Further, CAMLs ≥50µ...

Combining circulating tumor cells and circulating cancer associated macrophage-like cells for accurately predicting responsiveness of new line therapies in late stage cancers.

12032Background: The discovery of cancer associated macrophage like cells (CAMLs) as independent prognostic indicators of survival has highlighted the need for more in depth analysis of blood based...