Residual Disease In Patients Research Articles

Ex-vivo models of post-surgical residual disease in human glioblastoma

Background Glioblastoma is a highly infiltrative, currently incurable brain cancer. To date, translation of novel therapies for glioblastoma from the laboratory into clinical trials has relied heavily on in vitro cell culture and murine (subcutaneous and orthotopic) xenograft models using cells derived from the main bulk of patient tumours. However, it is the residual cells left-behind after surgery that are responsible for disease progression and death in the clinic. A lack of substantial improvements in patient survival for decades suggests commonly used murine xenograft models, a key step before clinical trials, do not reflect the biology of residual disease in patients. Methods To address this, we have developed the ‘Sheffield Protocol’ to generate ex vivo models that reflect both resected, and post-surgical residual disease from the same patient. The protocol leverages parallel derivation of inherently treatment-resistant glioblastoma stem cells (GSCs) from ‘core’ and distant ‘edge’ regions through careful macrodissection of a large en bloc specimen, such as from a partial lobectomy for tumour, followed by tissue dissociation and propagation in serum-free media. Opportunistic en bloc specimen use can liberate the most distant infiltrative cells feasibly accessible from living patients. Results We provide an example illustrating that resected and residual disease models represent spatially divergent tumour subpopulations harbouring distinct transcriptomic and cancer stem cell marker expression profiles. We also introduce the ‘Sheffield Living Biobank’ of glioma models (SLB) that incorporates over 150 GSC lines from 60+ patients, including 44+ resected and residual models, which are available for academic use via MTA. Conclusions These models provide a novel tool to reduce animal xenograft usage by improving candidate drug triage in early preclinical studies and directly replacing animal studies for some therapies that are post-Phase 1+ clinical trial for other cancers/conditions to, ultimately, deliver more effective treatments for post-surgical residual disease in glioblastoma.

Circulating tumor DNA fraction predicts residual cancer burden post-neoadjuvant chemotherapy in triple negative breast cancer

PurposePathologic response after preoperative/neoadjuvant chemotherapy (NAC) is a continuum that can range from complete pathologic response (pCR) to extensive residual disease (RD). We hypothesized that post-NAC plasma circulating tumor DNA (ctDNA) fraction (TF) reflect pathologic response as continuum measured by the residual cancer burden (RCB) score. MethodsctDNA was assessed using the PredicineBEACON assay, that interrogates up to 50 personalized tumor variants and 500 hot-spot mutations, in 3 mL archived plasma isolated from EDTA tubes collected post-NAC but before surgery from 44 patients with stage I/III triple negative breast cancer (TNBC) who received durvalumab and weekly nab-paclitaxel followed by doxorubicin/cyclophosphamide on a clinical trial (NCT02489448). Circulating free tumor DNA methylation profiling was performed using PredicineEPIC assay in paired pre- and post-NAC plasma (N=30). Youden’s J-statistics was used to define optimal thresholds. ResultsWe observed a significant positive correlation (r=0.45, p=0.004) between RCB scores and post-NAC TF. The median TF was significantly lower in pCR (RCB0) compared to RD patients (0.06% vs. 0.3%, p=0.02). Using a TF positivity threshold of ≥0.05%, PredicineBEACON had 58% sensitivity at 83% specificity for identifying RD. TF was higher in patients who experienced recurrence (n=9) compared to those without recurrence (n=35) (0.17% vs. 0.05% TF, p=0.029). There was significant decrease in methylation signal in post- compared to pre-NAC samples, but post-treatment methylation signal was lower in cases with pCR vs RD. ConclusionsPost-NAC plasma tumor fraction and change in tumor-derived methylation signal predict the extent of RD and recurrence in TNBC patients.

226TiP CLEAR-Me: Interception trial to detect and clear molecular residual disease in patients with high-risk melanoma

226TiP CLEAR-Me: Interception trial to detect and clear molecular residual disease in patients with high-risk melanoma

Risk factors for positive post-transplantation measurable residual disease in patients with acute lymphoblastic leukemia.

The level of measurable residual disease (MRD) before and after transplantation is related to inferior transplant outcomes, and post-hematopoietic stem cell transplantation measurable residual disease (post-HSCT MRD) has higher prognostic value in determining risk than pre-hematopoietic stem cell transplantation measurable residual disease (pre-HSCT MRD). However, only a few work has been devoted to the risk factors for positive post-HSCT MRD in patients with acute lymphoblastic leukemia (ALL). This study evaluated the risk factors for post-HSCT MRD positivity in patients with ALL who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT). A total of 1683 ALL patients from Peking University People's Hospital between January 2009 and December 2019 were enrolled to evaluate the cumulative incidence of post-HSCT MRD. Cox proportional hazard regression models were built for time-to-event outcomes. Multivariate analysis was performed to determine independent influencing factors from the univariate analysis. Both in total patients and in T-cell ALL or B-cell ALL, pediatric or adult, human leukocyte antigen-matched sibling donor transplantation or haploidentical SCT subgroups, positive pre-HSCT MRD was a risk factor for post-HSCT MRD positivity (P<0.001 for all). Disease status (complete remission 1 [CR1] vs. ≥CR2) was also a risk factor for post-HSCT MRD positivity in all patients and in the B cell-ALL, pediatric, or haploidentical SCT subgroups (P=0.027; P=0.003; P=0.035; P=0.003, respectively). A risk score for post-HSCT MRD positivity was developed using the variables pre-HSCT MRD and disease status. The cumulative incidence of post-HSCT MRD positivity was 12.3%, 25.1%, and 38.8% for subjects with scores of 0, 1, and 2-3, respectively (P<0.001). Multivariate analysis confirmed the association of the risk score with the cumulative incidence of post-HSCT MRD positivity and relapse as well as leukemia-free survival and overall survival. Our results indicated that positive pre-MRD and disease status were two independent risk factors for post-HSCT MRD positivity in patients with ALL who underwent allo-HSCT.

Development and validation of a circulating tumor DNA-based optimization-prediction model for short-term postoperative recurrence of endometrial cancer

BACKGROUND Endometrial cancer (EC) is a common gynecological malignancy that typically requires prompt surgical intervention; however, the advantage of surgical management is limited by the high postoperative recurrence rates and adverse outcomes. Previous studies have highlighted the prognostic potential of circulating tumor DNA (ctDNA) monitoring for minimal residual disease in patients with EC. AIM To develop and validate an optimized ctDNA-based model for predicting short-term postoperative EC recurrence. METHODS We retrospectively analyzed 294 EC patients treated surgically from 2015-2019 to devise a short-term recurrence prediction model, which was validated on 143 EC patients operated between 2020 and 2021. Prognostic factors were identified using univariate Cox, Lasso, and multivariate Cox regressions. A nomogram was created to predict the 1, 1.5, and 2-year recurrence-free survival (RFS). Model performance was assessed via receiver operating characteristic (ROC), calibration, and decision curve analyses (DCA), leading to a recurrence risk stratification system. RESULTS Based on the regression analysis and the nomogram created, patients with postoperative ctDNA-negativity, postoperative carcinoembryonic antigen 125 (CA125) levels of &lt; 19 U/mL, and grade G1 tumors had improved RFS after surgery. The nomogram’s efficacy for recurrence prediction was confirmed through ROC analysis, calibration curves, and DCA methods, highlighting its high accuracy and clinical utility. Furthermore, using the nomogram, the patients were successfully classified into three risk subgroups. CONCLUSION The nomogram accurately predicted RFS after EC surgery at 1, 1.5, and 2 years. This model will help clinicians personalize treatments, stratify risks, and enhance clinical outcomes for patients with EC.

Evolution and prognostic significance of HER-2 changes from primary to residual disease in patients with HER-2–negative breast cancer receiving neoadjuvant chemotherapy.

583 Background: HER-2 changes during the neoadjuvant chemotherapy has not been thoroughly evaluated. The current study aimed to analyze the HER-2 status changes from primary to residual disease in HER-2 negative patients after neoadjuvant chemotherapy and evaluate its impact in survival outcomes. Methods: HER-2 negative breast cancer patients with residual disease after NACT who had paired pre- and post-therapeutic HER2 testing results were retrospectively analyzed. Data on clinical and pathological characteristics, treatment information, and survival outcomes were collected for all eligible patients. HER2 changes from the primary to residual disease were described and logistic regression were performed to identify factors associated with HER2 changes. The Kaplan–Meier method and multivariate Cox regression were used for survival analysis. Results: 571 HER-2 negative breast cancer patients with residual disease after NACT were enrolled, including HER2-zero (35.2%, n=201) and HER2-low (64.8%, n=370) patients before NAC. The overall rate of HER-2 changes rate was 32.4% (negative to positive 1.1%, low to zero 15.4%, zero to low 15.9%). Among the patients with HER-2-zero before NAC, patients with HER-2 changes (zero to low) were likely to be hormone receptor positive (68.1% vs 36.7%, P&lt;0.0001) and had a higher proportion of patients with Ki-67≤20 (Ki-67≤20 27.5% vs 15.6%, P=0.035). Among the patients with HER2-low before NAC, a higher proportion of hormone receptor negative were observed in HER2 changes patients (low to zero) (31.8% vs 18.4%, P=0.008) and the Ki-67 is relatively high in HER-2 changes patients (Ki-76 &gt; 20 73.9% vs 65.0% P=0.030). Multivariable logistic regression analyses showed patients with hormone receptor positive before NAC were related to underwent HER2-zero transferring to HER2-low (OR, 3.436, P&lt;0.0001). The median follow-up time was 50.0 months. In the HER-2 zero group, HER2 changes (zero to low) was significantly associated with better DFS comparing with constant HER2-zero (HR0.49, P=0.01) after adjustment, with 4-year DFS rate was 80.1%,55.7% (Log-rank P =0.033). Subgroup analysis showed that in the hormone receptor positive subgroup, patients with HER2 zero to low has a significant better DFS than patients with constant HER2-zero (Log-rank P=0.037). In the HER2-low group, no significant differences in survival outcomes between constant HER2-low and HER2 low to zero (HR1.07, P=0.778). Conclusions: HER2 status showed instability from primary tumor to residual disease after neoadjuvant treatment. HER2 changes (zero to low) was associated with better DFS in HER-2 zero patients.

Utilization of personalized tumor-specific methylation haplotypes of circulating DNA to monitor minimal residual disease in patients with hepatocellular carcinoma undergoing curative resection.

e16245 Background: Minimal residual disease (MRD) is considered an essential factor leading to early relapse after radical surgery, which is challenging to be detected by conventional imaging. The majority of MRD studies in HCC are based on mutation detection. Simultaneously, in certain cancers such as lung and colorectal cancers, the use of methylation detection for MRD has been demonstrated to possess significant detection capabilities. In this study, we present a personalized methylation haplotype (MHP) method for MRD detection in HCC patients underwent curative resection. Methods: Tumor and paracancerous tissue samples were obtained from HCC patients undergoing surgical resections. WBC and plasma samples (T0) were collected before surgery, while plasma samples (T1) were collected in one month after surgery, and subsequent follow-up plasma samples were obtained every three months. All samples were tested using the GutSeer Panel. A personalized tumor-informed MRD detection approach, named TORNADO (Tumor-infORmed DNAm hAplotype DetectiOn), was then developed. Individual seed MHPs were pinpointed based on their presence in cancer tissue or plasma samples collected before surgery (T0 plasma), excluding those in para-cancer tissue and blood cells. The relapse risk score was determined by assessing the proportion of these MHPs detectable in follow-up evaluations. Results: We enrolled 39 early-stage HCC patients who have undergone surgical resections. All the patients have completed at least one year of regular clinical follow-up. Among them, 13 out of 39 patients (33.3%) were identified as MRD-positive based on predictions from T1. Of the MRD-positive patients, 6 experienced tumor relapse. In contrast, only 7.7% relapses were observed among the remaining 26 MRD-negative patients. When considering the T1 and T2 MRD cumulative risk score, 53.8% (21/39) of patients were classified as MRD-positive, and none of the MRD-negative patients experienced relapse. Regardless of whether based on T1 plasma alone or cumulative results, recurrence-free survival (RFS) was significantly correlated with MRD status. Kaplan-Meier analysis demonstrated that MRD-positive patients had significantly poorer RFS (p-value &lt; 0.05). Cox regression analysis further revealed that MRD status was a significant variable for predicting recurrence-free survival (HR=16.00, 95% CI 2-130, P=0.009). Conclusions: The MRD status detected byTORNADOmethod emerged as a significant prognostic indicator for disease relapse in early-stage HCC patients undergoing curative resection, underscoring the clinical relevance of MHP-based MRD detection guided by tumor information. These discoveries pave the way for the development of effective postoperative cancer surveillance strategies specifically tailored for early-stage liver cancer.

Longitudinal circulating tumor DNA monitoring for detection of molecular residual disease in patients with advanced germ cell tumors.

e17022 Background: Germ cell malignancies, commonly manifesting as testicular cancer, is one of the most common malignancies in men 15 to 45 years of age, with the incidence doubling over the past 40 years. When identified promptly and treated with a multidisciplinary approach, germ cell tumors have an excellent prognosis with cure rate &gt;90% and 5-year survival rate &gt;95%. Treatment involves radical orchiectomy for diagnostic and therapeutic purposes. Further management includes active surveillance, adjuvant chemotherapy, retroperitoneal lymph node dissection (RLND), and radiation therapy. While AFP, B-HCG, and LDH are good biomarkers, they are not sensitive or specific for detecting molecular residual disease (MRD). B-HCG is detected in &lt;30% of individuals with seminoma and B-HCG and AFP are detected in 50–60% of patients with non-seminomatous tumors. We describe the use of circulating tumor DNA (ctDNA) for detection of MRD in advanced germ cell malignancy, especially when baseline biomarkers are negative. Methods: This is a retrospective analysis of 20 patients with testicular cancer treated at Rush University Medical Center who monitored closely for MRD. Signatera (Natera Inc.), a personalized tumor-informed ctDNA assay, was utilized for detection and quantification of ctDNA in patients’ plasma samples, measured at MRD (1-12 weeks post-orchiectomy) and regular intervals thereafter, regardless of treatment modality. Event free survival (EFS) was determined, defined as the period from radical orchiectomy to date of radiographic progression of disease. Results: There was a total of 20 patients (35% seminoma, 65% non-seminoma) with 2 of the patients having extragonadal disease. The mean age was 32.2 years old with IQR (28-36). Post operatively, 20% underwent RLND, 10% were on surveillance, 53% received post-orchiectomy chemotherapy, 6.7% of patients underwent autologous stem cell transplant. The median follow up time is 1.14 years. Two patients had positive ctDNA despite negative baseline traditional biomarkers. There were 23.3% (7/30) of patients that had evidence of radiographic progression. Of the patients with progression, serial ctDNA monitoring had high rates of concordance (85%, 6/7) of ctDNA positivity with biomarker positivity and radiographic progression. Conclusions: Overall, ctDNA correlated with, and sometimes even preceded, traditional biomarkers and radiographic findings of disease progression. The ctDNA positivity of 2 patients in this cohort despite negative baseline AFP, B-HCG, and LDH, highlights the sensitivity and specificity of ctDNA for MRD compared to more traditional biomarkers. Serial monitoring of ctDNA offers valuable insights into germ cell tumor disease status, including presence of MRD, and there is a need for prospective studies to guide treatment escalation and de-escalation in germ cell tumors.

Comprehensive analysis of plasma methylome reveals distinct patterns of methylation changes between responders and non-responders to neoadjuvant chemotherapy in breast cancer

The potential of cell-free DNA (cfDNA) methylation profiling in oncology is increasingly recognized for its ability to offer a comprehensive assessment of tumor biology. In this report, we explore the plasma methylome dynamics in patients with early breast cancer (BC) undergoing neoadjuvant chemotherapy (NACT) using an agnostic genome-wide approach with cell-free DNA immunoprecipitation and sequencing (cfMeDIP-seq). Our cohort comprised seven women with triple-positive BC, assessed for cfDNA methylation changes at multiple time points during their treatment course. Genome-wide analysis revealed distinctive patterns of differential methylation in patients achieving pathological complete response (pCR), with no significant epigenomic modifications in patients with residual disease (RD). Gene set enrichment analysis highlighted dynamic variations in functional pathways associated with therapy response, including metabolism, immune response, and response to xenobiotics, with a notable reversibility post-treatment. Principal component analysis of the differentially methylated regions demonstrated a clear distinction between pCR and RD patients, indicating the potential of cfMeDIP-seq for the non-invasive assessment of therapy response. Such findings suggest that cfMeDIP-seq could complement ctDNA offering broader insights into the global modifications induced by chemotherapy. Although future research is needed to validate and expand on our findings, we offer a proof-of-principle of the potential utility of cfMeDIP-seq in the personalized management of BC.

Role of biomarkers in the diagnosis and management of HCC.

For many cancers, biomarkers have served as an important tool across the cancer care continuum from risk stratification and early detection to diagnosis and treatment. Alpha fetoprotein (AFP) remains one of the few validated biomarkers for patients with hepatocellular carcinoma (HCC). Although AFP has shown potential for each of these steps, its performance when used alone has often been suboptimal. There continues to be discordant recommendations about AFP's value when combined with ultrasound for surveillance as well as its role in diagnostic algorithms. Conversely, high AFP levels are associated with aggressive tumor biology and survival, so it remains a key factor for selection of liver transplant candidates. There have been immense efforts to identify and validate additional biomarkers for each of these steps in the HCC care continuum. Indeed, biomarker panels have shown promising data for HCC risk stratification and surveillance among patients with cirrhosis, as well as prognostication and detection of minimal residual disease in patients undergoing HCC treatment. Several large prospective studies to evaluate the role of these emerging biomarkers in clinical practice are currently ongoing.

Abstract PO3-13-09: Personalized ctDNA testing for detection of molecular residual disease in patients with localized HR+ breast cancer: temporal dynamics and impact on clinical outcomes

Abstract Background: Molecular residual disease (MRD) describes detectable circulating tumor DNA (ctDNA) after definitive surgery and is an expanding field in breast oncology. Multiple studies have demonstrated detectable MRD to be poorly prognostic, and clinical trials are now underway assessing the efficacy of therapeutic interception. However, most retrospective studies did not obtain baseline imaging at the time of MRD detection, such that the proportion of patients with detectable ctDNA and concurrent radiographically evident recurrent disease is not known. Furthermore, the role of serial ctDNA testing in the adjuvant setting remains unclear. Meanwhile, tumor-informed assays such as the SignateraTM platform are now covered by Medicare. To address these outstanding clinical questions, we aimed to determine the temporal dynamics of MRD detection in HR+/HER2- breast cancer, association with radiographic recurrence, and impact on clinical outcomes. Methods: Patients with HR+/HER2- breast cancer who underwent MRD testing either as part of a clinical trial or via an expanded access program were reviewed for MRD positivity and assessed alongside clinically annotated data. All patients completed surgical resection prior to MRD testing. Whole exome sequencing of primary tumor tissue and personalized ctDNA primers were designed for multiplex PCR targeting up to 16 single nucleotide variants for MRD testing via the SignateraTM platform. Clinical data was determined through focused chart review. Results: Among 185 patients with HR+/HER2- breast cancer who underwent MRD testing, 37 patients (20%) did not complete testing due to factors including tumor tissue not meeting pathology parameters, insufficient quantity of primary tissue, or tumor/normal discordance. 14 patients (8%) had detectable ctDNA (1, 5, and 8 patients had stage I, II, and III breast cancer, respectively). Among the 6 patients who did not have stage III disease, 3 had other high-risk features (Oncotype 40; grade 3 disease x2). Overall, 71% (n=10) of the +MRD population tested positive at the first test, while 29% (n=4) tested positive only on serial sampling. Median time from surgery to positive test was 3.5 years (range: 1 month – 10.2 years). Twelve patients (86%) were on adjuvant endocrine therapy at the time of detectable MRD. Among the 14 +MRD patients, 9 underwent staging scans shortly after positive MRD testing, and among these patients, 3 (33%) had radiographic evidence of asymptomatic metastatic recurrence (2 bone-only; 1 visceral); two of these pts had 1 MRD test, while the other had a negative test followed by a positive test 11 months later. The other 3 patients with radiographic recurrences had scans prompted only by clinical symptoms &amp;gt;6 months after +MRD. Among the 4 patients with serial data after first +MRD testing, none experienced subsequent ctDNA clearance. Serial testing occurred over a median of 11.5 months (range 1.1-21.7 months). With median follow up of 8.6 months, 43% (n=6) of patients with +MRD had distant disease recurrence. Conclusions: Personalized tumor-informed ctDNA testing identified patients with high risk of recurrence, including patients without classic high-risk clinical, pathologic, or genomic features, suggesting a complementary role of MRD testing for risk assessment. Serial monitoring was necessary to identify MRD in more than a quarter of patients. Furthermore, one-third of patients with +MRD disease had asymptomatic metastatic recurrence, highlighting the importance of staging scans in studies and clinical trials assessing therapeutic interception in the adjuvant setting. Citation Format: Arielle Medford, Elyssa Denault, Zanta Ephrem, Douglas Micalizzi, Yael Bar, Rachel Abelman, Amy Comander, Justine Knape, Minetta Liu, Angel Rodriguez, Steven Isakoff, Beverly Moy, Laura Spring, Aditya Bardia. Personalized ctDNA testing for detection of molecular residual disease in patients with localized HR+ breast cancer: temporal dynamics and impact on clinical outcomes [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-13-09.

Abstract PO3-07-06: Correlation between residual microcalcification and in-breast pathologic response in relation to MRI response and the subtypes after neoadjuvant chemotherapy

Abstract Background The significance of residual microcalcifications after neoadjuvant chemotherapy (NAC) in determining the extent of surgical intervention in breast cancer remains unclear. This study aimed to assess whether residual microcalcifications after NAC correlate with the residual disease in patients with breast cancer stratified by subtypes, particularly in excellent radiologic responders. Methods We conducted a retrospective study of patients with breast cancer who underwent NAC followed by curative surgery between January 2007 and December 2022. We evaluated the presence of suspicious microcalcifications on pre-NAC mammography and residual microcalcifications on post-NAC mammography. In the patients who had residual microcalcifications on post-NAC mammography, the rates of breast pathologic complete response (pCR), defined as the absence of invasive cancer (ypT0/is) or complete breast pCR defined as the absence of invasive cancer and in-situ cancer in the breast (ypT0), was assessed according to the MRI response and subtypes. We defined complete MRI response as the resolution of suspicious enhancement in the breast and axilla after NAC. Results Of the 1078 patients, 554 (51.4%) had suspicious microcalcifications at baseline, of which 518 (93.5%) had residual microcalcifications after NAC. Among the 518 patients with residual microcalcifications, 93 (18.0%) achieved a complete MRI response. We observed a difference in breast pCR rates depending on MRI response and hormone-receptor (HR) status. In the patients without complete MRI response, about one-third of patients (139 of 425 [32.7%]) achieved a breast pCR, irrespective of HR status (19.3% [47 of 244] in HR-positive breast cancer and 50.8% [92 of 181] in HR- negative breast cancer). Of the 93 patients who achieved a complete MRI response, the breast pCR rates were 64.3% (27 of 42) in HR-positive breast cancer and 96.1% (49 of 51) in HR-negative breast cancer, respectively. Moreover, the breast complete pCR rate was 54.8% (23 of 51) in HR-positive breast cancer and 88.2% (45 of 51) in HR-negative breast cancer, including 87.2% (34 of 39) in HR-HER2+ breast cancer and 91.7% (11 of 12) in triple-negative breast cancer. Conclusion Our results suggest that the clinical relevance of the residual microcalcifications differs according to the breast cancer subtypes. For patients with HR-positive breast cancer, removal of the residual microcalcifications should be considered to ensure complete cancer removal, regardless of MRI response. In contrast, de-escalating surgery may be considered in patients with HR-negative breast cancer who achieve complete MRI response after NAC. Citation Format: Soong June Bae, Seung Ho Baek, Seung Eun Lee, Min Ji Kim, Sohyun Moon, Junghyun Kim, Yoonwon Kook, Joon Jeong, Sung Gwe Ahn. Correlation between residual microcalcification and in-breast pathologic response in relation to MRI response and the subtypes after neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-07-06.

CtDNA as an Adjunct to Posttreatment PET for Head and Neck Cancer Recurrence Risk Assessment.

Circulating tumor DNA (ctDNA) detection is an emerging technique that identifies minimal residual disease in patients with solid tumors. ctDNA can act as an adjunct method to help overcome the limitations of positron emission tomography (PET) and select patients who are at high risk for recurrence. Retrospective Single Institutional Study. University Hospital Setting. Twenty-nine patients who underwent definitive treatment for squamous cell carcinoma of the head and neck (HNSCC) from 8/2021 to 01/2023 had ctDNA levels analyzed at 1 to 3, 6, 9, and 12 months after definitive treatment. A personalized, tumor-informed, multiplex polymerase chain reaction (PCR) next-generation sequencing (NGS) assay was used to detect the ctDNA levels. The primary outcome was recurrence-free probability (RFP), and the secondary outcomes were overall survival (OS), sensitivity, specificity, and the test's negative (NPV) and positive predictive values (PPV). The median age of patients was 65 years (interquartile range: 56-69), with majority being males (n = 22, 76%). The primary sites were larynx (n = 12), oropharynx (n = 10), and oral cavity (n = 6). Posttreatment ctDNA was detected in 7 patients, all of whom had disease recurrence. ctDNA detection after definitive treatment was associated with a higher risk of disease recurrence (hazard ratio: 9.94, 95% confidence interval: 1.56-63.3, P = .015). ctDNA identified recurrence with 100% specificity and 78% sensitivity. The NPV and PPV were 91% and 100%. PET had 78% sensitivity but only 68% specificity with 86% NPV, and 54% PPV. Based on our data, ctDNA can be an excellent adjunct test for posttreatment PET and can help guide physicians in cases where PET results are inconclusive and difficult to interpret.

Abstract 1221: Selective translational activation of PBK (PDZ- binding kinase) reveals a molecular vulnerability of drug-tolerant persister cells to targeted therapies in EGFR-mutant lung adenocarcinoma

Abstract In lung adenocarcinoma (LUAD), EGFR-targeted therapies often result in incomplete tumor responses followed by disease progression caused by acquired resistance. As EGFR mutations are truncal (i.e., present in every cell of a tumor), it remains unclear why a fraction of tumor cells can survive in the presence of drug treatment. It is increasingly recognized that drug-tolerant persister cells (DTPCs) can escape selective drug pressure and are important drivers of therapy failure and tumor relapse. The mechanisms underlying tumor cell adaptation and survival of DTPCs during therapy are not fully understood.Using EGFR-mutant LUAD cell lines treated with osimertinib, we unexpectedly observed that DTPCs display uncoupled mRNA and protein variations of PBK (PDZ Binding Kinase), a spindle assembly serine/threonine protein kinase. Mechanistically we discovered that although global protein synthesis is decreased upon osimertinib treatment, PBK translation is selectively maintained in DTPCs.The establishment and survival of DTPCs following EGFR inhibition is critically dependent on PBK, as genetic depletion of PBK reduces persistence to osimertinib treatment in vitro. PBK knockout leads to suppression of ERK1/2 reactivation following EGFR inhibition, and results in decreased expression of ZEB1 and Vimentin, key effectors of epithelial-to-mesenchymal transition (EMT), a hallmark of the DTPC phenotype. Consistently, combined treatment with osimertinib and OTS514 (PBK inhibitor) in vitro, reduces the DTPC fraction and decreases the enrichment of EMT-related transcriptional signatures. Similar results were obtained in LUAD patient-derived organoids, used to recapitulate the phenotype of residual disease in patients. In PDX treated with osimertinib, PBK is activated at both residual disease and acquired resistance. Concordantly, combined EGFR/PBK pharmacological inhibition was also effective in reverting osimertinib acquired resistance in vitro. Finally, in matched diagnosis and relapse samples from 7 EGFR-mutant LUAD patients treated with EGFR inhibitors, we observed increased PBK activation at the time of acquired resistance in 5 cases, including 3 EGFRT790M-positive cases, suggesting that PBK activation may co-occur with genetic resistant alterations.Our findings unveil a novel mechanism by which EGFR-mutant lung cancer DTPCs selectively translate PBK mRNA as an adaptive survival mechanism to persist during osimertinib treatment; and provide a therapeutic target of non-genetic drug resistance otherwise unlooked-for in routine transcriptomic analyses. A deeper understanding on the mRNA translational reprogramming in DTPCs is currently performed to uncover therapeutic vulnerabilities to reduce residual disease and delay or revert the emergence of acquired resistance. Citation Format: Camille Leonce, Anne-Laure Désage, Nour El-Houda Mourksi, Geneviève De Souza, Béatrice Vanbervliet, Camille Charrondière, Constance Nicq, Jérémy Néri, Aurélie Swalduz, Laurie Tonon, Eric Cumunel, Nicolas Gadot, Gabriel Ichim, Jean-Jacques Diaz, Christine Lovly, Julien Mazières, Mayeul Tabutin, Maurice Pérol, Sylvie Lantuejoul, Olivier Calvayrac, Fabien Forest, Virginie Marcel, Pierre Saintigny, Sandra Ortiz-Cuaran. Selective translational activation of PBK (PDZ- binding kinase) reveals a molecular vulnerability of drug-tolerant persister cells to targeted therapies in EGFR-mutant lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1221.

Longitudinal circulating tumor DNA monitoring for detection of molecular residual disease in patients with penile squamous cell carcinoma.

11 Background: Penile squamous cell carcinoma (SCC) is an extremely rare, aggressive disease, representing 0.4-0.6% of all malignancies in males in the U.S. and Europe. Early diagnosis is imperative, as lymphatic spread is associated with a dismal prognosis. The estimated 5-year survival rate in patients with pelvic lymph node involvement is 0%. Moreover, there are limited treatment options that carry significant toxicity risks. Accurate biomarkers to evaluate treatment response and guide treatment to improve outcomes are needed. Molecular residual disease monitoring with circulating tumor DNA (ctDNA) may be helpful in these scenarios. Methods: This is a retrospective analysis of a cohort of 7 patients with advanced HPV-unassociated penile SCC treated at Rush University Medical Center who monitored closely for molecular residual disease (MRD). Signatera (Natera Inc.), a personalized tumor-informed ctDNA assay, was utilized for detection and quantification of ctDNA in patients’ plasma samples, measured at regular intervals. Progression of disease was based on radiographic imaging. Results: In our cohort, personalized, tumor-informed ctDNA assays were designed for 100% (7/7) of the patients. ctDNA was detectable in at least one time point in all of the patients. One patient underwent partial penectomy and lymph node (LN) dissection with 1/11 LN involved. He is being monitored without adjuvant treatment and has sustained ctDNA clearance. Another patient underwent surgery, and had positive post-operative ctDNA. He was treated with adjuvant TIP (paclitaxel, ifosfamide, and cisplatin) with subsequent ctDNA clearance. In 3 of the patients, ctDNA was checked at regular intervals and rising ctDNA prompted imaging studies that showed progression. These patients proceeded to second line treatment options. The remaining 2 patients are non-surgical candidate with LN positivity, who will begin treatment with TIP and monitoring of ctDNA after each cycle of chemotherapy. Twenty-eight percent (2/7) of patients remain alive with sustained ctDNA clearance. Twenty-eight percent (2/7) of patients remain alive with ctDNA positivity, planned to initiate treatment and monitoring of ctDNA at regular intervals. Forty-three percent (3/7) of patients who had persistent ctDNA positivity are now deceased. Conclusions: Our data suggests that a personalized, tumor-informed ctDNA assay is not only useful for MRD monitoring, but also highly predictive of treatment response and imaging-based progression in patients with penile SCC. In our sample, MRD status correlated with both radiographic progression and clinical outcomes. Prospective studies are needed to better understand how ctDNA can be used to guide neoadjuvant and adjuvant approaches, including treatment intensification or de-escalation, in patients with penile SCC.

Deciphering stage 0 hematogones by flow cytometry in follow-up bone marrow samples of pediatric B-Acute lymphoblastic leukemia cases: A potential mimicker of residual disease after anti CD19 therapy.

CD19 is frequently targeted for immunotherapy in B cell malignancies, which may result in loss of CD19 expression in leukemic cells as an escape mechanism. Stage 0 hematogones (Hgs) are normal CD19-negative very early B cell precursors that can be potentially mistaken for CD19 negative residual leukemic cells by flow cytometry (FCM) in B cell acute lymphoblastic leukemia (BCP-ALL) cases treated with anti CD19 therapy. Our main objective was to characterize and study the incidence of stage 0 hematogones in follow-up bone marrow samples of pediatric BCP-ALL cases. We analyzed the flow cytometry standard files of 61 pediatric BCP-ALL cases treated with conventional chemotherapy and targeted anti-CD19 therapy, for identifying the residual disease and normal B cell precursors including stage 0 Hgs. A non-CD19 alternate gating strategy was used to isolate the B cells for detecting the residual disease and stage 0 Hgs. The stage 0 Hgs were seen in 95% of marrow samples containing CD19+ Hgs. When compared with controls and posttransplant marrow samples, the fraction of stage 0 Hgs was higher in patients receiving anti CD19 therapy (p = 0.0048), but it was not significant when compared with patients receiving chemotherapy (p = 0.1788). Isolated stage 0 Hgs are found in samples treated with anti-CD19 therapy simulating CD19 negative residual illness. Our findings aid in understanding the stage 0 Hgs and its association with CD19+ Hgs in anti CD19 therapy and conventional chemotherapy. This is crucial as it can be potentially mistaken for residual disease in patients treated with anti CD19 therapy.

Monitoring Minimal Residual Disease in Patients with Multiple Myeloma by Targeted Tracking Serum M-Protein Using Mass Spectrometry (EasyM).

We investigated both the clinical utilities and the prognostic impacts of the clonotypic peptide mass spectrometry (MS)-EasyM, a blood-based minimal residual disease (MRD) monitoring protocol in multiple myeloma. A total of 447 sequential serum samples from 56 patients with multiple myeloma were analyzed using EasyM. Patient-specific M-protein peptides were sequenced from diagnostic samples; sequential samples were quantified by EasyM to monitor the M-protein. The performance of EasyM was compared with serum immunofixation electrophoresis (IFE), bone marrow multiparameter flow cytometry (MFC), and next-generation flow cytometry (NGF) detection. The optimal balance of EasyM sensitivity/specificity versus NGF (10-5 sensitivity) was determined and the prognostic impact of MS-MRD status was investigated. Of the 447 serum samples detected and measured by EasyM, 397, 126, and 92 had time-matching results for comparison with serum IFE, MFC-MRD, and NGF-MRD, respectively. Using a dotp >0.9 as the MS-MRD positive, sensitivity was 99.6% versus IFE and 100.0% versus MFC and NGF. Using an MS negative cutoff informed by ROC analysis (<1.86% of that at diagnosis), EasyM sensitivity remained high versus IFE (88.3%), MFC (85.1%), and NGF (93.2%), whereas specificity increased to 90.4%, 55.8%, and 93.2%, respectively. In the multivariate analysis, older diagnostic age was an independent predictor for progression-free survival [PFS; high risk (HR), 3.15; 1.26-7.86], the best MS-MRD status (MS-MRD negative) was independent predictor for both PFS (HR, 0.25; 0.12-0.52) and overall survival (HR, 0.16; 0.06-0.40). EasyM is a highly sensitive and minimal invasive method of MRD monitoring in multiple myeloma; MS-MRD had significant predictive ability for survival outcomes.

Impact of pretransplant minimal residual disease in patients with multiple myeloma and a very good partial response or better receiving autologous hematopoietic stem cell transplantation.

The prognostic significance of minimal residual disease (MRD) status before autologous hematopoietic stem cell transplantation (autoHCT) in patients with multiple myeloma (MM) has not been clearly elucidated. Retrospective single-center study of adult MM patients who achieved ≥very good partial response (VGPR) after induction therapy from 2015 to 2021 received upfront autoHCT and had available pretransplant MRD status by next-generation flow cytometry. The cohort was divided into pretransplant MRD-negative (MRDneg) and MRD-positive (MRDpos) groups. A total of 733 patients were included in our analysis; 425 were MRDneg and 308 MRDpos at autoHCT. In the MRDpos group, more patients had high-risk cytogenetic abnormalities (48% vs. 38%, respectively; p=.025), whereas fewer patients achieved ≥CR before autoHCT (14% vs. 40%; p<.001). At day 100 after autoHCT, 37% of the MRDpos versus 71% of the MRDneg achieved ≥CR, and at best posttransplant response 65% versus 88% achieved ≥CR, respectively. After a median follow-up of 27.6months (range, 0.7-82.3), the median PFS was significantly shorter for patients in the MRDpos group compared to the MRDneg group: 48.2months (95% confidence interval [CI], 0.3-80.5) versus 80.1months (95% CI, 0.5-80.1), respectively (p<.001). There was no significant difference in overall survival between the two groups (p=.41). Pretransplant MRDpos status was predictive of shorter PFS in multivariate analysis (hazard ratio, 1.80; 95% CI, 1.31-2.46; p<.001). The impact of pretransplant MRD status was retained in most of the examined subgroups. In patients achieving ≥VGPR to induction, pretransplant MRDpos status was associated with a lower CR rate after autoHCT and a shorter PFS.

Prediction of disease recurrence or residual disease after primary endoscopic resection of pT1 colorectal cancer—results from a large nationwide Danish study

PurposeRisk assessment of disease recurrence in pT1 colorectal cancer is crucial in order to select the appropriate treatment strategy. The study aimed to develop a prediction model, based on histopathological data, for the probability of disease recurrence and residual disease in patients with pT1 colorectal cancer.MethodsThe model dataset consisted of 558 patients with pT1 CRC who had undergone endoscopic resection only (n = 339) or endoscopic resection followed by subsequent bowel resection (n = 219). Tissue blocks and slides were retrieved from Pathology Departments from all regions in Denmark. All original slides were evaluated by one experienced gastrointestinal pathologist (TPK). New sections were cut and stained for haematoxylin and eosin (HE) and immunohistochemical markers. Missing values were multiple imputed. A logistic regression model with backward elimination was used to construct the prediction model.ResultsThe final prediction model for disease recurrence demonstrated good performance with AUC of 0.75 [95% CI 0.72–0.78], HL chi-squared test of 0.59 and scaled Brier score of 10%. The final prediction model for residual disease demonstrated medium performance with an AUC of 0.68 [0.63–0.72].ConclusionWe developed a prediction model for the probability of disease recurrence in pT1 CRC with good performance and calibration based on histopathological data. Together with lymphatic and venous invasion, an involved resection margin (0 mm) as opposed to a margin of ≤ 1 mm was an independent risk factor for both disease recurrence and residual disease.

Monitoring Minimal Residual Disease in Patients with Multiple Myeloma By Tracking Serum M-Protein Using Mass Spectrometry

Objective： We performed this retrospective real-world study to evaluate the clinical utility of blood-based mass spectrometry for longitudinal M-protein monitoring during the disease course of patients of multiple myeloma（MM）. Methods：We initially analyzed serial serum samples of 56 patients with MM by clonal peptide MS Method named EasyM MS, then we compared the MS results with those determined using serum immunofixation electrophoresis (IFE),multiparameter flow cytometry (MFC), next-generation flow cytometry (NGF), to establish the concordance between different disease-monitoring protocols. A sensitivity for MFC MRD negativity of at least 1×10 -4 anda sensitivity for NGF MRD negativity of at least 1×10 -5.Additionally, we determined the optimal cutoff value of EasyM MS negativity to satisfy both the sensitivity and specificity between MS and NGF, and explored the prognostic impact of MS-MRD status. Results：The median diagnostic age of 56 NDMM patients was 56 years old, including 34 males (60.7%). There were 38 patients with IgG type MP and 18 patients with IgA type MP. 47 patients (83.9%) chose to receive proteasome inhibitor - based induction therapy, and a total of 44 patients (78.6%) received first-line ASCT treatment.Among 56 MM patients，38 patients with IgG type MP can further identify IgG subclasses through mass spectrometry. Additionally, 56 patients with MM have successfully used MS to identify their unique clonal peptides of MP. 56 patients provided a total of 447 samples of serum and all sequential serum samples were successfully measured by using the MS method. The initial comparison of the performance of blood-based MS-MRD with that of the conventional M-protein diagnostic method IFE used . Among the 397 comparable results collected at similar timepoints, 70.8% (281/397) of the samples were both M-MRD- and IFE-positive, 3.3% (13/397) were both MS-MRD- and IFE-negative, 25.7% (102/397) were MS-MRD-positive and IFE-negative.Flow cytometry MRD was performed on 218 bone marrow aspirates in this cohort, including 126 analyzed with MFC and 92 analyzed with NGF. MS-MRD detection was also performed on comparable serum acquired at the same time points. The sensitivity of MS-MRD was 100.0% in comparison with both MFC and NGF, with specificity of 1.9% and 22.2%, respectively. Considering the overly high sensitivity of EasyM at a cutoff of dotp &amp;gt;0.9, we used the sensitivity of NGF as the standard for detection (i.e., a detection sensitivity ≤10 -5) and set the EasyM MS negative cutoff value using ROC analysis, determined to be &amp;lt;1.86% of that at first diagnosis. Using these assumptions, we again performed the aforementioned analyses comparing the sensitivity and specificity between EasyM and IFE, MFC, and NGF. Under this scenario, the sensitivity of EasyM remained high in comparison with IFE (88.3%), MFC (85.1%), and NGF (93.2%), but the specificity was also greatly increased to 90.4%, 55.8%, and 93.2%, respectively. We then divided patients into MS-positive and MS-negative groups according to whether they achieved MS-MRD negativity, and median PFS and OS were calculated for each group. The median PFS of the MS-positive and MS-negative groups was 20.6 (95% CI: 16.6, 24.6) and 61.8 (39.7, 83.9) months, respectively . The median OS was 55.7 (95% CI:31.8, 79.5) months for the MS-positive group and not reached (NR) for the MS-negative group ,Both mPFS and mOS were significantly longer among the MS-negative group compared with the MS-positive group (both p &amp;lt; 0.001). Conclusion：EasyM exhibits high sensitivity and minimally invasive technological advantages, and also has important significance in efficacy evaluation and prognosis evaluation. In the future, EasyM MS detection can be used as a minimally invasive MRD monitoring method based on peripheral blood in clinical practice.