Aggressive Disease Course Research Articles

Methylation Signature Implicated in Immuno-Suppressive Activities in Tubo-Ovarian High-Grade Serous Carcinoma.

Better understanding of prognostic factors in tubo-ovarian high-grade serous carcinoma (HGSC) is critical, as diagnosis confers an aggressive disease course. Variation in tumor DNA methylation shows promise predicting outcome, yet prior studies were largely platform-specific and unable to evaluate multiple molecular features. We analyzed genome-wide DNA methylation in 1,040 frozen HGSC, including 325 previously reported upon, seeking a multi-platform quantitative methylation signature that we evaluated in relation to clinical features, tumor characteristics, time to recurrence/death, extent of CD8+ tumor-infiltrating lymphocytes (TIL), gene expression molecular subtypes, and gene expression of the ATP-binding cassette transporter TAP1. Methylation signature was associated with shorter time to recurrence, independent of clinical factors (N = 715 new set, hazard ratio (HR), 1.65; 95% confidence interval (CI), 1.10-2.46; P = 0.015; N = 325 published set HR, 2.87; 95% CI, 2.17-3.81; P = 2.2 × 10-13) and remained prognostic after adjustment for gene expression molecular subtype and TAP1 expression (N = 599; HR, 2.22; 95% CI, 1.66-2.95; P = 4.1 × 10-8). Methylation signature was inversely related to CD8+ TIL levels (P = 2.4 × 10-7) and TAP1 expression (P = 0.0011) and was associated with gene expression molecular subtype (P = 5.9 × 10-4) in covariate-adjusted analysis. Multi-center analysis identified a novel quantitative tumor methylation signature of HGSC applicable to numerous commercially available platforms indicative of shorter time to recurrence/death, adjusting for other factors. Along with immune cell composition analysis, these results suggest a role for DNA methylation in the immunosuppressive microenvironment. This work aids in identification of targetable epigenome processes and stratification of patients for whom tailored treatment may be most beneficial.

Colorectal adenosquamous carcinoma: genomic profiling of a rare histotype of colorectal cancer

Colorectal adenosquamous carcinoma (ASC) is exceedingly rare, comprising less than 0.1% of all colorectal malignancies, and is characterized by an aggressive disease course, with a higher metastatic rate and worse outcome than conventional colorectal adenocarcinoma. A comprehensive molecular profile of this group of neoplasms is still lacking. A total of 22 cases of colorectal ASCs (with 22 primary lesions and 7 metastases matched with 4 primaries) were subject to NGS targeting 67 cancer-related genes (VariantPlex solid tumor; Archer). Mismatch repair (MMR), p53, and V600EBRAF status were also investigated by immunohistochemistry. In 28 of 29 (96.6%) ASC samples, at least one single-nucleotide variant (SNV) or copy number variation (CNV) was detected. Among the 22 primary tumors, the most frequently mutated genes were TP53 (59.1%), APC (40.9%), KRAS (27.3%), BRAF (13.6%), and GNAS (9.1%). Only 1/22 (4.5%) primary ASC was MMR-deficient (MMRd) and harbored a BRAF mutation. Limited differences in SNVs were observed between primary and metastatic diseases. This study sheds light on the molecular landscape of colorectal ASCs. According to our data, the genomic profile of colorectal ASC is similar to that of conventional colorectal carcinoma, with significant druggable genetic alterations. Further studies are required to understand the more aggressive clinical behavior of this neoplasm.

Characteristics, treatment, and outcome of patients with bowel perforation after immune checkpoint inhibitor exposure.

Exposure to immune checkpoint inhibitors (ICIs) can predispose to immune-related adverse events (irAEs) involving the gastrointestinal tract. The association between ICIs and bowel perforation has not been well studied. We aimed to describe the clinical course, complications, treatment, and outcomes of patients experiencing bowel perforation during or after ICI treatment. This retrospective, single-center study included adult cancer patients with bowel perforation that occurred between the first dose of ICI treatment and up to 1year thereafter between 1/1/2010 and 4/30/2021. Patients' clinical course, imaging, treatment, and outcomes related to bowel perforation were collected and analyzed. Of the 13,991 patients who received ICIs during the study period, 90 (0.6%) met the inclusion criteria. A majority were male (54.4%), the most common cancer type was melanoma (23.3%), and most patients had received PD-1/L1 inhibitor treatment (58.8%). Onset of perforation occurred after a median of four ICI treatment cycles. The most common symptom was abdominal pain (95.5%). The colon was the most common location for the perforation (37.7%). Evidence of diverticulitis, enterocolitis, or appendicitis was seen in 32 (35.6%) patients, and 6 (6.6%) patients had luminal cancer involvement at the time of perforation. The overall hospitalization rate related to perforation was 95.5%, with mortality of 15.5% during the same admission. Antibiotics were given in 95% of our sample; 37.8% of patients also required surgical/interventional radiology intervention. Forty-six patients (51.1%) had perforation-related complications (e.g., sepsis, fistula, abscess), which were associated with a higher mortality rate (30%). Our findings suggest a low incidence of bowel perforation after ICI treatment (0.6%), with 40% of patients having coexisting bowel inflammation as a potential contributing factor. Patients with bowel perforation had an aggressive disease course and high rates of hospitalization, complications, and mortality. Early recognition and prompt intervention is critical to improve patient outcomes. Future studies are warranted to further investigate the cause, predictive markers, and optimal treatment for this patient population.

Abstract C065: Kinesin family member C1 (KIFC1/HSET) underlies aggressive disease course in androgen receptor-low triple negative breast cancers

Abstract Quadruple negative breast cancer (QNBC), which refers to triple negative breast cancers (TNBCs) lacking androgen receptor (AR) expression, is now considered to be a highly aggressive BC subtype, with no actionable therapeutic targets. QNBC disproportionately afflicts and impacts patients of African descent. Kinesin family member C1 (KIFC1/HSET), a centrosome clustering protein that prevents cancer cells from undergoing centrosome amplification-induced apoptosis, has been reported to be upregulated in TNBCs and African-American (AA) TNBCs, compared to non-TNBCs and European-American (EA) TNBCs. Herein, we analyzed KIFC1 mRNA levels and their associations with clinical features and outcomes among AR-low and AR-high TNBC patients in three distinct publicly-available gene expression datasets and the Breast cancer gene expression database (bc-GenExMiner). We discovered that KIFC1 levels were significantly higher (p<0.05) in AR-low and basal-like TNBCs compared to AR-high and luminal androgen receptor TNBCs, irrespective of stage, grade, tumor size, and lymph node status. We also observed that KIFC1 levels were upregulated in AR-low versus AR-high black and premenopausal TNBC patients (p<0.05). Furthermore, AR levels negatively correlated with KIFC1 levels among TNBC and basal-like TNBC patients (p<0.0001). High KIFC1 levels conferred significantly shorter overall survival (OS), disease-free survival, and distant metastasis-free survival among AR-low and basal-like TNBC patients in Kaplan-Meier analyses (p<0.05). In conclusion, KIFC1 levels may be upregulated in QNBCs and specifically QNBCs of African descent, wherein it may promote poorer outcomes. In addition, therapeutically targeting KIFC1 in these patient subpopulations could significantly mitigate racial disparities in TNBC outcomes. Citation Format: Nikita Wright, Yuan Yate-Ching, Tijana Talisman, Rama Natarajan, Mark LaBarge, Padmashree Rida, Victoria Seewaldt. Kinesin family member C1 (KIFC1/HSET) underlies aggressive disease course in androgen receptor-low triple negative breast cancers [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C065.

Structural and microstructural neuroimaging signature of C9orf72-associated ALS: A multiparametric MRI study

BackgroundALS patients with hexanucleotide expansion in C9orf72 are characterized by a specific clinical phenotype, including more aggressive disease course and cognitive decline. Computerized multiparametric MRI with gray matter volumetry and diffusion tensor imaging (DTI) to analyze white matter structural connectivity is a potential in vivo biomarker. ObjectiveThe objective of this study was to develop a multiparametric MRI signature in a large cohort of ALS patients with C9orf72 mutations. The aim was to investigate how morphological features of C9orf72-associated ALS differ in structural MRI and DTI compared to healthy controls and ALS patients without C9orf72 mutations. MethodsAtlas-based volumetry (ABV) and whole brain-based DTI-based analyses were performed in a cohort of n = 51 ALS patients with C9orf72 mutations and compared with both n = 51 matched healthy controls and n = 51 C9orf72 negative ALS patients, respectively. Subsequently, Spearman correlation analysis of C9orf72 ALS patients‘ data with clinical parameters (age of onset, sex, ALS-FRS-R, progression rate, survival) as well as ECAS and p-NfH in CSF was performed. ResultsThe whole brain voxel-by-voxel comparison of fractional anisotropy (FA) maps between C9orf72 ALS patients and controls showed significant bilateral alterations in axonal structures of the white matter at group level, primarily along the corticospinal tracts and in fibers projecting to the frontal lobes. For the frontal lobes, these alterations were also significant between C9orf72 positive and C9orf72 negative ALS patients. In ABV, patients with C9orf72 mutations showed lower volumes of the frontal, temporal, and parietal lobe, with the lowest values in the gray matter of the superior frontal and the precentral gyrus, but also in hippocampi and amygdala. Compared to C9orf72 negative ALS, the differences were shown to be significant for cerebral gray matter (p = 0.04), especially in the frontal (p = 0.01) and parietal lobe (p = 0.01), and in the thalamus (p = 0.004). A correlation analysis between ECAS and averaged regional FA values revealed significant correlations between cognitive performance in ECAS and frontal association fibers. Lower FA values in the frontal lobes were associated with worse performance in all cognitive domains measured (language, verbal fluency, executive functions, memory and spatial perception). In addition, there were significant negative correlations between age of onset and atlas-based volumetry results for gray matter. ConclusionsThis study demonstrates a distinct pattern of DTI alterations of the white matter and ubiquitous volume reductions of the gray matter early in the disease course of C9orf72-associated ALS. Alterations were closely linked to a more aggressive cognitive phenotype. These results are in line with an expected pTDP43 propagation pattern of cortical affection and thus strengthen the hypothesis that an underlying developmental disorder is present in ALS with C9orf72 expansions. Thus, multiparametric MRI could contribute to the assessment of the disease as an in vivo biomarker even in the early phase of the disease.

Review of the Developing Landscape of Prostate Biopsy and Its Roles in Prostate Cancer Diagnosis and Treatment.

Prostate cancer remains a significant global health challenge. Traditionally anchored by the Gleason score/Grade Group (GS/GG), the landscape of prostate cancer diagnosis is undergoing transformative steps, particularly in the domain of biopsy procedures. GS/GG continues to be pivotal in malignancy grading, but recent technological strides have augmented the diagnostic relevance of biopsies. Integral to this progression is the adoption of advanced imaging techniques, especially magnetic resonance imaging, which has refined biopsy accuracy and efficiency. A deep understanding of prostate cancer pathology reveals a cribriform pattern and intraductal carcinoma of the prostate as independent forms of malignancy, suggesting a potentially aggressive disease course. Furthermore, the distinct behaviour of ductal adenocarcinoma and small cell carcinoma of the prostate, compared with acinar adenocarcinoma, necessitates their accurate differentiation during biopsy. The genomic era ushers in a renewed emphasis on tissue samples obtained from prostate biopsies, especially as mutations in genes, such as BRCA1/2, and paves the way for precision medicine. This review encapsulates the evolving dynamics of prostate biopsy, from technological advancements to the profound implications on prostate cancer management and therapy.

Palliative care and hepatobiliary malignancies: say no to late referral.

Hepatobiliary malignancies (HBMs), primarily hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), share the common traits of having a generally poor prognosis, late presentation, and high symptom burden related to both the disease process itself and underlying poor liver function. The incidences of both malignancies have been rising in recent decades for unclear reasons. Curative options remain limited given the general aggressive disease course despite advances in diagnosis, therapies, and surgery. Early integration of palliative care into the routine care of patients with HBMs is an essential, but underutilized, component of care to improve the functional and symptomatic quality of the lives of patients and their families. While formal guidelines for its integration are currently lacking, palliative care can and should be provided in parallel to disease specific care at any stage to address the physical, emotional, and spiritual needs of patients with HBMs. In this review, the special needs of this patient population are examined ranging from early symptom management at the time of diagnosis all the way through to end-oflife care. Key barriers that prevent the early provision of palliative care for patients with HBMs are identified and discussed and recommendations provided on how to improve early integration.

Development of the B cell cancer vaccine HER-vaxx for the treatment of her-2 expressing cancers.

Her-2/neu is a tumor-associated protein that is overexpressed in a number of malignancies, including advanced cancer of the stomach, and has been proposed as a human cancer vaccine target. Overexpression of Her-2/neu in human breast and gastric carcinomas correlates with a more aggressive course of disease that results in poorer overall survival rates and shorter times to disease progression than in patients with tumors without overexpression of Her-2/neu. Cancer vaccines have the ability to stimulate the native immune system and in particular engineered B cell epitopes can elicit high affinity polyclonal antibodies with similar efficacy to Her-2 monoclonal antibodies such as trastuzumab (Roche). HER-Vaxx is under development as a therapeutic B cell vaccine for the treatment of gastric cancer in patients with Her-2/neu overexpressing metastatic or advanced adenocarcinoma of the stomach or gastroesophageal junction, referred to as advanced cancer of the stomach. P467-CRM197, the vaccine's immunogenic component, contains a single peptide antigen composed of 3 individual linear B cell epitope peptide sequences selected from the oncoprotein Her-2/neu that induce the patient's own B cells to produce endogenous anti-Her-2/neu antibodies. This review provides results from comprehensive preclinical studies encompassing primary and secondary pharmacodynamics, biodistribution and safety studies. These studies were performed to support clinical development of HER-Vaxx. Results from the GLP toxicology study in rodents showed that the vaccine did not produce any observable adverse effects suggesting that the doses proposed for the clinical trial should be well tolerated in patients.

High or low? Assessing disease risk in multiple myeloma

Based upon the development of highly effective therapies such as immunomodulatory drugs, proteasome inhibitors, and monoclonal antibodies that target plasma cell biology, a dramatic improvement in overall survival has been observed for most patients with multiple myeloma (MM) over the past 2 decades. Although it is now commonplace for many patients with myeloma to live in excess of 10 years after diagnosis, unfortunately a large subset of patients continues to experience an aggressive disease course marked by substantial morbidity and early mortality. Many clinical biomarkers and staging systems in use today can help with prognostication, but accurate risk assessment can be difficult due to the presence of many different biomarkers with variable prognostic value. Furthermore, with the implementation of novel therapies and unprecedented rates of deep and durable responses, it is becoming apparent that risk assessment is best envisioned as a dynamic process that requires ongoing reevaluation. As risk and response-adapted approaches are becoming more commonplace, it is essential that clinicians understand the biological and prognostic implications of clinical, genomic, and response-based biomarkers in order to promote management strategies that will help improve both survival and quality of life for patients across the risk spectrum.

Tau‐derived Locus coeruleus degeneration as a driver for sleep‐wake alterations and neuropsychiatric symptoms in early and late‐onset Alzheimer’s Disease

AbstractBackgroundEarly‐onset Alzheimer’s Disease presentations (EOAD, under 65) frequently present with atypical phenotypes and a more aggressive disease course with a higher burden of neuropsychiatric symptoms than late‐onset AD (LOAD). Current treatments for sleep and behavioral disturbances are still non‐specific, causing side effects (e.g., sedation, falls). Identifying the underlying changes driving behavioral differences between EOAD and LOAD is crucial to developing tailored treatment avenues. The noradrenergic locus coeruleus (LC), one of the first sites of tau deposition in AD, has been implicated in sleep‐wake patterns and mood regulation. We aim to test the hypothesis that the LC is more affected in EOAD than LOAD by comparing LC volume (neuromelanin‐sensitive MRI) and sleep‐behavioral symptoms in biomarker‐confirmed EOAD and LOAD cohorts.MethodFifty‐four subjects with AD biomarker‐based diagnosis (20 EOAD, 34 LOAD) were recruited at the Hospital Clínic de Barcelona. All participants and informants completed the Epworth Sleepiness Scale (ESS), Pittsburgh Sleep Quality Index (PSQI), and Neuropsychiatric Inventory (NPI) questionnaires to assess the severity of sleep‐wake alterations and neuropsychiatric symptoms. In addition, they underwent a 3T turbospin‐echo MRI to measure LC volume. We compared mean values of LC volume, ESS, PSQI, and NPI between EOAD and LOAD. Furthermore, linear regression models controlling by cognitive status (MMSE) were performed.ResultEOAD and LOAD had similar cognition (MMSE 21.3±5 vs. 22.6±4, respectively), functional status (CDR 0.92±0.1 vs. 0.68±0.1), and prevalence of amnestic phenotype (57 vs. 70%). EOAD compared to LOAD, trended towards higher scores for ESS (7.4±1 vs. 5.1±1, respectively), PSQI (7.3±2 vs. 5.6±1), NPI (21.6±8 vs. 14.6±8), and caregiver distress (9.4±3 vs. 4.7±3). LC volume was lower in EOAD according to preliminary MRI data [n=18, 9 EOAD (21.8±3 mm3), 9 LOAD (29.5±3 mm3)]. Linear regression models showed that MMSE did not influence the EOAD/LOAD effect on LC volume (coef. 7.7, p=0.032).ConclusionThe current preliminary study suggests that LC degeneration is greater in EOAD than LOAD. This difference may explain the EOAD‐associated worse sleep‐wake dysfunction and neuropsychiatric symptoms. Deep phenotyping/comparison of EOAD and LOAD can inform tailored treatment strategies for these behavioral symptoms.

NOVEL VIRTUAL REALITY‐ BASED METHOD FOR OBJECTIVE MEASUREMENT OF EMOTIONAL REACTIVITY IN PATIENTS WITH ALZHEIMER'S DISEASE

AbstractBackgroundDiminished emotional reactivity in cognitively normal (CN) older adults and in patients with Alzheimer's disease (AD) is associated with higher burden of neuropathology and predicts faster cognitive decline, stressing its potential to serve as a behavioral screening tool for individuals at high AD‐risk and for a more aggressive disease course. Its measurement is currently based on questionnaires which are prone to subjectivity biases. We present preliminary results of a study aimed to examine the feasibility of using autonomic nervous system (ANS) reactivity to emotional stimuli presented in a virtual reality (VR) environment, as an objective measurement tool of emotional reactivity. Specifically, we test the hypothesis that emotional reactivity, as expressed by galvanic skin response (GSR), will be reduced in patients with AD compared to CN individuals.MethodParticipants are patients with mild‐ moderate AD and cognitively normal age matched controls. Cognitive function is assessed via the Montreal Cognitive Assessment (MoCA) battery. Participants are exposed in the VR‐ environment to emotionally‐ laden stimuli (positive, neutral and aversive, 12 repetitions per stimulus type) while measuring GSR. Response weighted mean was calculated for each participant for each stimulus type. Non parametric group comparisons (AD vs. CN) were performed (U‐test) for each stimulus type separately (Bonferroni corrected).ResultSo far, 30 patients with AD and 21 CN individuals have been recruited. Participants' mean age is 75.6 years, 60.8 % females (Table 1). Patients with AD had lower scores on the MoCA (p<0.001), but did not differ from CN in demographic variables. VR‐based evaluation was well accepted by the participants with no adverse events. CN participants showed higher GSR reactivity as compared to AD patients for aversive stimuli (p<0.05) but not for neutral or positive stimuli (Figure 1).ConclusionGSR reactivity to emotional stimuli presented in the VR environment is a feasible method to study emotional reactivity in CN older adults and in participants with AD. GSR reactivity to aversive stimuli is reduced in AD participants compared to CN. These preliminary findings should be further established in larger populations while detecting the optimal type of stimulus required to differentiate between clinically distinguished cohorts.

Functional Drug Screen Identifies Thiostrepton, NAMPT Inhibitors and Metformin As Potential Candidates to Target the Macrophage Tumor Microenvironment in Acute Myeloid Leukemia

While it is increasingly becoming clear that cancer cells reside in symbiosis with diverse stromal and immune cells, the tumor supportive microenvironment (TSM) in acute myeloid leukemias (AML) remains poorly understood. We and others previously identified that the presence of tumor-supportive macrophages (often with an M2-like phenotype) was associated with poor prognosis and a more aggressive course of disease in vivo using various AML models. Therefore, tools to target the tumor supportive microenvironment, or to convert an M2-polarized TSM into a tumor suppressive one, are of great interest. Here, we investigated the effects of therapeutic drugs on AML blasts and their TSM specifically focusing on the macrophage compartment. Drug screens using 20 candidate compounds including metabolic inhibitors (targeting glycolysis and mitochondrial respiration) were performed on human AML cell lines (n=16), primary AML samples (n=52), and CD34+ cells from healthy cord blood donors (n=5). Experiments were performed in co-culture using MS5 stromal cell as a supportive feeder. Effects on cell viability and mitochondrial membrane potential (MMP) were evaluated by flow cytometry on blasts (CD45+CD34+) as well as on M1 or M2 macrophages (CD80+ or CD163+CD206+ within the CD45+CD34- fraction). Effects on glycolysis and oxygen consumption were evaluated by Seahorse. Human primary monocytes isolated from healthy donors were also used for co-culture experiments and M1 and M2 macrophages were generated as previously described (Weinhäuser et al, 2022). We identified that conventional drugs such as cytarabine and venetoclax had significant cytotoxic effects on AML blasts, albeit with heterogeneous efficacies dependent on AML subtype, while M2 macrophages remained largely unaffected. Functionally, M2 populations displayed higher MMP levels compared to AML blasts, presumably contributing to their increased drug resistance. Among all the evaluated compounds, only thiostrepton (an antibiotic that also inhibits FOXM1), KPT-9274, and metformin displayed cytotoxic effects on both the AML blasts and M2 macrophages, associated with reductions of MMP levels in both compartments. Various studies have aimed to identify compounds that would repolarize tumor supportive M2 macrophages into tumor suppressive M1 macrophages, including thiostrepton. Using healthy M2 macrophages, we could not detect consistent signs of re-polarization (evaluated by flow cytometry and gene expression profile) upon thiostrepton treatment. In fact, none of the 20 tested compounds was able to efficiently repolarize M2 into M1 macrophages. Furthermore, when we evaluated the effects of thiostrepton pretreated M2 macrophages on short-term and long-term AML cultures, we could not detect a significant reduction in AML cell viability or proliferation as observed for M1 co-cultures. However, we did notice a pronounced cytotoxic effect on the M2 macrophages themselves when a higher dose of thiostrepton (>5 µM) was administered. Metabolically, thiostrepton-treated macrophages also displayed decreased mitochondrial respiration. Next, we determined the ED50 of a large panel of AML cell lines (n=16), which ranged from 1.91-38.67 µM suggesting that tumor cells themselves display considerable sensitivity to thiostrepton. Cell line specific ED50 values were correlated to their transcriptomes and gene set enrichment studies revealed that thiostrepton resistance was associated with the term "ACTIVATION OF GENE EXPRESSION BY SREBF and SREBP", suggesting that fatty acid metabolism can be targeted to enhance thiostrepton efficiency. Surprisingly, the expression of its target FOXM1 did not correlate with the cytotoxic effects induced by thiostrepton in AML cells but was down-regulated in M2-macrophages upon treatment. Thiostrepton-treated healthy CD34+ cells remained largely unaffected in terms of cell viability and differentiation capacity, suggesting that thiostrepton may have a favorable therapeutic window. In conclusion, our screening setup allows for the identification of compounds that can target both leukemic blasts as well as their tumor supportive microenvironment, and thiostrepton, KPT-9274 and metformin emerged as potentially promising candidates. We hope that (combinatorial) targeted drug treatments that also take the tumor microenvironment into account can help to improve AML treatment and prevent relapse. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Rorɣ/SREBP2 Pathway Is a Master Regulator of Cholesterol Metabolism and Serves As Potential Therapeutic Target in T(4;11) Leukemia

Chromosomal translocations involving the KMT2A gene (KMT2Ar leukemia) occur in nearly 5 to 10% of acute leukemia and are associated with an aggressive course of disease and poor prognosis. The most common translocation partner of the KMT2A gene is the AF4 gene inducing a t(4;11) leukemia. It is known that a variety of cancer cells change their cholesterol homeostasis among other metabolic reprogramming leading to increased proliferation and survival. Therefore, dysregulated cholesterol metabolism promotes not only tumorigenesis and chemotherapy resistance, but also constitutes a new hallmark of cancer. The importance of understanding the connection between metabolic changes and leukemia development is necessary to identify potential targets and to develop new therapeutic strategies. By using our recently established CRISPR/Cas9-generated t(4;11) model, we identified the sterol regulatory element-binding protein 2 (SREBP2) as key transcription factor being highly upregulated. RAR-related orphan receptor gamma (RORɣ) has been reported to be a possible upstream regulator of SREBP2 but less is known about its function in the metabolic development of KMT2Ar leukemia and its role as therapeutic target. SREBF2 gene expression, measured via RT-qPCR, showed a strong upregulation in leukemic blasts of KMT2Ar patients, cell lines (SEM, RS4;11, MV4-11) and in our t(4;11) model. Furthermore, we found that the significant upregulation of SREBP2 in KMT2Ar leukemia correlates with poor patient survival (gent2, bloodspot.eu). To test whether RORɣ plays a role in hematopoiesis and leukemogenesis we examined intranuclear protein expression via flow cytometry. Cells of our t(4;11) leukemia model generally showed a stable RORɣ expression around 75%, while protein expression in CD34+ hematopoietic stem and progenitor cells (HSPCs) decreased upon differentiation in myeloid culture. We detected anti-leukemic effects of the SREBP2 inhibitor fatostatin (FS), HMGCR inhibitor atorvastatin (ATV) and the newly developed RORɣ antagonist XY018. All compounds induced leukemic cell death in a dose- and time-dependent manner while significantly changing cholesterol-target gene expression (HMGCR, HMGCS1, LDLR, ABCA1, SQLE, MSMO1, FDFT1, SC5D). Strikingly, the strongest effect was observed after XY018 treatment. Moreover, FS and ATV also affected cell growth of healthy HSPCs while anti-proliferative effects of XY018 were restricted to t(4;11). In addition to reduced cell viability and increased apoptosis upon RORɣ inhibition, we observed significant changes in the cell cycle. Genome-wide transcriptomic analysis by RNA-Seq identified SREBF2 as upstream regulator of these effects. Ongoing studies will address the molecular mechanism by which RORɣ and its downstream target SREBP2 regulate t(4;11) leukemogenesis. Combinatorial treatment studies of t(4;11) leukemia cells with XY018 showed promising additive effects (CI=0.90) together with the chemotherapeutic cytarabine and synergistic effects (CI=0.44) were achieved by adding ATV. In summary, our studies showed that t(4;11) leukemia cells exhibit an activated SREBP2-mediated cholesterol metabolism strongly linked to the nuclear receptor RORɣ. Moreover, we demonstrated that this metabolic reprogramming could serve as a therapeutic target with co-treatment of both cholesterol-modulating agents and common cytostatic drugs to improve the poor outcome of KMT2Ar leukemia patients.

Progression of Disease within 5 Months of Adult T-Cell Leukemia-Lymphoma (ATL5) As a Clinical Endpoint for High-Risk Mortality Outcomes in the Real-World Setting: A Multinational Cohort Analysis

Introduction Adult T-cell leukemia-lymphoma (ATL) is an aggressive hematological malignancy associated to poor outcomes. Most of these patients experience a rapid and aggressive disease course characterized by early progression and death. Previous studies have identified clinical and laboratory factors of prognostic value. However, their incorporation in the clinical setting has not been validated due to unstandardized management practices. Risk stratification based on time to progression has not been explored in patients with ATL. Therefore, we develop a clinical endpoint to evaluate whether patients that relapse early have a higher risk of death. Methods We designed a multicenter retrospective cohort study among treated patients with a serological and pathology confirmation of ATL. Included patients with acute or lymphomatous ATL subtypes received systemic treatment (e.g., chemotherapy; zidovudine plus interferon). The training cohort consists of patients from endemic regions treated in tertiary care centers across Latin America and the United States. A validation set of 193 patients from Japan was used for reproducibility. Our exploratory analysis identified that at least 20% of relapsed patients experienced this event within 5 months after diagnosis in both training and validation cohorts. Therefore, we used a 5-month landmark for patients with ATL (ATL5) to define early progression. Median follow-up (reverse Kaplan-Meier) was 48 and 40 months in the training and validation cohorts, respectively. Overall survival was defined from date progression for early-ATL5, and from the 5-month landmark for non-early-ATL5 (reference group). Patients that died without relapse within 5 months (n=62 training, n=54 validation) or those alive with less than 5 months of follow-up time (n=13 training, n=3 validation) were excluded from the analysis. We plotted Kaplan-Meier curves and used the log-rank test to compare groups across the datasets. Univariate and multivariate Cox regression analyses were employed to model the effect of ATL5 on all-cause mortality. Missing values were added as a missing category in the adjusted models. Results Of the 253 patients in the training cohort, 38% (n=96) relapsed within 5 months after diagnosis, and 62% (n=157) did not (reference group). There were 96 (38%) with acute (early-ATL5: n=55, reference: n=41) and 157 (62%) with lymphoma (early-ATL5: n=41, reference: n=116) ATL subtype. The median age at diagnosis was similar between both groups (early relapse: 55 years, reference: 54 years; p=0.831). Patients with early-ATL5 had poor performance status (ECOG 2-4 68% vs. 49%, p=0.009), higher frequency of acute ATL (57% vs. 26%, p<0.001), B symptoms (83% vs. 63%, p=0.034), advanced stage (III-IV 97% vs. 84%, p=0.002), hypercalcemia (51% vs. 35%, p=0.023), and higher values of lactate dehydrogenase (96% vs. 77%, p<0.001). Overall survival at 3 years was poorer among patients with early-ATL5 (12% vs. 30%; p=0.0001; Figure 1A). The univariate Cox analysis identified a hazard ratio (HR) of 2.02 (95% confidence interval [CI] 1.49-2.74) for early progression. After adjusting for ATL subtype, cancer stage, skin involvement, ECOG score, extranodal involvement, and hypercalcemia, patients with early-ATL5 remained with a higher risk of death (adjusted HR: 2.02, 95% CI 1.43-2.86). In the validation cohort, the 23 (12%) patients with early progression were deceased within 18 months after the landmark basal time (Figure 1B). The risk of death was higher among individuals with early-ATL5 in the validation set (adjusted HR: 1.84, 95% CI 1.14-2.95, Figure 1B). Conclusions Patients with ATL who relapse within 5 months after diagnosis had a higher risk of death. Treatment of ATL varies between and within countries, limiting the comparison and validation of current prognostic factors. Therefore, we developed a time-based marker to evaluate the prognosis of these patients. Our findings remain robust in the validation set, suggesting that ATTL5 is a clinically relevant endpoint in the real-world setting. Future studies should validate its applicability in prospective studies and determine its relevance for risk-based treatment in clinical trials. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Assessment of the Molecular International Prognostic Scoring System (IPSS-M) Risk Stratification Model in Therapy-Related Myelodysplastic Syndromes

Background Therapy-related myelodysplastic syndromes (t-MDS) represent a heterogenous group of myeloid neoplasms with distinctive clinical, molecular, and cytogenetic features associated with less favorable clinical outcomes relative to their primary counterpart (p-MDS). The original International prognostic scoring system (IPSS) and its revised version IPSS-R development cohorts did not include t-MDS patients. The Molecular IPSS (IPSS-M) is a newly developed clinical-molecular risk stratification model for myelodysplastic syndromes (MDS) that expands on the original IPSS-R by including data on somatic oncogenic mutations. Here we aimed to assess the validity of this tool in the risk stratification of t-MDS using a large cohort of patients treated at a tertiary referral center. Methods A total of 475 t-MDS patients with annotated clinical and molecular data treated at Moffitt Cancer Center were analyzed. For estimation of the IPSS-M score, R version 4.2.1 was used to run batch-calculation using syntax code provided by the Papaemmanuil lab at MSKCC. The mean IPSS-M score was used as reference. Time-to-event analyses were estimated using the Kaplan-Meier method and groups were compared by the log-rank test. We used Cox proportional hazards models for survival endpoints. Results We identified at least 737 driver point mutations involving up to 98 genes across 475 patients. We identified at least one gene mutation in 81% of patients (n=389), and 2 or more in 43% (n=206). Median overall survival (OS) was 1.5 years (95%CI, 1.3-1.8). Median leukemia-free survival (LFS) was 1.1 years (0.9-1.3). Median follow-up was 3.9 years (2.9-4.8). 475 patients (100%) were classified. The most prevalent mutations were TP53 (n=182, 38%), TET2 (n=79, 17%), DNMT3A (n=67, 14%), SF3B1 (n=43, 9%) and U2AF1 (n=35, 7%). Using the IPSS-M risk stratification schema, pts were classified as Very Low (2%, n=8), Low (13%, n=63), Moderate Low (14%, n=64), Moderate High (10%, n=48), High (22%, n=105) and Very High (39%, n=187) (Fig 1). The IPSS-M categories showed significant separation across all examined endpoints. Median LFS was NR, 3.7, 2.1, 1.7, 1.1 and 0.5 years from Very Low to Very High-risk categories (Fig 1). Conversely, median OS was NR, 4.4, 2.9, 3.5, 1.5 and 0.9 years from VL to VH subgroups respectively (Fig 1). A five-to-five mapping between the IPSS-R and IPSS-M risk subgroups (by merging moderate-low (ML) and moderate-high (MH) into moderate), resulted in the restratification of 45% of patients (n=209) (Fig 2). More than 3/4 of patients classified as IPSS-R very-low (VL) were upstaged (majority to IPSS-M low). More than half of patients classified as IPSS-R intermediate shifted: 50% (n=38) were reclassified as IPSS-M high/very high (H/VH). Similarly, 40% of patients classified as IPSS-R high were upstaged to IPSS-M VH. More than 20% of VH risk patients were downstaged (majority to high risk). Most significant was the reclassification of 9.4% (n=44) of the cohort from IPSS-R lower and intermediate risk categories into IPSS-M higher risk strata. Contextually, the restratification of patients from IPSS-R intermediate to higher risk strata in IPSS-M was associated with notable differences in survival outcomes which were as poor as 0.9 yrs and as high as 4.4 yrs for those reclassified as LR (relative to 2.1 yrs). There was significance across all IPSS-M risk strata between t-MDS and p-MDS. Stage to stage t-MDS was associated with worse outcomes compared to p-MDS, except for the very low risk IPSS-M subgroup. Conclusion To our knowledge this is the first external validation of IPSS-M in a large cohort of t-MDS patients. Use of the IPSS-M model for the risk stratification of patients with t-MDS led to reclassification of almost half of MDS patients and refined prognosis estimates. In general, outcomes of t-MDS are worse than p-MDS even when compared stage to stage using IPSS-M. The model can clearly discriminate patients with more indolent or aggressive disease course compared to the IPSS-R and better guide treatment decisions. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinical Characterization and Outcomes of Patients with NPM1-Mutated Myelodysplastic Syndromes or Chronic Myelomonocytic Leukemia

Background: Nucleophosmin (NPM1) gene mutations are rarely detected in patients with non-acute myeloid neoplasms (MN), such as myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML). Retrospective case series suggested that they are typically associated with an aggressive disease course and progress into acute myeloid leukemia (AML) in a short period of time, representing a distinct clinicopathological entity. However, the long-term outcomes and prognostic factors of these MNs in the era of modern therapies remain not well defined given their rarity. Methods: In the current study, we retrospectively reviewed clinical features, treatments, and outcomes of patients with NPM1-mutated MDS or CMML seen at Moffitt Cancer Center between 2009 and 2022. In comparison, AML patients with confirmed prior histories of MDS or CMML (sAML) that had NPM1 mutations detected at their AML diagnoses were used as the control group. Results: We identified 22 patients with NPM1-mutated MNs (median age 66 years at diagnosis and 54.5% male), including 16 cases of MDS (68.8% excess blasts) and 6 cases of CMML (50% CMML-2). Most patients had a normal karyotype (72.7%), and the median variant allele frequency of NPM1 mutation was 30.7%. Compared to NPM1-mutated sAML (n = 31), patients with NPM1-mutated MNs had lower bone marrow blast counts as expected and a trend for less IDH1/2 and FLT3-ITD/TKD mutations (Table). The majority of NPM1-mutated MNs (77.3%) were treated with upfront hypomethylating agents, and treatment response was unsatisfactory, with only one complete response. Four patients ultimately underwent allogeneic stem cell transplant. In contrast, most NPM1-mutated sAML (71.0%) received intensive chemotherapy as the frontline therapy, which resulted in a higher rate of complete response (81.8%), and 12 patients (38.7%) subsequently underwent allogeneic stem cell transplant. The overall survival was significantly inferior in NPM1-mutated MNs compared to sAML (median 22.1 vs. 41 months, p = 0.041; p = 0.023 if data censored at the time of transplant), which remained significant after adjusted by age (hazard ratio 2.15, 95% confidence interval 1.07-4.34, p = 0.032). During follow-up, 14 patients (63.6%) with NPM1-mutated MNs transformed into AML with a median time to transformation of 21.1 months. Among them, nine patients were able to receive intensive chemotherapy as induction for AML, and four achieved complete responses. In patients with mutational data available at both MN and subsequent AML phases, NPM1 mutations remained detectable in 75% of cases, and gain of FLT3-ITD was commonly seen (41.7%) at the AML phase. In terms of prognostic factors, concurrent baseline DNMT3A mutations conferred a trend of inferior outcomes (median 16.0 vs. 30.7 months; p = 0.116), and patients who received transplant showed improved outcomes (median 59.7 vs. 18.1 months; p = 0.029). Conclusion: Our findings suggest that NPM1-mutated MNs are an aggressive clinicopathological entity and support the recent WHO proposal to reclassify these MNs into AML irrespective of the blast counts, as they may benefit from more intensive treatment options. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Age and Sex Differences in the Eμ-TCL1 Adoptive Transfer Mouse Model of CLL

The Eμ-TCL1 model is the most widely used syngeneic mouse model of chronic lymphocytic leukemia (CLL) and has been extensively used to understand the pathogenesis of select genes, effect of the immune microenvironment and for pre-clinical drug development studies. Recently there has been an increasing awareness of the impact of age and sex differences on the development of cancers along with the efficacy and toxicity of specific cancer therapies. However, despite the predominance of older males in CLL patient demographics, Eμ-TCL1 adoptive transfer (AT) studies have used almost exclusively young female mice. We therefore sought to understand how recipient age and sex impact the development and survival of the Eμ-TCL1 model in order to allow for age and sex diverse pre-clinical studies using this model. First, a primary AT mouse experiment was performed to assess the impact of recipient sex on engraftment and survival. We found that in primary AT, sex matched recipients had faster engraftment and higher leukemia burden in the peripheral blood at 1- and 3-months post-AT. However, while disease burden was correlated with survival, young female recipients had prolonged survival compared to young male mice regardless of donor sex. Notably, sex differences were more prominent in recipients with relatively indolent disease with a median survival of median survival 178 and 132 days respectively as compared with more aggressive donors where sex differences were not apparent and both male and female recipients had a median survival of 87 days. A subsequent secondary AT using splenocytes isolated from primary AT recipients showed a more aggressive disease course (median survival 56 days) in which sex differences were not present. Additional secondary transfer experiments were performed to determine the impact of recipient age. Three age cohorts of male and female recipient mice (young 3-6 months) middle aged (9-12 months) and old (18-22 months) received a secondary AT. These ages were selected to correspond to approximate human ages of 20-30 years, 40-45 years, and 56 to 65 years old. While recipient age did not have a significant effect on the rate of disease establishment or survival in female mice, old male mice had a significantly decreased rate of Eμ-TCL1 tumor engraftment, and higher rates of failure to engraft, but similar overall survival to younger cohorts. In conclusion, we show that age and sex differences are present in the Eμ-TCL1 model, with female mice having prolonged survival compared to male mice particularly in indolent disease presentations. These findings suggest that age and sex differences must be considered in the experimental design using the Eμ-TCL1 adaptive transfer model of CLL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

MAF Translocations Are Enriched in NDMM Patients with Elevated Levels of Circulating Tumor Cells Suggesting a Genetic Basis for Aggressive Disease Course

Introduction A subset of Multiple Myeloma (MM) patients responds poorly to treatment, highlighting the need to understand the pathobiology of high-risk MM. The correlation between aggressive disease and elevated levels of circulating tumor cells (CTCs) has been recognized. We recently identified a subset of high-risk newly diagnosed MM (NDMM) patients based on a transcriptomic classifier derived from patients with high levels of CTC, indicating that CTC levels are a relevant reflection of tumor cell biology. Here, we hypothesized that comparing bone marrow plasma cells (BM PCs) of MM patients with high and low CTC levels will provide an opportunity to identify mechanistic drivers of aggressive disease. Results We first set out to exclude the presence of a circulating aggressive subclone of MM cells in patients with high levels of CTCs. Paired single cell transcriptomes from BM and peripheral blood MM cells of 7 NDMM patients were generated. Analysis of 55,230 CTCs and BM PCs revealed patient specific clustering, but with large transcriptional overlap between individual paired CTCs and BM PCs. Since only 11 genes were significantly upregulated in the BM PCs (P < 0.05) compared to paired CTCs, we excluded the presence of circulating aggressive subclones in patients with high CTC levels. Next, we aimed to identify differences in MM cell pathobiology between patients with high and low levels of CTCs, we generated single cell transcriptomes from 89,092 BM MM cells from 8 NDMM patients with high (≥0.5%) and 14 patients with low (0.004-0.006%) percentages of CTCs. Single cell data integration across all patients led to the identification of 8 distinct PC clusters. Gene set enrichment analysis between both patient groups revealed upregulation of cell cycle pathways in MM cells of patients with high CTC levels. Based on cell cycle analyses an increase in cycling PCs (53% vs 32%, p=0.007) was associated with high CTC levels (Figure 1). Ki67 labeling of BM biopsies validated this finding. Interestingly, proliferation was increased across all transcriptomically-defined plasma cell clusters, suggesting a common cell-intrinsic driver of increased proliferation. To analyze whether the prevalence of genetic lesions previously associated with proliferation was increased in patients with high CTC levels, WGS was performed on the same 22 patients. This revealed an elevated mutation burden and a predilection for MAF translocations, as 50% (4/8) of the patients with high CTC levels carried MAF translocations versus none in the patients with low CTC levels (0/14). We also observed increased transcription of cyclin D2 (CCND2), CCR1 and IntegrinB7 (ITGB7) in patients with high CTC levels. These proliferation or tumor survival genes have been shown to be increased in patients with t(14;16), a translocation also resulting in overexpression of MAF. To validate these findings in a larger patient cohort, clinical, genomic and CTC data were retrieved from the MMRF-CoMMpass database (n=725). Strikingly, 21% of patients with CTC levels above 0.3% showed MAF translocations vs only 3% in patients with CTC levels below 0.3% (p<0.001), solidifying the correlation between MAF translocations and high levels of CTCs in NDMM patients with a high-risk disease profile. Conclusion Single cell RNA sequencing and WGS of MM cells from patients with high and low CTC levels identified a correlation between CTC levels and overrepresentation of MAF translocations in NDMM patients. This finding suggests that MAF target genes and downstream proliferation could be drivers of aggressive disease and might be used for future patient stratification. Figure 1. Based on cell cycle analyses in single cell transcriptomics an increase in cycling PCs was associated with high CTC levels. a) UMAP of cell cycle analysis of 89,902 BM MM cells from 22 patients with high (n=8) and low (n=14) CTC levels. Colors represent cell cycle phase. b) Bar graph representing percentage of cycling cells. Data points are individual patients (high CTC levels, n=8; low CTC levels, n=14). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Antibody-drug conjugates targeting Trop-2: Clinical developments in early breast cancer therapy

Although breast cancer has a good prognosis compared with various cancers, metastatic breast cancer has an aggressive disease course and remains incurable. Therefore, treatment of early breast cancer to prevent recurrence and metastasis is crucial. Recently, the development of anti-cancer drugs, such as targeted agents and immuno-oncology, has been accelerating. Antibody-drug conjugates (ADCs) are also building a new paradigm. Particularly, ADCs targeting Trop-2 were approved for their efficacy in metastatic triple-negative breast cancer patients who received ≥2 prior systemic therapies and showed significant results in heavily pretreated hormone receptor-positive/HER2-negative breast cancer. In this brief review, we provide an overview of ongoing clinical trials of ADCs targeting Trop-2 in early breast cancer, specifically sacituzumab govitecan.

Cross-species efficacy of enzyme replacement therapy for CLN1 disease in mice and sheep.

CLN1 disease, also called infantile neuronal ceroid lipofuscinosis (NCL) or infantile Batten disease, is a fatal neurodegenerative lysosomal storage disorder resulting from mutations in the CLN1 gene encoding the soluble lysosomal enzyme palmitoyl-protein thioesterase 1 (PPT1). Therapies for CLN1 disease have proven challenging because of the aggressive disease course and the need to treat widespread areas of the brain and spinal cord. Indeed, gene therapy has proven less effective for CLN1 disease than for other similar lysosomal enzyme deficiencies. We therefore tested the efficacy of enzyme replacement therapy (ERT) by administering monthly infusions of recombinant human PPT1 (rhPPT1) to PPT1-deficient mice (Cln1–/–) and CLN1R151X sheep to assess how to potentially scale up for translation. In Cln1–/– mice, intracerebrovascular (i.c.v.) rhPPT1 delivery was the most effective route of administration, resulting in therapeutically relevant CNS levels of PPT1 activity. rhPPT1-treated mice had improved motor function, reduced disease-associated pathology, and diminished neuronal loss. In CLN1R151X sheep, i.c.v. infusions resulted in widespread rhPPT1 distribution and positive treatment effects measured by quantitative structural MRI and neuropathology. This study demonstrates the feasibility and therapeutic efficacy of i.c.v. rhPPT1 ERT. These findings represent a key step toward clinical testing of ERT in children with CLN1 disease and highlight the importance of a cross-species approach to developing a successful treatment strategy.