Citations

Abstract Cytotoxic CD8+ T cells play a key role in response to anti-PD-1/PD-L1 immune checkpoint inhibitors (ICI) therapy. However, the exact effector molecules that are regulated by PD-1 signals in cytotoxic CD8+ T cells have not been completely defined. Lack of this knowledge is a crucial problem in addressing how to overcome the low clinical response rate to ICI therapy. When exploring why certain patients not responding to ICI therapy, we found non-responders did not have an elevated NKG7 expression in CD8+ T cells as responders to ICI therapy. The difference in NKG7 expression in CD8+ T cells between non-responders and responders to anti-PD-1 therapy prompted us to investigate the potential role of PD-1 signaling in regulating NKG7 expression. To that end, we generated a CD8+ T cell-specific Pdcd1 conditional knockout mice (CD8 E8ICre-Pdcd1fl/fl, also known as CD8-PD-1 cKO) where Pdcd1 is deleted after thymic selection of single positive CD8+ T cells. We observed a delayed tumor growth and prolonged survival in CD8-PD-1 cKO mice compared to control mice after B16-OVA melanoma tumor challenge. On Day 12, when the CD8-PD-1 cKO mice demonstrated their ability to control tumor growth, we conducted single cell RNA sequencing on isolated tumor infiltrating CD8+ T cells, revealing a significant increase of Nkg7 expression in effector CD8+ T cells in the Pdcd1 deficient CD8+ T cells compared to the control wild type T cells. Pdcd1-deficient effector CD8+ T cells also had increased expression of genes linked to NFAT/Calcineurin signaling pathway including Nfatc1, S100a4, Tbet and Tox, suggesting Nkg7 could be regulated by Pdcd1 via the NFAT pathway. To determine the impact of increased NKG7 expression in CD8+ T cells, we used OVA protein as a surrogate tumor antigen in vaccination. We found a complete B16-OVA tumor rejection in CD8-PD-1 cKO mice compared to control mice when tumor cells were injected on day 7 after vaccination with OVA protein and poly (I:C) (as an adjuvant), but only a partial rejection on day 21. The CD8-PD-1 cKO mice that were tumor-free for 30-40 days rejected 2nd tumor challenge with B16-OVA tumors. This long-term protection is tumor antigen (OVA) specific because these mice did not reject B16F10 tumor cells that do not express OVA antigen. We also found effector memory CD8+ T cells with high cytotoxicity increased in the spleen of CD8-PD-1 cKO mice after OVA antigen immunization compared to control. In conclusion, we found NKG7 is one of the effector molecules in cytotoxic CD8+ T cells, which can be regulated by PD-1 signals through NFAT signaling. Targeting the PD-1-NFAT pathway may be a new option to overcome low responses of T cells to ICI therapy in treatment of refractory cancer. Citation Format: Zhiming Mao, Joanina Gicobi, Jacob Hirdler, Xin Liu, Michelle Hsu, Emilia Dellacecca, Wenjing Zhang, Fabrice Lucien-Matteoni, Hai dong. PD-1 signals regulate NKG7 that is required for CD8+ T cells to sustain cytotoxicity and durable antitumor memory responses [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 3979.

Abstract Introduction: Porcine myeloperoxidase (pMPO) is a cationic enzyme secreted by granulocytic leukocytes capable of selectively binding cancer cells due to their negative surface charge. In the presence of hydrogen peroxide (H2O2) and sodium chloride (NaCl), MPO catalyzes the production of electronically excited singlet molecular oxygen, which exerts focused cytotoxic activity against cancer cells. Methods: Human bladder non-cancer (SV-HUC1) and cancer cells (T24 and 5637) were used to assess differential cytotoxic activity of pMPO. pMPO was combined with associated reagents, including H2O2, NaCl, and L-proline and glycine for 45 minutes to support the generation of singlet oxygen. MTS assay was used to assessed cell viability at day 3 post-treatment. Non-cancer and cancer cells were labeled with GFP and mCherry, respectively, via infection with lentivirus and cultured in tandem to allow for simultaneous treatment of both cell types. After the treatment, flow cytometry and microscopy were used to monitor the growth of the cells. Results: No cytotoxicity was noted when SV-HUC1 cells were cultured with individual reagents pMPO, NaCl, L-Proline, or Glycine, and limited cytotoxicity was observed with H2O2. Notably, cell viability was nearly maximally preserved for SV-HUC1 cells. The combination of pMPO with associated reagents resulted in killing of 5637 and T24 cells, with tumoricidal activity maintained across increasing tumor cell density, with cell viability preserved for SV-HUC1 cells. Within the cell co-culture system, simultaneous treatment of both cell types with the pMPO treatment resulted in selective killing of tumor cells and preservation of SV-HUC1 cells. Mechanistic studies confirm specificity of pMPO binding to tumor cells, and further, high levels of expression of gammaH2AX and phospho-ATM in 5637 and T24 cells following treatment. Conclusion: pMPO demonstrates selective and efficient killing of bladder cancer cells with minimal cytotoxicity against non-cancer urothelial cells. Given its reliance on direct surface contact with tumor cells for activity, pMPO may be developed as an intravesical therapy for treatment of non-muscle invasive bladder cancers. Acknowledgements: This work was partially supported by Mayo funds provided to Dr. Haojie Huang. Citation Format: Junnan Liu, Robert C. Allen, Jackson T. Stephens, Paras Shah, Haojie Huang. Investigation of anti-tumor activity of porcine myeloperoxidase for urothelial carcinoma of the bladder [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 2029.

Abstract Background: Although targeting myeloid cells has become one of the next generation targets for cancer immunotherapy, there is a lack of therapeutic tools to specifically target these cells. Our group has developed a novel humanized PD-L1-targeting antibody, H1A, that induces PD-L1 degradation via blockade of PD-L1 and CMTM6, therefore limiting PD-L1 intrinsic signaling while also preventing interactions with PD-1. In the immune cell compartment, CMTM6 expression is restricted to myeloid cells, making these cells the primary target of H1A. Recently, we unexpectedly found that targeting intrinsic PD-L1 in myeloid cells via H1A resulted in increased release of CCL2 from myeloid cells. In this study, we examined the role of CCL2/CCR2 signaling in CD8 T cell function using human peripheral blood-derived immune cells and evaluated the therapeutic potential of H1A antibody using our humanized PD-L1/PD-1 mouse model. Methods: Human PBMCs derived from healthy donors were treated with H1A or Atezolizumab, a blocking PD-L1 antibody, followed by mass cytometry and flow cytometry to evaluate immune cell phenotype and functional states. PD-L1 expressing myeloid cells isolated from human PBMCs were treated with H1A or Atezolizumab and assessed for CCL2 secretion using multiplex immunoassays. CCL2 production and transcription were confirmed by flow cytometry and single-cell RNA-sequencing. CCR2 blockade was used on human PBMCs to evaluate the effects of CCL2/CCR2 signaling on T cell function alongside H1A treatment. Finally, in vivo antitumor activity of H1A and Atezolizumab were compared in two humanized PD-L1 tumor models. Results: H1A reduced the frequency of regulatory T cells and increased effector T cells, in whole PBMCs upon global T cell activation with anti-CD3/CD28. H1A enhanced the secretion and production of CCL2 from myeloid cells, which was confirmed at a transcript level. CCR2 blockade diminished CD8 T cells’ ability to differentiate into effector cells in H1A treated PBMCs. H1A demonstrated comparable therapeutic effects with Atezolizumab in treatment of immunogenic and ICI-responsive mouse tumor MC38, which is CD8 T cell-dependent, but showed superior therapeutic effects in treatment of ICI-insensitive mouse tumor E0771 compared to Atezolizumab. Both treatments elicited durable memory specific immune responses upon E0771 tumor rechallenge. Conclusion: H1A demonstrated a superior antitumor activity compared to Atezolizumab in our preclinical models via a new mechanism by which H1A caused degradation of intrinsic PD-L1 in myeloid cells. This causes more release of CCL2 to improve T cell function via CCR2 upon T cell activation. Thus, H1A is emerging as a new immunotherapy tool to target PD-L1/CMTM6 in myeloid cells for treatment of cancers that are refractory to current immune checkpoint inhibitor therapy. Citation Format: Michelle Alexandria Hsu, Xin Liu, Ying Li, Whitney Barham, Kevin Pavelko, Jake Hirdler, Fabrice Lucien, Haidong Dong. Targeting PD-L1/CMTM6 in myeloid cells results in elevated release of CCL2 to improve CD8 T cell-mediated antitumor response [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 2664.

Abstract Background: Previous studies demonstrated that obesity is associated with poor outcomes and significantly worse pathological complete response (pCR) in triple-negative breast cancer (TNBC) treated with conventional chemotherapy. Emerging studies described a phenomenon termed the obesity paradox, which referred to improved outcomes observed in obese patients treated with immune checkpoint inhibitors (ICIs). Our study aimed to determine distinct immune features in TNBC across body mass index (BMI). Methods: Characteristics and outcomes of TNBC patients treated with neoadjuvant pembrolizumab-based chemotherapy at Mayo Clinic were collected. Normal BMI was defined as 18-25 kg/m2, overweight 25-30, and obese BMI ≥ 30. NanoString CosMx, a spatial multi-omics single-cell imaging (SMI), was performed in 75 samples from the Mayo Clinic TNBC cohort (Leon-Ferre BCRT 2018). Differential expression as log 2-fold change (FC) was estimated from the linear mixed model with significance defined as two-sided p< 0.05. Results: Between 2021-2023, there were 108 TNBC patients treated with neoadjuvant pembrolizumab. 25.9% were obese, 32.4% were overweight, and 41.7% had normal BMI. In contrast to previous studies with conventional chemotherapy, obese patients numerically had a higher pCR rate of 71.4% with pembrolizumab-based chemotherapy compared to 55.6% in normal and 62.9% in overweight patients. However, this difference did not reach statistical significance (obese vs. normal weight, OR 2.26, 95%CI 0.89-6.06, p 0.094). We further evaluated spatially-resolved immune features associated with obesity using CosMx SMI in 75 samples from the MC TNBC cohort. Obese patients had significantly higher intratumoral B cells (log2FC 1.88, p 0.017), stromal B cells (log2FC 2.79, p 0.001), stromal plasmablasts (log2FC 1.94, p 0.047), and stromal macrophages (log2FC 1.80, p 0.017) compared to patients with normal BMI. Obese patients also had significantly lower intratumoral myeloid dendritic cells (mDC) (log2FC -1.27, p 0.037). Similar trends were observed after standardizing cell counts per 100 tumor or stromal cells, including higher intratumoral B cells (log2FC 3.00, p 0.001), intratumoral plasmablasts (log2FC 2.00, p 0.006), intratumoral natural killer (NK) cells (log2FC 1.66, p 0.044), and stromal B cells (log2FC 1.50, p 0.008). Differential gene analyses for intratumoral B cells revealed that genes (CD74, HLA-C, HLA-DRB1, HLA-DRA, HLA-DRB) associated with the positive regulation of type 2 immune or chemokine response were upregulated. Conclusions: Using in-depth analysis with spatially defined context, our study showed that obese TNBC patients had significantly higher B cell infiltration, which is associated with improved outcomes with ICIs. Future research on how obesity alters the immune microenvironment is needed to identify potential pathways to improve outcomes with ICIs in TNBC. Citation Format: Songhan Pang, Yi Liu, Yaohua Ma, Zhuo Li, Roberto A. Leon-Ferre, David Zahrieh, David W. Hillman, Judy C. Boughey, James N. Ingle, Krishna R. Kalari, Fergus J. Couch, Matthew P. Goetz, Keith L. Knutson, Saranya Chumsri, E. Aubrey Thompson. Tumor-immune microenvironment associated with obesity in triple-negative breast cancer in the Mayo Clinic cohort [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 1162.

Abstract Melanoma is a uniquely difficult cancer to treat due to complex tumor heterogeneity and resistance, where upon dissemination, survival drops to less than 17%. Drug resistance has arisen for the current standard of care and combinatorial therapies, expediting the need for new efficacious agents. The lack of physiologically relevant preclinical models has impeded the development of these therapeutics. Potential melanoma drugs are typically assayed using cell lines on tissue culture plastic, then tested for in vivo efficacy and side effects in animals. This paradigm is inherently unsuitable for the generation of effective therapies as typical cell culture platforms fail to replicate the complex microenvironments known to play critical roles in tumor pathology and rodent models are prohibitively expensive for optimizing novel and combination therapies. Thus, our goal is to synthesize tissue-engineered melanoma models using biomimetic polymers that recapitulate patient tumor characteristics to assess combination and novel therapies that attenuate resistance and disrupt metastatic processes. In this study, patient melanoma cells, which were unable to recapitulate drug responses on traditional cell culture platforms, were cultured in a controlled spatial and temporal manner on polyacrylamide hydrogels. The hydrogel scaffolds feature stiffnesses spanning that of physiological tissue, with microcontact printed human fibronectin for biomimetic protein presentation to create a microtumor array. Cells cultured on the hydrogel showed a 2.5-fold increase in expression of the cancer stem cell biomarkers, CD271 and ABCB5, which play a key role in melanoma survival and recurrence, compared to cells cultured on plastic or glass substrates. Further study identified BRAF mutant, drug resistant patient cells, regained resistance to standard of care targeted therapies, trametinib and dabrafenib. Through hydrogel microarrays featuring physiological similarities, the technology has enabled the exploration of heterogeneous cell populations with novel and combinatorial therapeutics. Citation Format: Yi L. Liu, Justyna Gleba, Chantal Kopecky, Matthew Pawlush, John A. Copland, Kristopher A. Kilian. Biomimetic hydrogels at physiological stiffness reprogram patient derived melanoma cells [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 6896.

Abstract Background: Neoadjuvant immunotherapy (IO) has become standard of care for early-stage triple negative breast cancer (TNBC). I-SPY2 was the first randomized trial to examine the efficacy of IO therapy in high-risk HR+HER2- breast cancer, where most IO arms showed improved efficacy relative to control. We also previously showed immune-gene expression signatures associate with pathologic complete response (pCR) in HR+HER2- treated with IO; and developed a clinically applicable Immune classifier (ImPrint) predicting response to IO that is now being used in I-SPY2.2 as part of the Response Predictive Subtypes. Here we report the performance of ImPrint in HR+HER2- patients from 5 IO arms. Methods: 204 HR+HER2- (MammaPrint high risk) patients from 5 IO arms (anti-PD1, anti-PDL1/PARPi, anti-PD1/TLR9 dual-IO, and anti-PD1 +/- LAG3 dual-IO) and 191 patients from the control arm were included in this analysis. Patients were classified ImPrint+ (likely sensitive) vs. ImPrint- (likely resistant), by Agendia, Inc, using pre-treatment mRNA. Performance of ImPrint for predicting pCR to IO was characterized and compared to that of MammaPrint (ultra) High2 risk (MP2) and tumor grade (III). Based on the DRFS of pCR vs. non-pCR HR+HER2-/ImPrint+ patients (median follow up ~4.5yr), we predicted the DRFS in the IO vs. control arms using an exponential model as a function of the pCR rates. Results: Overall, the pCR rate over the 5 IO arms was 33%. 28% of HR+HER2- patients were ImPrint+; and pCR rates with IO were 76% in ImPrint+ vs. 16% in ImPrint-, with the highest pCR rate >90% in a dual-IO arm. In the control arm, pCR rates were 33% in ImPrint+ and 8% in ImPrint-. Based on the pCR rates in the IO vs. control (76% vs. 33%), the predicted DRFS of HR+HER2-/ImPrint+ patients are 91% vs 80% at 5 years, respectively. This translates to a risk reduction of 52% for the IO arm relative to standard chemotherapy in HR+HER2-/ImPrint+ patients. We also compared MP2 and tumor grade (III) to ImPrint+ as pCR predictors, as these markers have been used or proposed as selection markers for neoadjuvant IO trials in HR+HER2-. The pCR rates for MP2, Grade III, at 56%, 45%, respectively, are much smaller than that for ImPrint+ at 75% pCR; thus ImPrint appears to be a more precise predictive biomarker for neoadjuvant IO therapy in the HR+HER2- breast cancers. Conclusion: These results suggest that a subset of high risk HR+HER2- breast cancers is highly sensitive to immunotherapy, and by using a specific and sensitive selection strategy patients could achieve pCR rates similar to what is seen with best neoadjuvant therapies in TNBC and HER2+ (i.e., pCR rate > 65-70%). ImPrint, an FDA IDE-enabled assay currently being further evaluated in I-SPY2, may represent the way to identify patients for IO that best balances likely benefit vs risk of serious immune-related adverse events. Citation Format: Denise M. Wolf, Christina Yau, Lajos Pusztai, Rita Nanda, Jo Chien, Erica Stringer-Reasor, Rebecca Shatsky, Claudine Isaacs, MInetta Liu, Hyo Han, Hatem Soliman, Michael Campbell, Annuska Glas, Andrei Barcaru, Lorenza Mittempergher, Midas Kuilman, Lamorna Brown Swigart, Gillian Hirst, Hongmei Yu, Philip Beineke, Amrita Basu, I-SPY2 Investigators, Douglas Yee, W. Fraser Symmans, Angela DeMichele, Jane Perlmutter, Amy Delson, Laura Huppert, Hope Rugo, Amy Clark, Nola Hylton, Paula Pohlmann, Laura Esserman, Laura van 't Veer. Immune subtyping identifies a subset of HR+HER2- early-stage breast cancer patients with a very high likelihood of response to neoadjuvant immunotherapy (IO): Results from 5 IO arms of the I-SPY2 TRIAL [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 1207.

ImportanceFinding a reliable diagnostic biomarker for the disorders collectively known as synucleinopathies (Parkinson disease [PD], dementia with Lewy bodies [DLB], multiple system atrophy [MSA], and pure autonomic failure [PAF]) is an urgent unmet need. Immunohistochemical detection of cutaneous phosphorylated α-synuclein may be a sensitive and specific clinical test for the diagnosis of synucleinopathies.ObjectiveTo evaluate the positivity rate of cutaneous α-synuclein deposition in patients with PD, DLB, MSA, and PAF.Design, Setting, and ParticipantsThis blinded, 30-site, cross-sectional study of academic and community-based neurology practices conducted from February 2021 through March 2023 included patients aged 40 to 99 years with a clinical diagnosis of PD, DLB, MSA, or PAF based on clinical consensus criteria and confirmed by an expert review panel and control participants aged 40 to 99 years with no history of examination findings or symptoms suggestive of a synucleinopathy or neurodegenerative disease. All participants completed detailed neurologic examinations and disease-specific questionnaires and underwent skin biopsy for detection of phosphorylated α-synuclein. An expert review panel blinded to pathologic data determined the final participant diagnosis.ExposureSkin biopsy for detection of phosphorylated α-synuclein.Main OutcomesRates of detection of cutaneous α-synuclein in patients with PD, MSA, DLB, and PAF and controls without synucleinopathy.ResultsOf 428 enrolled participants, 343 were included in the primary analysis (mean [SD] age, 69.5 [9.1] years; 175 [51.0%] male); 223 met the consensus criteria for a synucleinopathy and 120 met criteria as controls after expert panel review. The proportions of individuals with cutaneous phosphorylated α-synuclein detected by skin biopsy were 92.7% (89 of 96) with PD, 98.2% (54 of 55) with MSA, 96.0% (48 of 50) with DLB, and 100% (22 of 22) with PAF; 3.3% (4 of 120) of controls had cutaneous phosphorylated α-synuclein detected.Conclusions and RelevanceIn this cross-sectional study, a high proportion of individuals meeting clinical consensus criteria for PD, DLB, MSA, and PAF had phosphorylated α-synuclein detected by skin biopsy. Further research is needed in unselected clinical populations to externally validate the findings and fully characterize the potential role of skin biopsy detection of phosphorylated α-synuclein in clinical care.

Background: Peripheral nerve injury (PNI) following revision total hip arthroplasty (rTHA) can be a devastating complication. This study assessed the frequency of and risk factors for postoperative PNI following rTHA. Methods: Patients who underwent rTHA from 2003 to 2015 were identified using the National Inpatient Sample (NIS). Demographics, medical history, surgical details, and complications were compared between patients who sustained a PNI and those who did not, to identify risk factors for the development of PNI after rTHA. Results: Overall, 112,310 patients who underwent rTHA were identified, 929 (0.83%) of whom sustained a PNI. Univariate analysis found that younger patients (p < 0.0001), females (p = 0.025), and those with a history of flexion contracture (0.65% vs. 0.22%, p = 0.005), hip dislocation (24.0% vs. 18.0%, p < 0.001), and spine conditions (4.8% vs. 2.7%, p < 0.001) had significantly higher rates of PNI. In-hospital complications associated with PNI included postoperative hematoma (2.6% vs. 1.2%, p < 0.0001), postoperative seroma (0.75% vs. 0.30%, p = 0.011), superficial wound dehiscence (0.65% vs. 0.23%, p = 0.008), and postoperative anemia (36.1% vs. 32.0%, p = 0.007). Multivariate analysis demonstrated that a history of pre-existing spine conditions (aOR: 1.7; 95%-CI: 1.3–2.4, p < 0.001), prior dislocation (aOR 1.5; 95%-CI: 1.3–1.7, p < 0.001), postoperative anemia (aOR 1.2; 95%-CI: 1.0–1.4, p = 0.01), and hematoma (aOR 2.1; 95%-CI: 1.4–3.2, p < 0.001) were associated with increased risk for PNI. Conclusions: Our findings align with the existing literature, affirming that sciatic nerve injury is the prevailing neuropathic complication after total hip arthroplasty (THA). Furthermore, we observed a 0.83% incidence of PNI following rTHA and identified pre-existing spine conditions, prior hip dislocation, postoperative anemia, or hematoma as risk factors. Orthopedic surgeons may use this information to guide their discussion of PNI following rTHA, especially in high-risk patients.

Vascular pathology is the second leading cause of cognitive impairment and represents a major contributing factor in mixed dementia. However, biomarkers for vascular cognitive impairment and dementia (VCID) are under-developed. Here we aimed to investigate the potential role of CO2 Cerebrovascular Reactivity (CVR) measured with phase-contrast quantitative flow MRI in cognitive impairment and dementia. Forty-five (69 ± 7 years) impaired (37 mild-cognitive-impairment and 8 mild-dementia by syndromic diagnosis) and 22 cognitively-healthy-control (HC) participants were recruited and scanned on a 3 T MRI. Biomarkers of AD pathology were measured in cerebrospinal fluid. We found that CBF-CVR was lower (p = 0.027) in the impaired (mean±SE, 3.70 ± 0.15%/mmHg) relative to HC (4.28 ± 0.21%/mmHg). After adjusting for AD pathological markers (Aβ42/40, total tau, and Aβ42/p-tau181), higher CBF-CVR was associated with better cognitive performance, including Montreal Cognitive Assessment, MoCA (p = 0.001), composite cognitive score (p = 0.047), and language (p = 0.004). Higher CBF-CVR was also associated with better physical function, including gait-speed (p = 0.006) and time for five chair-stands (p = 0.049). CBF-CVR was additionally related to the Clinical-Dementia-Rating, CDR, including global CDR (p = 0.026) and CDR Sum-of-Boxes (p = 0.015). CBF-CVR was inversely associated with hemoglobin A1C level (p = 0.017). In summary, CBF-CVR measured with phase-contrast MRI shows associations with cognitive performance, physical function, and disease-severity, independent of AD pathological markers.