Advanced Quantification Pipeline Reveals New Spatial and Temporal Tumor Characteristics in Preclinical Multiple Myeloma

Advanced Quantification Pipeline Reveals New Spatial and Temporal Tumor Characteristics in Preclinical Multiple Myeloma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Advanced Quantification Pipeline Reveals New Spatial and Temporal Tumor Characteristics in Preclinical Multiple Myeloma Zhixin Sun, Jacqueline Godbe, Alexander Zheleznyak, Brad Manion, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6596974/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 Jul, 2025 Read the published version in EJNMMI Research → Version 1 posted 5 You are reading this latest preprint version Abstract Background. Radiological imaging plays an indispensable role in both preclinical and clinical studies of multiple myeloma (MM). However, manual quantification in longitudinal small animal PET/CT is limited by annotator bias, signal artifacts from urinary/fecal excretion, and voxel misalignment due to non-rigid registration. To address these challenges and improve characterization of tumor biology, we developed a semi-automated PET/CT quantification pipeline targeting defined regions of interest (ROIs) within the bone marrow-rich mouse skeleton, achieving sub-organ spatial resolution, including in anatomically complex sites such as the pelvis. We applied this MM-specific preclinical pipeline to analyze tumor distribution in a longitudinal molecular PET study using an immunocompetent mouse model of skeletally disseminated MM. An Attention U-Net was trained to segment the thoracolumbar spine, pelvis and pelvic joints, sacrum, and femurs from 2D CT slices. A custom algorithm masked spillover signal from physiological excretion, and a PCA-based projection was used to map tumor distribution along the skeletal axis. Quantification metrics included mean and maximum standardized uptake values (SUV mean , SUV max ) from PET and Hounsfield Units (HU) from CT to assess tumor burden, spatiotemporal tumor distribution, and bone involvement. Results. Tumor burden localized preferentially to skeletal regions near joints. Using precise CT-based alignment (DICE = 0.966 ± 0.005), we detected early disease progression and aggressive phenotypes. A marked increase in tumor uptake was observed by day 18 post-implantation, with significant SUV mean increases in the spine (p = 0.012), left/right femurs (p = 0.007/0.006), pelvis and pelvic joints (p = 0.018), and sacrum (p = 0.02). Notably, sex-based differences were identified: female mice showed greater bone loss near the hip joint at later stages, with significant HU mean reductions at days 25 (p = 0.008) and 32 (p = 0.002). Conclusions. This pipeline enables reproducible, anatomically precise quantification of region-specific trends in MM progression, including joint-specific lesion tropism and sex-based differences, from longitudinal PET/CT scans. By mitigating common challenges such as excretion artifacts and inconsistent mouse positioning, our approach overcomes limitations of manual analysis and enhances evaluation of tumor biology and treatment response in preclinical models of bone-involved cancers. Multiple Myeloma (MM) Imaging Bone Segmentation PET/CT Quantification Skeletal Lesions Full Text Supplementary Files 20250508SupplementalFileEJNMMISun2025.pdf Cite Share Download PDF Status: Published Journal Publication published 31 Jul, 2025 Read the published version in EJNMMI Research → Version 1 posted Editorial decision: Minor Revision 06 Jul, 2025 Reviewers agreed at journal 16 May, 2025 Reviewers invited by journal 09 May, 2025 Editor assigned by journal 09 May, 2025 First submitted to journal 08 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6596974","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":454417085,"identity":"d6445235-5c4b-4d0b-851a-766d5bcb8862","order_by":0,"name":"Zhixin Sun","email":"","orcid":"","institution":"Washington University In Saint Louis: Washington University in St Louis","correspondingAuthor":false,"prefix":"","firstName":"Zhixin","middleName":"","lastName":"Sun","suffix":""},{"id":454417086,"identity":"8fff1b97-e7f7-4200-8bf3-2809925893b1","order_by":1,"name":"Jacqueline Godbe","email":"","orcid":"","institution":"Washington University School of Medicine in Saint Louis: Washington University in St Louis School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jacqueline","middleName":"","lastName":"Godbe","suffix":""},{"id":454417087,"identity":"734e5390-5339-46b8-a5e3-be1fc9020884","order_by":2,"name":"Alexander Zheleznyak","email":"","orcid":"","institution":"Washington University School of Medicine in Saint Louis: Washington University in St Louis School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Alexander","middleName":"","lastName":"Zheleznyak","suffix":""},{"id":454417088,"identity":"1d2824d2-b332-4a19-a59b-c348098956b7","order_by":3,"name":"Brad Manion","email":"","orcid":"","institution":"Washington University School of Medicine in Saint Louis: Washington University in St Louis School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Brad","middleName":"","lastName":"Manion","suffix":""},{"id":454417089,"identity":"a5048375-93f2-4a06-bd07-a5471db2f3fd","order_by":4,"name":"Junhao Hu","email":"","orcid":"","institution":"Washington University In St Louis: Washington University in St Louis","correspondingAuthor":false,"prefix":"","firstName":"Junhao","middleName":"","lastName":"Hu","suffix":""},{"id":454417090,"identity":"86d1a58d-361e-4dc0-8a0e-4a9c90eb0cb3","order_by":5,"name":"Julie Prior","email":"","orcid":"","institution":"Washington University School of Medicine in Saint Louis: Washington University in St Louis School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Julie","middleName":"","lastName":"Prior","suffix":""},{"id":454417091,"identity":"22e83b2f-f529-47d8-85d0-28d5b150a655","order_by":6,"name":"Kathleen Duncan","email":"","orcid":"","institution":"Washington University School of Medicine in Saint Louis: Washington University in St Louis School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Kathleen","middleName":"","lastName":"Duncan","suffix":""},{"id":454417092,"identity":"f179bdc5-1cc8-4535-9221-fb56518c9b01","order_by":7,"name":"Ulugbek S. Kamilov","email":"","orcid":"","institution":"Washington University In St Louis: Washington University in St Louis","correspondingAuthor":false,"prefix":"","firstName":"Ulugbek","middleName":"S.","lastName":"Kamilov","suffix":""},{"id":454417093,"identity":"9835bb2e-1b4e-4294-9a8d-43e322c2b16b","order_by":8,"name":"Monica Shokeen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYDACCQZmMM0PJguA+ACxWiQbQKoNSNFicIBYLfKzmw8bfKi4E218I8f48wcDBjm+Gwn4tRjcOZacOOPMs9xtN3LMJIC2GEsS1CKRY3yYt+1w7rbbOWYghyVuIKRFfgZQy99/h3M3z84x/gDUUk9QCwPQC8mMDYdzN0jnGIAclmBA0GE30pINe44dzp1x/1mZxBkDCcOZZx4QcljyYYkfNYdz+3sOb/5QUWEjz3eckMPQgARpykfBKBgFo2AUYAcAlc1MMX2q4YkAAAAASUVORK5CYII=","orcid":"","institution":"Washington University School of Medicine in Saint Louis: Washington University in St Louis School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Monica","middleName":"","lastName":"Shokeen","suffix":""}],"badges":[],"createdAt":"2025-05-05 19:28:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6596974/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6596974/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13550-025-01291-x","type":"published","date":"2025-07-31T16:12:59+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88268408,"identity":"7c572c3d-11ae-4bb0-a2f6-f5dd3eed5621","added_by":"auto","created_at":"2025-08-04 16:51:34","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1353723,"visible":true,"origin":"","legend":"","description":"","filename":"20250508FinalEJNMMISun2025.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6596974/v1_covered_1032b7b0-ee99-4bc4-a2c8-df36dd0bf206.pdf"},{"id":82740812,"identity":"a89848ae-195e-4e86-968c-97d7ba4e4d93","added_by":"auto","created_at":"2025-05-14 17:02:01","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":1488765,"visible":true,"origin":"","legend":"","description":"","filename":"20250508SupplementalFileEJNMMISun2025.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6596974/v1/b86d38dc8ecf6b0c9ec54496.pdf"}],"financialInterests":"","formattedTitle":"Advanced Quantification Pipeline Reveals New Spatial and Temporal Tumor Characteristics in Preclinical Multiple Myeloma","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"ejnmmi-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejre","sideBox":"Learn more about [EJNMMI Research](http://ejnmmires.springeropen.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ejre/default.aspx","title":"EJNMMI Research","twitterHandle":"@officialEANM","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Multiple Myeloma (MM) Imaging, Bone Segmentation, PET/CT Quantification, Skeletal Lesions","lastPublishedDoi":"10.21203/rs.3.rs-6596974/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6596974/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground.\u003c/h2\u003e \u003cp\u003eRadiological imaging plays an indispensable role in both preclinical and clinical studies of multiple myeloma (MM). However, manual quantification in longitudinal small animal PET/CT is limited by annotator bias, signal artifacts from urinary/fecal excretion, and voxel misalignment due to non-rigid registration. To address these challenges and improve characterization of tumor biology, we developed a semi-automated PET/CT quantification pipeline targeting defined regions of interest (ROIs) within the bone marrow-rich mouse skeleton, achieving sub-organ spatial resolution, including in anatomically complex sites such as the pelvis. We applied this MM-specific preclinical pipeline to analyze tumor distribution in a longitudinal molecular PET study using an immunocompetent mouse model of skeletally disseminated MM. An Attention U-Net was trained to segment the thoracolumbar spine, pelvis and pelvic joints, sacrum, and femurs from 2D CT slices. A custom algorithm masked spillover signal from physiological excretion, and a PCA-based projection was used to map tumor distribution along the skeletal axis. Quantification metrics included mean and maximum standardized uptake values (SUV\u003csub\u003emean\u003c/sub\u003e, SUV\u003csub\u003emax\u003c/sub\u003e) from PET and Hounsfield Units (HU) from CT to assess tumor burden, spatiotemporal tumor distribution, and bone involvement.\u003c/p\u003e\u003ch2\u003eResults.\u003c/h2\u003e \u003cp\u003eTumor burden localized preferentially to skeletal regions near joints. Using precise CT-based alignment (DICE\u0026thinsp;=\u0026thinsp;0.966\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005), we detected early disease progression and aggressive phenotypes. A marked increase in tumor uptake was observed by day 18 post-implantation, with significant SUV\u003csub\u003emean\u003c/sub\u003e increases in the spine (p\u0026thinsp;=\u0026thinsp;0.012), left/right femurs (p\u0026thinsp;=\u0026thinsp;0.007/0.006), pelvis and pelvic joints (p\u0026thinsp;=\u0026thinsp;0.018), and sacrum (p\u0026thinsp;=\u0026thinsp;0.02). Notably, sex-based differences were identified: female mice showed greater bone loss near the hip joint at later stages, with significant HU\u003csub\u003emean\u003c/sub\u003e reductions at days 25 (p\u0026thinsp;=\u0026thinsp;0.008) and 32 (p\u0026thinsp;=\u0026thinsp;0.002).\u003c/p\u003e\u003ch2\u003eConclusions.\u003c/h2\u003e \u003cp\u003eThis pipeline enables reproducible, anatomically precise quantification of region-specific trends in MM progression, including joint-specific lesion tropism and sex-based differences, from longitudinal PET/CT scans. By mitigating common challenges such as excretion artifacts and inconsistent mouse positioning, our approach overcomes limitations of manual analysis and enhances evaluation of tumor biology and treatment response in preclinical models of bone-involved cancers.\u003c/p\u003e","manuscriptTitle":"Advanced Quantification Pipeline Reveals New Spatial and Temporal Tumor Characteristics in Preclinical Multiple Myeloma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-14 17:01:56","doi":"10.21203/rs.3.rs-6596974/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor Revision","date":"2025-07-06T11:54:11+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-05-16T18:32:42+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-09T15:29:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-09T04:23:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"EJNMMI Research","date":"2025-05-08T11:15:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"ejnmmi-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejre","sideBox":"Learn more about [EJNMMI Research](http://ejnmmires.springeropen.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ejre/default.aspx","title":"EJNMMI Research","twitterHandle":"@officialEANM","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4d82f931-7f89-4514-80cf-45580fc9d0ea","owner":[],"postedDate":"May 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-04T16:47:58+00:00","versionOfRecord":{"articleIdentity":"rs-6596974","link":"https://doi.org/10.1186/s13550-025-01291-x","journal":{"identity":"ejnmmi-research","isVorOnly":false,"title":"EJNMMI Research"},"publishedOn":"2025-07-31 16:12:59","publishedOnDateReadable":"July 31st, 2025"},"versionCreatedAt":"2025-05-14 17:01:56","video":"","vorDoi":"10.1186/s13550-025-01291-x","vorDoiUrl":"https://doi.org/10.1186/s13550-025-01291-x","workflowStages":[]},"version":"v1","identity":"rs-6596974","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6596974","identity":"rs-6596974","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}