4ceb16fba6aeca23c9b36c317f138199 pmc_10417313.pdf 7b812a5e8e01b2e6889fa030430faea892bb33d1 pmc_10417313.pdf 7b926b6612c93888cd5e40cff2c3de84526632f428061fbfc7a2f355a0ab454e pmc_10417313.pdf Title: Tracking Therapy Response in Glioblastoma Using 1D Convolutional Neural Networks Subject: Background: Glioblastoma (GB) is a malignant brain tumour that is challenging to treat, often relapsing even after aggressive therapy. Evaluating therapy response relies on magnetic resonance imaging (MRI) following the Response Assessment in Neuro-Oncology (RANO) criteria. However, early assessment is hindered by phenomena such as pseudoprogression and pseudoresponse. Magnetic resonance spectroscopy (MRS/MRSI) provides metabolomics information but is underutilised due to a lack of familiarity and standardisation. Methods: This study explores the potential of spectroscopic imaging (MRSI) in combination with several machine learning approaches, including one-dimensional convolutional neural networks (1D-CNNs), to improve therapy response assessment. Preclinical GB (GL261-bearing mice) were studied for method optimisation and validation. Results: The proposed 1D-CNN models successfully identify different regions of tumours sampled by MRSI, i.e., normal brain (N), control/unresponsive tumour (T), and tumour responding to treatment (R). Class activation maps using Grad-CAM enabled the study of the key areas relevant to the models, providing model explainability. The generated colour-coded maps showing the N, T and R regions were highly accurate (according to Dice scores) when compared against ground truth and outperformed our previous method. Conclusions: The proposed methodology may provide new and better opportunities for therapy response assessment, potentially providing earlier hints of tumour relapsing stages. Keywords: therapy response; glioblastoma; temozolomide; preclinical models; magnetic resonance spectroscopy; class activation mapping; Grad-CAM; convolutional neural networks; deep learning Author: Sandra Ortega-Martorell, Ivan Olier, Orlando Hernandez, Paula D. Restrepo-Galvis, Ryan A. A. Bellfield and Ana Paula Candiota Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Thu Aug 10 04:36:57 2023 CEST ModDate: Thu Aug 10 04:47:08 2023 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 20 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1814537 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- RIZNVS+VnURWPalladioL Type 1 Custom yes yes no 10 0 HSQVHS+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 BWKJOM+URWPalladioL-Roma Type 1 Custom yes yes no 20 0 YMKRBF+URWPalladioL-Ital Type 1 Custom yes yes no 24 0 LCJEVJ+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 43 0 JYUNWZ+CMSY10 Type 1 Builtin yes yes no 68 0 OQIVQT+EURM10 Type 1 Builtin yes yes no 72 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 87 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 91 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 95 0 BCDIEE+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 99 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 106 0 IWLJVN+PalatinoLinotype-Italic TrueType WinAnsi yes yes yes 134 0 RZYQRV+PalatinoLinotype-Bold TrueType WinAnsi yes yes yes 138 0 XQYVXJ+PalatinoLinotype-Roman TrueType WinAnsi yes yes yes 142 0 IWLJVN+PalatinoLinotype-Italic TrueType WinAnsi yes yes yes 155 0 RZYQRV+PalatinoLinotype-Bold TrueType WinAnsi yes yes yes 159 0 XQYVXJ+PalatinoLinotype-Roman TrueType WinAnsi yes yes yes 163 0 IWLJVN+PalatinoLinotype-Italic TrueType WinAnsi yes yes yes 175 0 RZYQRV+PalatinoLinotype-Bold TrueType WinAnsi yes yes yes 179 0 XQYVXJ+PalatinoLinotype-Roman TrueType WinAnsi yes yes yes 183 0 IWLJVN+PalatinoLinotype-Italic TrueType WinAnsi yes yes yes 198 0 RZYQRV+PalatinoLinotype-Bold TrueType WinAnsi yes yes yes 202 0 XQYVXJ+PalatinoLinotype-Roman TrueType WinAnsi yes yes yes 206 0 MDIKPC+CMBSY10 Type 1 Builtin yes yes no 214 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-01-18 02:28:29 CET RepresentationInformation: pmc_10417313.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2023-09-16 02:17:06 CEST Size: 1814537 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 520 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: MRI/MRSI Data Used in This Study Destination: subsection.2.1 Children: Item: Title: Preclinical Model Generation and Treatment Administration Destination: subsubsection.2.1.1 Item: Title: MR Data Acquisition Destination: subsubsection.2.1.2 Item: Title: MR Data Processing and Post-Processing Destination: subsubsection.2.1.3 Item: Title: Single-Point and Longitudinal Cases Used Destination: subsubsection.2.1.4 Item: Title: Data Used for the Development of the Models Destination: subsubsection.2.1.5 Item: Title: Model Development Destination: subsection.2.2 Children: Item: Title: Hierarchical Classification Approach Destination: subsubsection.2.2.1 Item: Title: Methods Used for Classification Destination: subsubsection.2.2.2 Item: Title: Hyperparameter Tuning Destination: subsubsection.2.2.3 Item: Title: Model Explainability Destination: subsection.2.3 Children: Item: Title: Class Activation Maps Destination: subsubsection.2.3.1 Item: Title: Colour-Coded Maps Destination: subsubsection.2.3.2 Item: Title: Evaluation of the Colour-Coded Maps Using a Dice Score Destination: subsubsection.2.3.3 Item: Title: Results Destination: section.3 Children: Item: Title: Selection of the Best Models Destination: subsection.3.1 Children: Item: Title: Training of the Models Destination: subsubsection.3.1.1 Item: Title: Comparison and Evaluation of the Different Algorithms Destination: subsubsection.3.1.2 Item: Title: Description of the Best-Performing Models Destination: subsubsection.3.1.3 Item: Title: Model Explainability Destination: subsection.3.2 Children: Item: Title: Class Activation Maps Destination: subsubsection.3.2.1 Item: Title: Colour-Coded Maps Destination: subsubsection.3.2.2 Item: Title: Evaluation of the Colour-Coded Maps Destination: subsubsection.3.2.3 Item: Title: Discussion Destination: section.4 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: section.6 Info: Title: Tracking Therapy Response in Glioblastoma Using 1D Convolutional Neural Networks Author: Sandra Ortega-Martorell, Ivan Olier, Orlando Hernandez, Paula D. Restrepo-Galvis, Ryan A. A. Bellfield and Ana Paula Candiota Subject: Background: Glioblastoma (GB) is a malignant brain tumour that is challenging to treat, often relapsing even after aggressive therapy. Evaluating therapy response relies on magnetic resonance imaging (MRI) following the Response Assessment in Neuro-Oncology (RANO) criteria. However, early assessment is hindered by phenomena such as pseudoprogression and pseudoresponse. Magnetic resonance spectroscopy (MRS/MRSI) provides metabolomics information but is underutilised due to a lack of familiarity and standardisation. Methods: This study explores the potential of spectroscopic imaging (MRSI) in combination with several machine learning approaches, including one-dimensional convolutional neural networks (1D-CNNs), to improve therapy response assessment. Preclinical GB (GL261-bearing mice) were studied for method optimisation and validation. Results: The proposed 1D-CNN models successfully identify different regions of tumours sampled by MRSI, i.e., normal brain (N), control/unresponsive tumour (T), and tumour responding to treatment (R). Class activation maps using Grad-CAM enabled the study of the key areas relevant to the models, providing model explainability. The generated colour-coded maps showing the N, T and R regions were highly accurate (according to Dice scores) when compared against ground truth and outperformed our previous method. Conclusions: The proposed methodology may provide new and better opportunities for therapy response assessment, potentially providing earlier hints of tumour relapsing stages. Keywords: therapy response; glioblastoma; temozolomide; preclinical models; magnetic resonance spectroscopy; class activation mapping; Grad-CAM; convolutional neural networks; deep learning Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Thu Aug 10 04:36:57 CEST 2023 ModDate: Thu Aug 10 04:47:08 CEST 2023 ID: 0x90c149257ce726c652b01218abccd887, 0x90c149257ce726c652b01218abccd887 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: BWKJOM+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 241 FontDescriptor: FontName: BWKJOM+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: JYUNWZ+CMSY10 FontSubset: true FirstChar: 0 LastChar: 6 FontDescriptor: FontName: JYUNWZ+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: MDIKPC+CMBSY10 FontSubset: true FirstChar: 6 LastChar: 6 FontDescriptor: FontName: MDIKPC+CMBSY10 Flags: Symbolic FontBBox: -26, -940, 1332, 825 FontFile: true Font: BaseFont: YMKRBF+URWPalladioL-Ital FontSubset: true FirstChar: 46 LastChar: 121 FontDescriptor: FontName: YMKRBF+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: OQIVQT+EURM10 FontSubset: true FirstChar: 13 LastChar: 22 FontDescriptor: FontName: OQIVQT+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: RIZNVS+VnURWPalladioL FontSubset: true FirstChar: 129 LastChar: 243 FontDescriptor: FontName: RIZNVS+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: HSQVHS+URWPalladioL-Bold FontSubset: true FirstChar: 2 LastChar: 122 FontDescriptor: FontName: HSQVHS+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 106, 281, 152, 290 Annotation: Subtype: Link Rect: 34, 269, 140, 278 Annotation: Subtype: Link Rect: 34, 164, 96, 186 Annotation: Subtype: Link Rect: 126, 90, 154, 99 Annotation: Subtype: Link Rect: 34, 78, 147, 87 Annotation: Subtype: Link Rect: 34, 756, 152, 792 Annotation: Subtype: Link Rect: 515, 756, 560, 792 Annotation: Subtype: Link Rect: 160, 663, 171, 676 Annotation: Subtype: Link Rect: 227, 663, 239, 676 Annotation: Subtype: Link Rect: 131, 650, 142, 664 Annotation: Subtype: Link Rect: 273, 650, 284, 664 Annotation: Subtype: Link Rect: 114, 17, 265, 28 Annotation: Subtype: Link Rect: 410, 17, 560, 28 Annotation: Subtype: Link Rect: 34, 366, 87, 385 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 192, 682, 199, 691 ActionDest: 227 Annotation: Subtype: Link Rect: 241, 620, 248, 629 ActionDest: 227 Annotation: Subtype: Link Rect: 379, 544, 386, 553 ActionDest: 227 Annotation: Subtype: Link Rect: 362, 444, 369, 453 ActionDest: 227 Annotation: Subtype: Link Rect: 370, 444, 376, 453 ActionDest: 227 Annotation: Subtype: Link Rect: 335, 393, 342, 403 ActionDest: 227 Annotation: Subtype: Link Rect: 455, 343, 462, 352 ActionDest: 227 Annotation: Subtype: Link Rect: 465, 343, 472, 352 ActionDest: 227 Annotation: Subtype: Link Rect: 463, 318, 475, 327 ActionDest: 227 Annotation: Subtype: Link Rect: 478, 318, 490, 327 ActionDest: 227 Annotation: Subtype: Link Rect: 206, 306, 218, 315 ActionDest: 227 Annotation: Subtype: Link Rect: 221, 306, 233, 315 ActionDest: 227 Annotation: Subtype: Link Rect: 303, 306, 315, 315 ActionDest: 227 Annotation: Subtype: Link Rect: 318, 306, 330, 315 ActionDest: 230 Annotation: Subtype: Link Rect: 530, 306, 542, 315 ActionDest: 230 Annotation: Subtype: Link Rect: 544, 306, 556, 315 ActionDest: 230 Annotation: Subtype: Link Rect: 189, 280, 201, 290 ActionDest: 230 Annotation: Subtype: Link Rect: 204, 281, 216, 290 ActionDest: 230 Annotation: Subtype: Link Rect: 405, 230, 416, 239 ActionDest: 230 Annotation: Subtype: Link Rect: 240, 142, 252, 151 ActionDest: 230 Annotation: Subtype: Link Rect: 388, 105, 400, 114 ActionDest: 230 Annotation: Subtype: Link Rect: 543, 92, 555, 101 ActionDest: 230 Annotation: Subtype: Link Rect: 286, 80, 298, 89 ActionDest: 230 Annotation: Subtype: Link Rect: 298, 80, 310, 89 ActionDest: 230 Annotation: Subtype: Link Rect: 457, 80, 469, 89 ActionDest: 230 Annotation: Subtype: Link Rect: 279, 67, 291, 76 ActionDest: 230 Annotation: Subtype: Link Rect: 406, 67, 418, 76 ActionDest: 230 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 472, 660, 479, 669 ActionDest: 227 Annotation: Subtype: Link Rect: 261, 635, 273, 644 ActionDest: 230 Annotation: Subtype: Link Rect: 220, 597, 227, 606 ActionDest: 227 Annotation: Subtype: Link Rect: 276, 585, 288, 594 ActionDest: 230 Annotation: Subtype: Link Rect: 260, 572, 272, 581 ActionDest: 230 Annotation: Subtype: Link Rect: 491, 535, 498, 544 ActionDest: 227 Annotation: Subtype: Link Rect: 427, 522, 439, 531 ActionDest: 230 Annotation: Subtype: Link Rect: 472, 429, 560, 441 Annotation: Subtype: Link Rect: 165, 417, 364, 429 Annotation: Subtype: Link Rect: 354, 395, 365, 403 ActionDest: 233 Annotation: Subtype: Link Rect: 297, 253, 560, 265 Annotation: Subtype: Link Rect: 165, 241, 230, 253 Annotation: Subtype: Link Rect: 245, 218, 252, 227 ActionDest: 227 Annotation: Subtype: Link Rect: 253, 218, 265, 227 ActionDest: 233 Annotation: Subtype: Link Rect: 219, 105, 226, 114 ActionDest: 227 Annotation: Subtype: Link Rect: 227, 106, 239, 114 ActionDest: 233 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 199, 683, 221, 695 ActionDest: 76 Annotation: Subtype: Link Rect: 239, 673, 246, 682 ActionDest: 227 Annotation: Subtype: Link Rect: 419, 534, 431, 543 ActionDest: 233 Annotation: Subtype: Link Rect: 210, 459, 222, 468 ActionDest: 233 Annotation: Subtype: Link Rect: 388, 447, 400, 455 ActionDest: 233 Annotation: Subtype: Link Rect: 446, 383, 458, 392 ActionDest: 227 Annotation: Subtype: Link Rect: 307, 371, 314, 380 ActionDest: 227 Annotation: Subtype: Link Rect: 314, 371, 321, 380 ActionDest: 227 Annotation: Subtype: Link Rect: 199, 356, 221, 368 ActionDest: 65 Annotation: Subtype: Link Rect: 483, 318, 490, 330 ActionDest: 76 Annotation: Subtype: Link Rect: 225, 295, 232, 305 ActionDest: 227 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 337, 735, 344, 744 ActionDest: 227 Annotation: Subtype: Link Rect: 508, 720, 515, 732 ActionDest: 79 Annotation: Subtype: Link Rect: 491, 404, 498, 416 ActionDest: 79 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 509, 720, 521, 729 ActionDest: 230 Annotation: Subtype: Link Rect: 190, 695, 202, 704 ActionDest: 233 Annotation: Subtype: Link Rect: 357, 695, 369, 704 ActionDest: 233 Annotation: Subtype: Link Rect: 467, 695, 479, 704 ActionDest: 233 Annotation: Subtype: Link Rect: 284, 682, 296, 691 ActionDest: 233 Annotation: Subtype: Link Rect: 259, 419, 270, 428 ActionDest: 233 Annotation: Subtype: Link Rect: 461, 393, 473, 403 ActionDest: 233 Annotation: Subtype: Link Rect: 302, 167, 314, 176 ActionDest: 233 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 456, 496, 468, 505 ActionDest: 233 Annotation: Subtype: Link Rect: 443, 269, 455, 279 ActionDest: 233 Annotation: Subtype: Link Rect: 255, 206, 267, 215 ActionDest: 233 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 210, 505, 217, 517 ActionDest: 79 Annotation: Subtype: Link Rect: 514, 507, 526, 516 ActionDest: 233 Annotation: Subtype: Link Rect: 195, 381, 202, 391 ActionDest: 121 Annotation: Subtype: Link Rect: 221, 381, 228, 391 ActionDest: 121 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 332, 708, 339, 720 ActionDest: 121 Annotation: Subtype: Link Rect: 358, 708, 365, 720 ActionDest: 121 Annotation: Subtype: Link Rect: 284, 645, 306, 657 ActionDest: 121 Annotation: Subtype: Link Rect: 264, 607, 271, 619 ActionDest: 124 Annotation: Subtype: Link Rect: 290, 607, 297, 619 ActionDest: 124 Annotation: Subtype: Link Rect: 524, 177, 531, 189 ActionDest: 127 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 368, 113, 375, 125 ActionDest: 148 Annotation: Subtype: Link Rect: 394, 113, 401, 125 ActionDest: 148 Annotation: Subtype: Link Rect: 553, 113, 560, 125 ActionDest: 192 Annotation: Subtype: Link Rect: 289, 88, 296, 100 ActionDest: 192 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 319, 420, 339, 432 ActionDest: 118 Annotation: Subtype: Link Rect: 319, 82, 339, 94 ActionDest: 118 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 537, 424, 558, 435 ActionDest: 118 Annotation: Subtype: Link Rect: 397, 375, 404, 389 ActionDest: 148 Annotation: Subtype: Link Rect: 421, 375, 428, 389 ActionDest: 148 Annotation: Subtype: Link Rect: 458, 277, 464, 284 ActionDest: 227 Annotation: Subtype: Link Rect: 252, 133, 259, 142 ActionDest: 227 Annotation: Subtype: Link Rect: 321, 118, 328, 130 ActionDest: 211 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 289, 711, 295, 719 ActionDest: 227 Annotation: Subtype: Link Rect: 309, 675, 315, 683 ActionDest: 227 Annotation: Subtype: Link Rect: 408, 364, 420, 373 ActionDest: 233 Annotation: Subtype: Link Rect: 420, 364, 432, 373 ActionDest: 233 Annotation: Subtype: Link Rect: 184, 101, 191, 110 ActionDest: 227 Annotation: Subtype: Link Rect: 191, 101, 203, 110 ActionDest: 230 Annotation: Subtype: Link Rect: 204, 101, 216, 110 ActionDest: 230 Annotation: Subtype: Link Rect: 216, 101, 228, 110 ActionDest: 233 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 278, 723, 290, 732 ActionDest: 233 Annotation: Subtype: Link Rect: 496, 685, 503, 694 ActionDest: 227 Annotation: Subtype: Link Rect: 497, 660, 504, 669 ActionDest: 227 Annotation: Subtype: Link Rect: 377, 647, 384, 657 ActionDest: 227 Annotation: Subtype: Link Rect: 227, 494, 234, 506 ActionDest: 121 Annotation: Subtype: Link Rect: 256, 494, 263, 506 ActionDest: 121 Annotation: Subtype: Link Rect: 497, 406, 504, 418 ActionDest: 148 Annotation: Subtype: Link Rect: 524, 406, 531, 418 ActionDest: 148 Annotation: Subtype: Link Rect: 474, 381, 481, 393 ActionDest: 148 Annotation: Subtype: Link Rect: 352, 369, 359, 381 ActionDest: 127 Annotation: Subtype: Link Rect: 438, 319, 445, 331 ActionDest: 127 Annotation: Subtype: Link Rect: 289, 246, 300, 255 ActionDest: 233 Annotation: Subtype: Link Rect: 419, 246, 431, 255 ActionDest: 233 Annotation: Subtype: Link Rect: 543, 208, 555, 217 ActionDest: 233 Annotation: Subtype: Link Rect: 298, 183, 310, 192 ActionDest: 233 Annotation: Subtype: Link Rect: 313, 183, 325, 192 ActionDest: 233 Annotation: Subtype: Link Rect: 427, 83, 439, 92 ActionDest: 233 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 343, 720, 350, 732 ActionDest: 148 Annotation: Subtype: Link Rect: 375, 635, 382, 644 ActionDest: 227 Annotation: Subtype: Link Rect: 452, 582, 459, 594 ActionDest: 127 Annotation: Subtype: Link Rect: 444, 509, 451, 519 ActionDest: 227 Annotation: Subtype: Link Rect: 286, 472, 298, 481 ActionDest: 233 Annotation: Subtype: Link Rect: 298, 472, 310, 481 ActionDest: 233 Annotation: Subtype: Link Rect: 468, 459, 480, 468 ActionDest: 233 Annotation: Subtype: Link Rect: 458, 434, 470, 443 ActionDest: 236 Annotation: Subtype: Link Rect: 471, 434, 483, 443 ActionDest: 236 Annotation: Subtype: Link Rect: 524, 409, 536, 418 ActionDest: 233 Annotation: Subtype: Link Rect: 272, 372, 284, 380 ActionDest: 236 Annotation: Subtype: Link Rect: 421, 321, 433, 330 ActionDest: 236 Annotation: Subtype: Link Rect: 521, 309, 528, 318 ActionDest: 227 Annotation: Subtype: Link Rect: 359, 233, 366, 242 ActionDest: 227 Annotation: Subtype: Link Rect: 242, 221, 254, 230 ActionDest: 236 Annotation: Subtype: Link Rect: 417, 208, 429, 217 ActionDest: 236 Annotation: Subtype: Link Rect: 276, 170, 288, 180 ActionDest: 236 Annotation: Subtype: Link Rect: 543, 133, 555, 142 ActionDest: 236 Annotation: Subtype: Link Rect: 286, 108, 298, 117 ActionDest: 236 Annotation: Subtype: Link Rect: 225, 83, 237, 92 ActionDest: 236 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 522, 733, 529, 745 ActionDest: 192 Annotation: Subtype: Link Rect: 245, 632, 252, 644 ActionDest: 211 Annotation: Subtype: Link Rect: 517, 585, 529, 594 ActionDest: 236 Annotation: Subtype: Link Rect: 529, 585, 541, 594 ActionDest: 236 Annotation: Subtype: Link Rect: 194, 484, 201, 493 ActionDest: 227 Annotation: Subtype: Link Rect: 540, 183, 560, 194 Annotation: Subtype: Link Rect: 165, 171, 262, 182 Annotation: Subtype: Link Rect: 444, 118, 560, 131 Annotation: Subtype: Link Rect: 165, 106, 422, 117 Page: Sequence: 17 Annotations: Annotation: Subtype: Link Rect: 287, 704, 456, 715 Annotation: Subtype: Link Rect: 95, 562, 133, 572 Annotation: Subtype: Link Rect: 139, 562, 175, 572 Annotation: Subtype: Link Rect: 263, 527, 300, 538 Annotation: Subtype: Link Rect: 306, 527, 342, 538 Annotation: Subtype: Link Rect: 104, 493, 142, 503 Annotation: Subtype: Link Rect: 148, 493, 184, 503 Annotation: Subtype: Link Rect: 95, 470, 133, 480 Annotation: Subtype: Link Rect: 139, 470, 175, 480 Annotation: Subtype: Link Rect: 226, 435, 263, 446 Annotation: Subtype: Link Rect: 131, 401, 168, 411 Annotation: Subtype: Link Rect: 475, 377, 512, 388 Annotation: Subtype: Link Rect: 314, 344, 351, 354 Annotation: Subtype: Link Rect: 414, 308, 452, 319 Annotation: Subtype: Link Rect: 509, 285, 547, 296 Annotation: Subtype: Link Rect: 243, 262, 280, 273 Annotation: Subtype: Link Rect: 440, 239, 477, 250 Annotation: Subtype: Link Rect: 355, 205, 393, 216 Annotation: Subtype: Link Rect: 59, 171, 96, 181 Annotation: Subtype: Link Rect: 59, 137, 96, 147 Annotation: Subtype: Link Rect: 389, 113, 426, 124 Annotation: Subtype: Link Rect: 353, 91, 391, 101 Annotation: Subtype: Link Rect: 266, 67, 304, 78 Page: Sequence: 18 Annotations: Annotation: Subtype: Link Rect: 262, 710, 300, 721 Annotation: Subtype: Link Rect: 473, 687, 511, 698 Annotation: Subtype: Link Rect: 59, 653, 96, 664 Annotation: Subtype: Link Rect: 102, 653, 138, 664 Annotation: Subtype: Link Rect: 519, 629, 557, 640 Annotation: Subtype: Link Rect: 59, 619, 95, 629 Annotation: Subtype: Link Rect: 221, 595, 258, 606 Annotation: Subtype: Link Rect: 264, 595, 301, 606 Annotation: Subtype: Link Rect: 59, 561, 96, 571 Annotation: Subtype: Link Rect: 338, 526, 376, 537 Annotation: Subtype: Link Rect: 388, 491, 425, 502 Annotation: Subtype: Link Rect: 322, 457, 359, 468 Annotation: Subtype: Link Rect: 202, 377, 240, 387 Annotation: Subtype: Link Rect: 243, 353, 280, 364 Annotation: Subtype: Link Rect: 377, 330, 415, 341 Annotation: Subtype: Link Rect: 108, 308, 146, 318 Annotation: Subtype: Link Rect: 204, 273, 242, 284 Annotation: Subtype: Link Rect: 292, 238, 330, 249 Annotation: Subtype: Link Rect: 271, 215, 309, 226 Annotation: Subtype: Link Rect: 277, 193, 315, 203 Annotation: Subtype: Link Rect: 90, 159, 128, 169 Annotation: Subtype: Link Rect: 86, 124, 123, 134 Annotation: Subtype: Link Rect: 95, 90, 132, 100 Annotation: Subtype: Link Rect: 332, 66, 369, 77 Page: Sequence: 19 Annotations: Annotation: Subtype: Link Rect: 383, 722, 421, 733 Annotation: Subtype: Link Rect: 427, 722, 463, 733 Annotation: Subtype: Link Rect: 227, 629, 265, 640 Annotation: Subtype: Link Rect: 271, 629, 307, 640 Annotation: Subtype: Link Rect: 186, 595, 223, 606 Annotation: Subtype: Link Rect: 286, 584, 324, 594 Annotation: Subtype: Link Rect: 358, 549, 396, 560 Annotation: Subtype: Link Rect: 402, 549, 438, 560 Annotation: Subtype: Link Rect: 264, 526, 301, 537 Annotation: Subtype: Link Rect: 127, 504, 164, 514 Annotation: Subtype: Link Rect: 342, 480, 380, 491 Annotation: Subtype: Link Rect: 492, 457, 530, 468 Annotation: Subtype: Link Rect: 467, 445, 505, 456 Annotation: Subtype: Link Rect: 149, 423, 186, 433 Annotation: Subtype: Link Rect: 303, 399, 340, 410 Annotation: Subtype: Link Rect: 77, 377, 114, 387 Annotation: Subtype: Link Rect: 170, 343, 208, 353 Annotation: Subtype: Link Rect: 360, 319, 397, 330 Annotation: Subtype: Link Rect: 362, 296, 400, 307 Annotation: Subtype: Link Rect: 331, 273, 368, 284 Annotation: Subtype: Link Rect: 493, 250, 531, 261 Annotation: Subtype: Link Rect: 179, 216, 217, 226 Annotation: Subtype: Link Rect: 323, 192, 360, 203 Annotation: Subtype: Link Rect: 136, 158, 174, 169 Annotation: Subtype: Link Rect: 59, 136, 96, 146 Annotation: Subtype: Link Rect: 102, 136, 138, 146 Annotation: Subtype: Link Rect: 304, 101, 342, 111 Annotation: Subtype: Link Rect: 348, 101, 384, 111 Annotation: Subtype: Link Rect: 519, 77, 557, 88 Annotation: Subtype: Link Rect: 59, 67, 95, 77 Page: Sequence: 20 Annotations: Annotation: Subtype: Link Rect: 95, 722, 133, 733 Annotation: Subtype: Link Rect: 139, 722, 175, 733 Annotation: Subtype: Link Rect: 516, 699, 554, 710 Annotation: Subtype: Link Rect: 59, 665, 96, 675 Annotation: Subtype: Link Rect: 59, 630, 96, 640 Annotation: Subtype: Link Rect: 461, 606, 498, 617 Annotation: Subtype: Link Rect: 135, 584, 173, 594 Annotation: Subtype: Link Rect: 104, 561, 142, 571 Annotation: Subtype: Link Rect: 497, 537, 535, 548 Annotation: Subtype: Link Rect: 156, 515, 193, 525 Annotation: Subtype: Link Rect: 344, 491, 382, 502 Annotation: Subtype: Link Rect: 178, 458, 216, 468 Checksum: 0225385b Type: CRC32 Checksum: 4ceb16fba6aeca23c9b36c317f138199 Type: MD5 Checksum: 7b812a5e8e01b2e6889fa030430faea892bb33d1 Type: SHA-1 Checksum: 7b926b6612c93888cd5e40cff2c3de84526632f428061fbfc7a2f355a0ab454e Type: SHA-256