ACRIN 6690 PATHOLOGY MANUAL

Transcription

1 ACRIN 6690 PATHOLOGY MANUAL

2 ACRIN 6690 Prepared by the American College of Radiology Imaging Network Original: December 15, 2010 Version Date: May 3, 2012 American College of Radiology Imaging Network 1818 Market Street, Suite 1600 Philadelphia, PA Phone: extension 4183 Michael Nalesnik, MD Professor of Pathology University of Pittsburgh School of Medicine Division of Transplantation Pathology University of Pittsburgh Medical Center Suite E Lothrop Street Pittsburgh, PA Mitchell D. Schnall, MD, PhD Network Chair University of Pennsylvania Health System Department of Radiology 3400 Spruce Street Philadelphia, PA Christoph Wald, MD, PhD Associate Professor of Radiology Tufts University Medical School Radiology Department Lahey Clinic Medical Center Burlington, MA Phone: Fax: Version 2.0: May 3, 2012 Page 2 of 28

3 ACRIN 6690 Table of Contents 1.0 Introduction: Regulatory Considerations Local Institution Identifies Participant Obtaining MR and CT Images and Reports Explant Liver Workup in Local Pathology Lab: Goals Explant Liver Workup and Radiographic Correlation in Local Pathology Lab: Implementation Digital Photography of Macroscopic Pathology: Submission Instructions AT THE SITE S REQUEST: Pathology Block and/or Slides Shipped for Central Pathology Review and Adjudication Questions and Contact Information Attachments 1) Research Patient List ) Reminder Sheet for Macroscopic Analysis ) Topics Regarding the Pathologic Diagnosis of Hepatocellular Carcinoma ) Pointers on Macroscopic Pathology Photography and Labeling.. 23 Version 2.0: May 3, 2012 Page 3 of 28

4 ACRIN Introduction: Regulatory Considerations This protocol is conducted according to United States and international standards of International Conference on Harmonisation [ICH] Guidelines/Good Clinical Practice [GCP], applicable government regulations (e.g. Title 45, Part 46 Code of Federal Regulations), and the American College of Radiology Imaging Network (ACRIN) research policies and procedures. Informed Consent Form/Human Subject Protection. Study participants are consented per informed consent procedures in accordance with Title 45 Code of Federal Regulations (CFR) Part 46 and Title 21 CFR Part 50. The institution-specific, IRB-approved informed consent form grants permission for study investigators to examine the participant s explant liver in comparison with study-related imaging. For this protocol, permission to perform this analysis must be obtained from the participant. Confidentiality. The confidentiality of the participant s identity will be maintained. All collected information will be protected per ACRIN policies and procedures and federal regulatory guidelines. Access to study data will be limited to the ACRIN 6690 staff and authorized representatives of ACRIN. All computer data will be maintained in a manner consistent with Title 21 CRF Part 11. In addition, access to the data management system will be limited to designated staff through use of individualized, confidential login ID and password. Designated staff must not share login IDs or passwords. ACRIN is not a covered entity according to the Privacy Rules of the 1996 Health Insurance Portability and Accountability Act (HIPAA); therefore, HIPAA regulations should be followed according to institutional standards. Safe Laboratory Practices. Pathology faculty and staff bear the responsibility to practice safe work habits per national regulatory and accrediting agencies policies for laboratory safety standards. Laboratory policy defers to institutional standards for clinical trials research in minimizing risk and ensuring management for biological, electrical, chemical, and fire and explosive hazards safety; radiation hazards; cryogenics; ergonomics and environment control; housekeeping and sanitation; storage and security; and manuals and record keeping. Record Maintenance. The data from this study will be maintained per protocol specifications and for a minimum of seven (7) years following completion of the study, or for a longer period should the data be required for research. Participating institutions will be notified if the data need to be maintained beyond minimum requirements. Upon notification, data will be destroyed per institutional policy and as required by the ACRIN Record Retention Policy and federal regulatory guidelines. ACRIN encourages sites to retain pathology specimens until the completion of the trial or the core laboratory assessment of the radiology-pathology correlation has been completed. Once the core lab assessment is completed, the sites will be informed that the original specimen is no longer needed. Pathology specimens will be destroyed per institutional policy. Version 2.0: May 3, 2012 Page 4 of 28

5 ACRIN Local Institution Identifies Participant and Radiologist and Pathologist Prepare for Rad/Path Analysis Not all patients transplanted in a participating center will be part of this study cohort. In fact, in most centers, approximately 75% of patients are transplanted with regular MELD points rather than HCC-exception points. Therefore, the majority of explant livers received by a pathology department in a participating center will not belong to a participant from this study cohort. We highly recommend that study coordinators provide a Research Participant List (see Attachment 1) with participant names and explant dates to the Anatomic Surgical Pathology (macroscopic) lab on a weekly basis to aid in identification of trial-related livers from participants. For each participant, CT and MR images no older than 90 days pre-explant need to be available via institutional PACS system for the pathology lab to access during liver processing. Whenever possible, radiologists should be present during dissection of the explant liver to aid with identification and rad-path correlation of all non-cancerous/optn Class 4 and HCC/OPTN Class 5 liver lesions that have been identified in a given participant in vivo prior to transplant. However, should coordinated analysis be impossible, the pathologist may proceed with analysis using the PA Form lesion reporting information (which may be pre-populated prior to radiology/pathology correlation) and images of the most recent CT and MR exams to aid rad-path correlation. 2.1 Site PIs and Coordinators: Identifying Participants On-Trial Upon receipt of an explant liver, the local pathologist or a designee familiar with the ACRIN 6690 protocol will check with the transplant team and/or trial study coordinator to see whether the patient is enrolled in this trial so the explant workup, including digital photographs for the trial, can be performed according to specifications. Alternatively, the study site principal investigator and study coordinator will inform the local pathologist of when the study participant went to transplant and the date of the most-recent CT and MR images (including the Eovist-enhanced MRI if the participant has consented to this sub-study and images are available). It is also ideal to provide an instructional memo that highlights the needs and specific requirements of the ACRIN 6690 trial. (See Pathology Macroscopic Analysis Overview provided as Attachment 2 and Pointers on Macroscopic Pathology Photography and Labeling provided as Attachment 4.) 2.2 Images Preparation Prior to Explant Analysis Local pathologists will be provided with multiplanar images per their preference for correlation and will be required to identify all Class 4 and 5 lesions described on presurgical imaging. It would be helpful for pathologists to talk to the site radiologist overseeing the imaging portion of this trial about the need for specific multiplanar images so they can be created and provided in PACS. Version 2.0: May 3, 2012 Page 5 of 28

6 ACRIN 6690 Do not conduct explant liver dissection until the results images and interpretation forms are available for the CT and MR studies completed most-recently prior to explant. (If liver analysis is completed prior to availability of images and results and/or prior to completion of forms, this will be considered a protocol violation.) 2.3 Processing the Explant Liver: An Overview Explant livers should ideally be processed on the day of explant. If this is not possible, the pathologist or designates should take appropriate steps to minimize tissue autolysis, which could interfere with accurate histopathologic assessment. Such steps should not interfere with subsequent radiologic-pathologic correlation. Lesions will be designated with unique identifiers (see Section 5.0 for details) to allow one-to-one correlation of radiologic and microscopic diagnoses on a per-lesion basis. In the unlikely event that no lesion is identified pathologically at the site of a radiologic lesion, the segmental area of the radiologic lesion will be photographed and extensively sampled according to the judgment of the local pathologist and this discrepancy noted on the PA Form for Local Pathology: Macroscopic Analysis. In the event an area of ablation has been identified with incomplete margins and no biopsy was conducted prior to ablation, the segmental area of the radiologic finding will be extensively sampled since residual tumor cells may allow diagnosis. 3.0 Obtaining MR and CT Images and Reports Available dictated reports, applicable data elements available through ACRIN (e.g., for the study-related complementary imaging assessment data since no formal dictated report will likely be generated), and corresponding images from most-recent CT and MRI scans (including the Eovist-enhanced MRI if the participant has consented to this sub-study and images are available) will be provided to the pathologist and macroscopic lab by the local site trial designated radiologist and/or the study coordinator. Again, analysis of the explant liver should never begin without most-recent images and interpretation forms. The following radiologist(s) and study coordinator should be notified as soon as the explant liver has been received, prior to the explant liver being processed: <<Site-Specific Contact Information may be included here.>> Site coordinators should make every effort to have participant historical data available to the pathology lab at the time of processing the explant liver. If the participant has undergone ablative therapy, any available pre-ablation biopsy reports should be provided to the pathologist. We request that the radiologist(s) be present during the macroscopic evaluation of the explant liver to assist in identification, photographing, sampling, and labeling of the Version 2.0: May 3, 2012 Page 6 of 28

7 ACRIN 6690 radiographically-identified lesion, in order to correlate between imaging and pathology. Alternatively, the PA Form may be pre-populated by the radiologist to correlate lesions across imaging modalities. At the time of explant liver evaluation, the corresponding most-recent MRI and CT images (including the Eovist-enhanced MRI if the participant has consented to this substudy and images are available) will need to be made available (through institutional PACS or with assistance from ACRIN), as well as imaging reports and interpretation forms, and should be reviewed to guide lesion search and 1:1 correlation between imaging and the pathology assessment. 4.0 Explant Liver Workup in Local Pathology Lab: Goals 4.1 Overview Take digital photographs of each side of all cut gross specimen liver sections. Take digital photographs of each identified and sampled lesion. Sample all suspected lesions. Correlate lesions to imaging findings and/or identify them as not being identified on imaging. 4.2 Additional Details Identify, photograph, sample, and label per protocol-specific parameters all lesions previously identified on imaging as either OPTN Class 4 or Class 5 and obtain 1:1 macroscopic and histopathologic correlation. Identify, photograph, sample, and label per protocol-specific parameters all other grossly suspicious focal liver lesions and report/record those which turn out to be HCC but were not recorded as such (false negative imaging findings). Report pathologic staging on a per-patient basis (American Joint Committee on Cancer, Cancer Staging Manual, 7 th ed.) based on results from the identification, sampling, and labeling of lesions as described above. The digital photographs of liver slices and macroscopic lesions will be submitted to the central trial data collection center repository. (See Section 6.0 below for submission instructions.) Photographs are uploaded to ACRIN and may be reviewed selectively by a central pathologist for quality control purposes, to ensure compliance with the protocol specifications, and to allow feedback to participating sites should the need arise, especially early in the trial process. Version 2.0: May 3, 2012 Page 7 of 28

8 ACRIN Explant Liver Workup and Radiographic Correlation in Local Pathology Lab: Macroscopic and Microscopic 5.1 Macroscopic Work: Identification, Sampling, Labeling, and Photographing Macroscopic inspection will include digital photographic documentation of each liver slice (photograph of each side) and a photograph of each of the areas of sampling (that is, of each lesion). Labeling of individual photographs should be performed in a manner that facilitates ordered reconstruction of images and avoids obstruction of physical labels used to identify areas of sampling. Photographs should include ruler or other means to allow accurate measurements of lesions directly from images. See Pointers on Macroscopic Pathology Photography and Labeling in Attachment 4 for examples of appropriate photographs for the trial. All lesions suspicious for HCC on macroscopic inspection of the liver sections will be documented by digital photographs, sampled, and histologically analyzed. Each lesion sampled that was not identified on imaging should be labeled to include, at a minimum: liver segment, consecutive number in series of lesions per case. Labeling by location will be consecutively numbered within each liver segment (see Figure 1). Figure 1. Schematic illustrates Couinaud segmental liver anatomy and the normal portal venous structures. Legend: IVC = inferior vena cava PV = portal vein Adapted from Madoff DC, et al. Radiographics. 2002;22: Version 2.0: May 3, 2012 Page 8 of 28

9 ACRIN 6690 Labeling needs to be consistently introduced from superior to inferior and anterior to posterior (anatomical reference). Lesions should be identified on pathology and imaging and labeled in tandem to ensure correlation. The goal of labeling is to unambiguously identify individual lesions that can be correlated to microscopic slides and radiographic images throughout the entire study process. Labeling of samples, for consistency across all participating study sites in the trial, is described below. A number of imaging and photographic systems incorporate software that allows image annotation, and this is acceptable. In the absence of this capability, physical labels such as arrows with numbers, etc., may be used to identify lesions providing that they do not obscure the sampled lesions. See Pointers on Macroscopic Pathology Photography and Labeling as Attachment 4 for samples of acceptable photographs for the trial. Pathologists should have the study-specific PA Form (Local Pathology: Macroscopic Analysis) for use during macroscopic examination of the explant liver. The PA Form is available online at under Data Forms in the left navigation column. Local pathologic analysis of explant liver will be performed by the cutting of liver, at a thickness of 10 mm or less. In addition, it will be ideal to slice the liver in the same direction the imaging was performed. Labeling for Lesion Correlation Between Pathology and Imaging Digital photography for the ACRIN 6690 trial is required to allow researchers to correlate pathology results with imaging findings. Labeling ultimately should describe the slice and location of lesions and reflect all sampled tissue for microscopic assessment. Sites should be able to obtain slides/slide information based on digital photograph labeling details. PHOTOGRAPHS BY SLICE Each liver slice will be photographed first on each side and labeled according to the slice number (see also the PA Form example on the next page for location of slice number on the ACRIN form) and side of the cut: ACRIN-assigned Case #; Slice # (from the pathology cuts in sequential order); Side of cut: cranial (superior) or caudal (inferior); anterior or posterior. ACRIN-CASE#.SLICE#.SIDEofSLICE e.g.: cranial or anterior for one side and caudal or posterior for the other side (See also Figure 1 in Attachment 4) Version 2.0: May 3, 2012 Page 9 of 28

10 PHOTOGRAPHS BY LESION ACRIN 6690 Pathologists will need to make every attempt possible to document (on paper and in digital photographs), sample, and histologically evaluate all lesions described by imaging as OPTN Class 4, 4-g, 5A, 5A-g, 5B, 5B-g, or 5T. Lesions not identified on imaging will also be reported on ACRIN data forms. If there is more than one lesion present in a specific segment, the running lesion number within that given segment is assigned in sequential order from most superior to inferior and anterior to posterior anatomic location. Note: If a lesion cannot be unequivocally assigned to just one specific segment, identify the lesion by the involved segment with the highest Arabic numeral (not to be confused with the segment with the greatest tumor burden). For example, a lesion seen involving segments 6, 7, and 8 would have a [main] segment number of 8 for labeling, and the additional involved segments would be identified on the appropriate data entry form. Should a lesion appear in segments 4a and 4b, then 4b would be used as the main segment, following the same logic (b>a). Pathologists also will record on the PA Form, Local Pathology: Macroscopic Analysis, if: a) A lesion diagnosed on imaging cannot be identified by pathology; b) Any lesion is found by pathology that was not identified by imaging. For photographic examples of adequate digital photography and labeling of images for submission to ACRIN, see Pointers on Macroscopic Pathology Photography and Labeling (see Attachment 4). Please print and provide the following page to the pathology department as an aid to lesion photo labeling in coordination with lesion identification on the PA Form. Version 2.0: May 3, 2012 Page 10 of 28

11 ACRIN 6690 Labeling of lesions for each digital photograph should include the following four elements for each lesion. The sample PA Form image below provides numbered arrows that match the location of elements on the PA Form that need to match labeling of the lesion photos. 1 st arrow: ACRIN-assigned Case #; 2 nd arrow: Slice # (from the pathology cuts in sequential order; a single lesion may be represented in multiple slices and photos from each slice will need to be included); 3 rd arrow: Highest-number liver segment #; and 4 th arrow: Running # (used if there is more than one lesion in a slice). 1st 3rd 4th 2nd ACRIN-CASE#.SLICE#.SEGMENT#.RUNNING# (See the PA Form sample above and Figure 8 in Attachment 4) EXAMPLE, One Lesion, More Than One Slice: e.g.: and for the same lesion seen in two slices of liver (more than one slice is indicated in the 2 nd arrow element of the PA Form example above); - the lesion s highest-number segment is still 6, but the lesion appears in two different slices of the explant liver. EXAMPLE, Two Lesions, Same Slice: e.g.: and for two lesions seen in slice 12, segment 6; - segment 6 is the highest-numbered segment that contains lesion (even though it may also appear in segments 4 and 5, for example); and - lesion #1 is more superior compared with lesion #2. Version 2.0: May 3, 2012 Page 11 of 28

12 ACRIN 6690 IMPORTANT: ABILITY TO ACCESS MICROPATHOLOGY SLIDES ACRIN will not be collecting or photographing slides. However, lesions seen and unseen on imaging may be reviewed to determine histology, so slides for this trial should be labeled to easily link cells on slides with the digital photographs and MRI/CT images. For example, slide identifiers could include the same elements as the lesion specimens labels on the photos, plus a running slide letter to identify sequential slides containing tissue from a single lesion. 5.2 Microscopic Work For ease of work flow, the trial team recommends that the pathologist determine whether biopsies have been performed pre-ablation and collect the biopsy specimens for microscopic evaluation prior to beginning the analysis of all lesions and associated CRF completion. Histopathologic Analysis of All Lesions Histopathologic analysis of all lesions identified during the macroscopic phase will be performed in the manner customarily employed at the individual institution to obtain an unambiguous diagnosis. Histopathologic summaries are provided (Attachment 3) discussing aspects of HCC and dysplastic hepatocellular nodules, hepatocellular adenoma, and focal nodular hyperplasia, as well as HCC variants and grading of HCC. Differential Diagnostic Procedures & Secondary Features A summary of immunocytochemical markers useful in the differential diagnosis of HCC is provided for reference (see Attachment 3). These are to be employed at the discretion of the pathologist to support the diagnosis. It is not necessary to perform all, or, indeed, any of these stains if the diagnosis is clear on routine histology. In situ hybridization and molecular analyses, while of interest from the pathologic standpoint, are only rarely necessary to resolve diagnostic dilemmas, and may themselves be open to interpretation. Local centers are encouraged to incorporate such techniques if already a part of their clinical diagnostic armamentarium. However, inclusion of these newer approaches is not mandated in this study, as they are not in widespread clinical use at this time. Fluorescent in situ hybridization and loss of heterozygosity analyses are used at the central pathology institution and are available if deemed necessary by the central pathologist for diagnostic resolution of an individual case. In addition to the diagnosis itself, tumors will be assessed individually for secondary features, such as: degree of fibrous encapsulation, angiolymphatic invasion, and degree of differentiation. Version 2.0: May 3, 2012 Page 12 of 28

13 Ablation Sampling ACRIN 6690 In the case of planned ablation of HCC, centers are strongly encouraged to obtain biopsies of liver lesions at the time of and/or immediately prior to percutaneous thermal ablation in those participants enrolled in the study. In many participants, this will permit tissue diagnosis prior to (possibly complete) ablation. For ablated areas in explant livers, lesions with diameters up to 3 cm will be entirely submitted for histologic analysis. For larger lesions, at least an entire cross-section will be submitted for analysis, up to a total of 10 one-cm 2 slides per tumor lesion. 5.3 Reporting: ACRIN Case Report Forms Pathologists will report lesion details on the PA Form (Local Pathology: Macroscopic Analysis); a printed version of the form may be useful during liver sampling. This case report form also will state concordance or discordance with imaging findings in addition to detailed results of the pathologic analysis: o Correlation with imaging criteria will be based on individual lesion diagnosis. o In the case of previously ablated tumors, the presence or absence of viable tumor will serve as the variable of comparison. o In the case of pathologic lesions not previously identified by radiologic imaging, that discrepancy will be noted here. o Conversely, in the unlikely event that a radiologic lesion has not been identified by pathologic examination, this will be noted here. 6.0 Digital Photography of Macroscopic Pathology: Submission Instructions Digital photographs, taken during the macroscopic assessment of the explant liver, from your institution to ACRIN must be transferred via TRIAD or media (CD/DVD). Photographs uploaded to ACRIN may be reviewed selectively by a central pathologist for quality control purposes, to ensure compliance with the protocol specifications, and to allow feedback to participating sites should the need arise, especially early in the trial process. Sites have two options for submitting the digital photographs to ACRIN s image archive: Using ACRIN s image transfer application (TRIAD); or Media submission (CD/DVD). 6.1 TRIAD Submission The preferred image transfer method is via TRIAD, a software application that ACRIN provides for installation on a site s PC. One or several computers of choice within the institutional firewall and on the institutional network may be equipped with TRIAD Version 2.0: May 3, 2012 Page 13 of 28

14 ACRIN 6690 software; Internet access is also required. The TRIAD application can then be configured as a DICOM destination on either scanner(s) and/or PACS system for direct network transfer of study-related images into the TRIAD directory. When properly configured, the TRIAD software anonymizes, encrypts, and performs a lossless compression of the images before they are transferred to the ACRIN image archive in Philadelphia. Once equipment-readiness has been determined, imaging personnel from ACRIN will coordinate installation and training for the software. For more information, contact: TRIAD-support@phila.acr.org or call Upon electronically submitting the digital photos, sites should fax the Pathology Transmittal Worksheet (PTW; see Pathology Transmittal Worksheet Instructions posted online at to the ACRIN core lab at or it to lcimino@acr.org. 6.2 Image Submission via CD/DVD In the event that images are sent via CD/DVD, please address as follows ACRIN Protocol 6690 Images American College of Radiology 1818 Market Street, Suite 1600 Philadelphia, PA Attn: ACRIN 6690 Imaging Specialist 6.3 General Digital Photograph Submission Procedures Each upload should contain the following, regardless of upload platform: File folder (or zip file): o Digital photographs submitted in jpg format. o Photographs from a single (1) explant liver assessment (one participant case per folder). Appropriate labeling: o Folders labeled according to local institutional standards and ACRIN-assigned case number (e.g., Case 23). o Slices. Photographs of each side of the slices should be labeled by ACRIN case number and slice # sequentially depending on the plane of reference and the side of the slice. For example, if a liver is dissected across the transverse plane, then slices would be labeled: 23.1.cranial and 23.1.caudal, 23.2.cranial and 23.2.caudal, etc. If the liver is sliced along the sagittal plane, then slices would be labeled: 23.1.anterior and 23.1.posterior, 23.2.anterior and 23.2.posterior, etc. Version 2.0: May 3, 2012 Page 14 of 28

15 ACRIN 6690 o Lesions. Photographs of each sampled lesion should be labeled according to ACRIN case number and protocol-specific macroscopic lesion-labeling conventions for slice of explant liver, highest-number segment, and running lesion sequence number by segment (e.g., b.1 and b.2; and ). See Section 5.1 for more details; the highest-number segment and running lesion sequence number by segment should match the Nodule ID numbers of the PA Form for Local Pathology: Macroscopic Analysis. A Pathology Transmittal Worksheet (PTW) o Completed electronically and included in the file folder for upload. o After the PTW has been completed electronically, print it out, and fax it to to inform the ACRIN core lab that a new file folder is available. 7.0 AT THE SITE S REQUEST: Pathology Block and/or Slides Shipped for Central Pathology Review and Adjudication Note: This is only required if the case review is problematic or deemed equivocal at the local site. Should the site encounter a case deemed problematic/equivocal by the local pathologist, sites may send slides and/or paraffin blocks of relevant lesions to the ACRIN 6690 Project Manager, Donna Hartfeil, to coordinate further examination by the central pathologist. ATTN: Donna Hartfeil, RN, BSN Senior Project Manager, ACRIN 6690 American College of Radiology Imaging Network 1818 Market Street, Suite 1600 Philadelphia, PA Phone: Fax: dhartfeil@acr.org This process has been designed to provide an additional layer of confidence in the gold standard of pathologic diagnosis across multiple participating centers. Any disagreements between central and local pathologists will be resolved by individual communication and, possibly, additional studies. In the unanticipated event that agreement cannot be reached concerning a given liver lesion, this will be reported as such, with the individual interpretations reported. If the specimens are not completely used during adjudication, the remaining tissue/block will be returned to the site of origin at its request. Instructions for submission will be provided upon request. Version 2.0: May 3, 2012 Page 15 of 28

16 ACRIN Questions and Contact Information For questions regarding completion of data forms, information required in shipment, and questions relating to the participant, please contact the ACRIN 6690 Project Manager (Donna Hartfeil, phone: ) or ACRIN 6690 Lead Data Manager (see under 6690 Contact Personnel for contact information). Version 2.0: May 3, 2012 Page 16 of 28

17 Attachment 1 HCC Research Trial: ACRIN 6690 Research Patient List Please review research patient list to identify explant livers for trial. The American College of Radiology Imaging Network (ACRIN), an NCI-sponsored research group, with the support of the United Network for Organ Sharing (UNOS) is conducting a research trial, called ACRIN 6690, to determine whether the false positive rate in malignant liver lesion diagnosis can be reduced with the use of a new liver imaging policy from the Organ Procurement and Transplantation Network (OPTN). This trial requires the explant liver to be correlated with CT and MR images from within 90 days prior to transplant. Please review the list of patients noted below. These patients are enrolled on this research trial (ACRIN 6690). There are study-specific guidelines and processes for the macroscopic evaluation of the explant livers for these research trial participants. These guidelines are detailed in the ACRIN 6690 (available online at IMPORTANT: If you are unsure whether the explanted liver belongs to a participant on ACRIN 6690, please contact the study radiologist, study site physician investigator (if different from radiologist), or study coordinator. Site Radiologist Name & Contact Number: Site Physician Investigator Name & Contact Number: Study Coordinator Name & Contact Number: PATIENTS ENROLLED IN ACRIN 6690 AS OF (DATE) Name Local ID/ Date of Date of Most-Recent Medical Record # Explant* CT and MR Images * Note that explant livers should not be pinned and should be preserved for no more than 3 days prior to pathologic assessment per ACRIN 6690 guidelines. Optimally, the pathologist and radiologist will be available to conduct the macroscopic pathologic assessment together. For all trial-related assessments, the most-recent CT and MR images and their accompanying dictated reports will be reviewed during pathologic sampling of all lesions, especially but not limited to those classified on imaging as Class 4 or 5 lesions per the new OPTN guidelines. Label this column with an E if the participant has consented to the optional Eovist sub-trial and Eovist-enhanced MR images will need to be available to pathology, as well.

18 Attachment 2 HCC Research Trial: ACRIN 6690 Reminder Sheet for Macroscopic Analysis of Trial Patient Explant Livers For additional information, contact: <<SITE RA CONTACT INFORMATION>> Preparation PROCESSING EXPLANT LIVER 1. Is liver from participant on ACRIN 6690? (Check ACRIN 6690 Research Participant List.) 2. Has the radiologist completed lesion identification for MRI and CT images (PA Form)? Or, is the radiologist available to conduct macroscopic analysis with pathologist? Steps should be taken to minimize autolysis and arrange for timely processing with the required imaging-related lesion identification as described in the ACRIN 6690 Pathology Manual ( 3. Are dictated reports and corresponding imaging (MR and CT images, including Eovist-enhanced images for enrolled participants) from within 90 days prior to explant available to you? Are pre-ablation biopsy reports available to you? If not, contact site study coordinator. 4. Have participant medical history documents been provided? If not, contact site study coordinator. Procedure All liver slices will be photographed digitally on each side for the trial. All lesions suspicious for hepatocellular carcinoma both identified on imaging and by the pathologist for areas not seen on imaging will be sampled, documented by digital photographs, labeled sequentially, and histologically analyzed. 1. Cut liver at thickness of 10 mm or less, ideally in the same direction as the imaging was performed. 2. Digitally photograph both sides of each slice. Photographs should include ruler or other means of allow accurate measurements of lesions directly from the images. 3. Use the PA Form, Local Pathology: Macroscopic Analysis, during processing of the liver. 4. Sample and photograph all lesions identified on the images and imaging reports as Class 4 or 5 lesions, as well as lesions found by pathology that do not appear on imaging. 5. Lesions will be designated with unique identifiers (see Labeling of Slices and Sample Lesions to the right) to allow one-to-one correlation with radiologic and microscopic diagnoses on a per-lesion basis. 6. If no lesion is identified pathologically at the site of a radiologic lesion, the segmental area of the radiologic lesion will be photographed and extensively sampled. 7. In the event an area of ablation has been identified with incomplete margins and no biopsy was conducted prior to ablation, the segmental area of the radiologic finding will be extensively sampled. IDENTIFYING LESIONS Suspicious Lesion? Identify Photograph Sample Label Report DIGITAL PHOTOS 1. Take digital photos of each side of all cut gross specimen liver sections. Include ruler or other means of measurement. 2. Take digital photos of all relevant macroscopic lesions. 3. Label digital photos (see ACRIN 6690 for instructions). 4. Submit photos to ACRIN via TRIAD or CD/DVD media. LABELING OF SLICES AND SAMPLED LESIONS Labeling of liver slices in gross lab: Each liver slice will be photographed first on each side and labeled according to the slice number and side of the cut: ACRIN-assigned Case #; Slice # (from the pathology cuts in sequential order); Side of cut: cranial (superior) or caudal (inferior); anterior or posterior. Labeling of lesions in gross lab: Labeling of lesions for each digital photograph should include the following four elements for each lesion (see PA Form). 1 st : ACRIN-assigned Case #; 2 nd : Slice # (from the pathology cuts in sequential order; a single lesion may be represented in multiple slices and photos from each slice will need to be included); 3 rd : Highest-number liver segment #; and 4 th : Running # (used if there is more than one lesion in a slice). Slides for each lesion should be identifiable for future reference. That is, sites are asked to develop a means to retrieve slides for specific lesions if requested for the trial. FOR COMPLETE INSTRUCTIONS For complete instructions on pathology work-up procedures, see the ACRIN 6690, available online at Version 1.5: April 17, 2012 Page 18 of 28

19 Attachment 3 ACRIN 6690 Topics Regarding the Pathologic Diagnosis of Hepatocellular Carcinoma This attachment summarizes several items related to histologic and immunophenotypic aspects of hepatocellular carcinomas (HCCs) and related hepatocyte lesions. It is meant to serve as a brief reference and is not intended to represent a comprehensive discussion of the pathology of these tumors. SMALL HEPATOCELLULAR LESIONS AND EARLY HEPATOCELLULAR CARCINOMA A recent report from the International Consensus Group for Hepatocellular Neoplasia 1 updated the previous consensus nomenclature and diagnostic criteria for hepatocellular nodular lesions that are less than 2 cm in diameter. The following discussion is based on that report. Four diagnostic categories were summarized as follows: Low grade dysplastic nodule: This premalignant lesion usually has a distinctly nodular appearance because of the condensation of fibrous stroma of the type that is typically seen surrounding cirrhotic nodules. On occasion the lesion may be vaguely nodular. There is a mild increase in cell density without atypia and generally with a monotonous pattern. Large cell change may be present. Atypical architectural features such as markedly thickened trabeculae or pseudogland formation are not seen. Isolated arteries can occasionally be present in small numbers. Diffuse increase in iron or copper may be present. These lesions do not show evidence of stromal invasion and do not have a nodule in nodule appearance. The panel felt that it was difficult or sometimes impossible to distinguish these nodules from large regenerative nodules but noted that this did not appear to have any significant consequences at present. High grade dysplastic nodule: These lesions may be distinctly or vaguely nodular and may show increased cell density up to twice that of the surrounding liver. Cytologic atypia is present and most often consists of small cell dysplasia (large cell change may also be present but is insufficient for this diagnosis). There may be an irregular trabecular pattern to the architecture. However, the cytologic and architectural changes are not of a magnitude sufficient to allow the diagnosis of HCC. Most lesions will show unpaired arteries but not in high numbers. Stromal invasion is not present (by definition). A nodule in nodule appearance may occasionally be present. In such cases, the entire nodule is classified by the worst component, which typically is represented by the sub-nodule. Early hepatocellular carcinoma (small well differentiated HCC of vaguely nodular type): This is one of two subdivisions of the original small HCC defined by the International Working Party of the World Congress of Gastroenterology in as a tumor less than 2 cm in diameter. Early HCC by definition shows evidence of stromal invasion, which distinguishes it from high grade dysplastic nodules. It also shows varying combinations of the following five (5) features: a) Increased cell density more than twice that of the surrounding tissue with an increased nuclear to cytoplasmic ratio and irregular thin trabecular pattern; b) May contain intratumoral portal tracts (which are helpful in evaluating stromal invasion); c) Pseudoglandular pattern; d) Diffuse fatty change; and e) Varying numbers of unpaired arteries. Version 1.5: April 17, 2012 Page 19 of 28

20 The consensus group stressed that these five (5) features may also be individually found in high grade dysplastic nodules, thereby emphasizing the importance of stromal invasion to differentiate early HCC. Progressed hepatocellular carcinoma: This tumor is also by definition less than 2 cm but is distinctly nodular and generally moderately differentiated with evidence of microvascular invasion. This form typically is not difficult to distinguish from the other above listed lesions. IMMUNOCYTOCHEMISTRY Some of the more commonly utilized immunophenotypic stains helpful for the diagnosis of HCC are given. The list is provided as a guide. It is not intended not as a comprehensive summary, nor is meant to imply that these stains must be performed for this study. However, recent recommendations 3 endorsed by the American Association for the Study of Liver Diseases state that tissue that is not clearly HCC should be stained by all the available markers including CD34, CK7, glypican 3, HSP-70, and glutamine synthetase to improve diagnostic accuracy. Frequency estimates of positive immunostains for individual tumors were obtained from PathIQ Immunoquery search ( Alpha-fetoprotein: Classical marker for HCC, positive in approximately 36% of cases. Also positive in fibrolamellar variant, hepatoblastoma, mixed hepatocellular/cholangiocarcinoma. Positive in some germ cell tumors and other tumors unlikely to be considered in the differential diagnosis. Glypican-3: Positive in an estimated 72% of HCCs. Staining may be focal. May also occur in 5% to 10% of benign conditions such as focal nodular hyperplasia or regenerative nodules, or in the setting of inflammation. Also positive in melanomas and some germ cell tumors. Heat shock protein 70 (HSP70): Estimated 70% sensitivity for detection of HCC in resected specimens. Preferentially expressed in malignant as opposed to premalignant conditions. Glutamine synthetase: Normally present in centrilobular regions. Diffuse pattern of staining may occur in HCC. Also expressed in tumors with activating molecular lesions of beta catenin, since it is a target of the gene. CD34: Endothelial marker useful for detecting diffuse capillarization of sinusoids in HCC. Occasional diffuse staining may also occur in hepatocellular adenoma or focal nodular hyperplasia. Alpha smooth muscle actin: Smooth muscle marker utilized to highlight unpaired arteries within hepatocellular lesions. Cytokeratin 7/cytokeratin 19: Keratin markers useful in the evaluation of stromal invasion. There is some overlap with markers used primarily in the subclassification of hepatocellular adenomas. Beta-catenin activation, histologically evidenced by nuclear translocation of the protein, defines a subtype of hepatocellular adenoma 4 and is also seen in a subset of carcinomas, possibly at a later stage of tumor progression. 5 Occasional carcinomas may also show activating mutations in the IL-6 receptor gp130, 6 thereby presumably leading to expression of phospho-stat 3 and C-reactive protein, markers currently used to define the inflammatory subtype of adenoma. Some adenomas may show diffuse sinusoidal capillarization on CD34 stain. 7 As always, immunophenotypic analysis should be interpreted in context with all of the immunostains and the histology. Version 2.0: May 3, 2012 Page 20 of 28

21 HISTOPATHOLOGIC GRADING OF HEPATOCELLULAR CARCINOMA Most studies that incorporate histologic grading of HCC refer to the Edmondson Steiner system. 8 This approach, first published in 1954, relied on six features and stressed cytoplasmic and architectural features in addition to nuclear features. This system was updated in the 2001 AFIP liver fascicle 9 (Ishak, Goodman, and Stocker) with a focus on the nuclear features. The four (4) grade system is summarized as follows: Grade 1: This is identical to the original grade 1 of Edmondson and Steiner. Cells are described as adenoma-like with abundant cytoplasm and minimal nuclear irregularity. Since the cytology is identical to hepatocellular adenoma, a diagnosis of malignancy is based on architecture, including the pattern of trabecular or pseudoglandular growth, as well as the presence of vascular invasion or metastatic disease (to this we might add the presence of stromal invasion). Grade 2: These tumors have more prominent nuclear hyperchromatism and some degree of irregularity of the nuclear membrane. In the provided photomicrographs, the nuclei remain fairly uniform as to their rounded or ovoid shapes, but have a uniform pattern of nuclear irregularities. Grade 3: These carcinomas were said to have greater nuclear pleomorphism and angulated nuclei. The provided photomicrograph shows a distinct difference in nuclear appearance compared to grade 1 or 2 lesions. In my own experience, the photomicrograph would represent what many pathologists would consider moderately to poorly differentiated carcinoma. Grade 4: These tumors had marked pleomorphism, hyperchromatism, and usually anaplastic giant cells. The authors pointed out that in the original classification such tumors were considered to represent grade 3 tumors. Another difference between the two systems related to the presence of tumor giant cells. In the original Edmondson Steiner approach, all tumors with giant cells or multinucleated cells were considered grade 4 tumors. In the modified approach, the nuclear appearance was used to grade the tumor, regardless of whether one or several nuclei were present within a tumor cell. Informally, one might approach this by considering that grade 1 tumors can resemble normal hepatocytes or hepatocellular adenoma, with grade 2 tumors being well differentiated. Grade 3 tumors would be moderately or moderately-to-poorly differentiated, whereas grade 4 tumors would be poorly differentiated to anaplastic. In practice, grades 1 and 2 can be combined into well-differentiated, grade 3 is moderately-differentiated, and grade 4 is poorly-differentiated. The Cancer Staging Manual of the American Joint Committee on Cancer (AJCC), 7th edition, 10 gives the option of using a two (2) grade, three (3) grade, or four (4) grade system. The number of grades in the system used at the individual center should be specified. HEPATOCELLULAR CARCINOMA VARIANTS On occasion, one of the recognized variants of HCC may be present in the explant liver. It is desirable to document this as part of the diagnosis. Fibrolamellar hepatocellular carcinoma typically occurs in a younger age group without cirrhosis and as such is unlikely to be encountered during this study. The cells possess abundant granular and Version 2.0: May 3, 2012 Page 21 of 28

22 eosinophilic cytoplasm and have characteristic prominent nucleoli. Prominent collagenous bands arranged as parallel lamellae are characteristic but may be sparse or even absent in some tumors. Clear cell hepatocellular carcinoma contains malignant hepatocytes with most showing clear or empty appearing cytoplasm due to intracellular glycogen or lipid. In most cases other areas are clearly diagnostic of hepatocellular carcinoma. Consideration of other clear cell tumors may be necessary. Scirrhous (sclerosing) hepatocellular carcinoma may contain extensive non-lamellar fibrosis and has a tendency to express cytokeratin 7. This tumor, may occur in older age groups, should be distinguished from both fibrolamellar carcinoma and cholangiocarcinoma. Combined hepatocellular/cholangiocarcinoma should show unequivocal differentiation into hepatocellular and cholangiocellular components. Occasional cytokeratin 7/19 positivity in an otherwise typical HCC does not qualify for inclusion into this category. The current AJCC Cancer Staging Manual indicates that such mixed tumors should be staged as intrahepatic cholangiocarcinomas. Sarcomatoid hepatocellular carcinoma may contain components of any type of sarcoma in addition to a malignant hepatocellular component. Sarcomatoid change may also occur in a mixed hepatocellular/cholangiocarcinoma, which should be considered in the differential diagnosis. OTHER LESIONS Hepatocellular adenomas are unlikely to be encountered in these cirrhotic livers. Individual features related to adenomas were briefly discussed in the section on immunocytochemistry, above. A recent summary of the current approach to hepatocellular adenoma classification is given in reference 4. Focal nodular hyperplasia or lesions resembling focal nodular hyperplasia within a cirrhotic liver may also be encountered. True FNH contains isolated arterial vessels with vascular dysplasia, i.e., irregular muscular thickening of the vascular wall. Ductular proliferation without true bile duct presence is typical but may be variable in individual fibrotic regions. If features of dysplasia are present (see above) such nodules should be evaluated in line with those features. REFERENCES 1. International_Consensus_Group_for_Hepatocellular_Neoplasia. Pathologic diagnosis of early hepatocellular carcinoma: a report of the international consensus group for hepatocellular neoplasia. Hepatology. 2009;49: International_Working_Party. Terminology of nodular hepatocellular lesions. Hepatology. 1995;22: Bruix J, Sherman M. Management of hepatocellular carcinoma. Hepatology (online update 2010);42: Bioulac-Sage P, Laumonier H, Laurent C, Zucman-Rossi J, Balabaud C. Hepatocellular adenoma: what is new in Hepatol Int. 2008;2: Sakamoto M, Effendi K, Masugi Y. Molecular diagnosis of multistage hepatocarcinogenesis. Jpn J Clin Oncol. 2010;40: Rebouissou S, Amessou M, Couchy G, et al. Frequent in-frame somatic deletions activate gp130 in inflammatory hepatocellular tumours. Nature. 2009;457: Coston WM, Loera S, Lau SK, et al. Distinction of hepatocellular carcinoma from benign hepatic mimickers using Glypican-3 and CD34 immunohistochemistry. Am J Surg Pathol. 2008;32: Edmondson HA, Steiner PE. Primary carcinoma of the liver: a study of 100 cases among 48,900 necropsies. Cancer. 1954;7: Ishak KG, Goodman ZD, Stocker JT, eds. Tumors of the Liver and Intrahepatic Bile Ducts. Washington, D.C.: American registry of Pathology, Armed Forces Institute of Pathology; Edge SB, Byrd DR, Compton CC, et al, eds. AJCC Cancer Staging Manual. 7 ed. New York, NY: Springer-Verlag; Version 2.0: May 3, 2012 Page 22 of 28

23 Attachment 4 ACRIN 6690 Pointers on Macroscopic Pathology Photography and Labeling Introduction Macroscopic photography is a crucial element allowing correlation between imaging diagnosis and reference explant pathology. To date, photographs received have generally been of good quality. On occasion, correlation is hampered by inattention to certain details. The purpose of the photography is to document the location, number, and size of tumors/lesions in the explant liver. Diagnosis of each lesion by subsequent histopathology is the basis for determining the true and false positive and negative rates of imaging studies. With this in mind, the technical requirements of photography are self-evident. Inadequate photography would include such things as: a) missing slices of liver explant or b) insufficient information to reconstruct the images in proper sequence on review. Difficulty in visualizing the liver parenchyma or lesion(s) would also fall under this category. Some examples are presented that show where photographs without certain details make it problematic to obtain all of the necessary information from individual liver slices. (The images are not derived from study cases but do in some cases reflect photographs that have been submitted for review.) The ruler was photographed with the liver; other markings were added to the images digitally. However, leeway is provided to investigators depending on the available setup. Pieces of paper with pencil markings or other approaches are acceptable if the following general guidelines are followed. Model Images (See Also Section 5.1 of the ACRIN 6690 for Label Details) For the sake of argument, we will use a liver slice that represents hypothetical ACRIN Case #23, slice #12, with a single tumor nodule in segment 6 of the right lobe. The liver is sectioned horizontally (transversely/axially) and the image represents the slice from above (cranial/superior aspect). In Figure 1, the figure label denotes ACRIN Case #23, slice #12, viewed from the cranial aspect (transversely sectioned). This slice happens to contain a tumor, but as part of a series of sequential liver slice photographs, the label focuses on slice information alone. (A separate close-up view of the tumor will be labeled differently to present tumor information, see below). Figure 1: cranial the photograph is acceptably labeled This photograph contains information to orient the viewer (anterior, right sides). At least one label is desirable, since both are not necessary for orientation if the direction of transection and view aspect (in this case transverse and cranial, respectively) are known. Also note that a ruler is provided for scale. This is stained, which is not good photographic form, but since it does not obscure the ability to measure, it is acceptable. Version 1.5: April 17, 2012 Page 23 of 28

24 Regarding figure labels, the following examples refer to this particular slice: Table 1. Figure labels of Case 23, Slice 12, transversely sectioned and viewed from the cranial aspect: Label Acceptable Comment cranial Yes Provides case#, slice #, surface orientation No Does not indicate surface from which the slice is viewed a Maybe Shortcuts are OK if the key (in this case, cranial vs. caudal) is clearly and unambiguously provided 12.cranial No Does not provide case # The following photographs demonstrate some technical issues that hamper interpretation. For convenience, the orientation labels are not included. Figure 2: Cropping. The liver is cropped on the left side of the photograph, leading to nonvisualized area. Figure 2. Unacceptable due to cropping Figure 3: Masking by Ruler. Figure 3 shows similar unacceptable masking, this time due to placement of the ruler on the liver itself. Figure 3. Unacceptable due to masking by ruler Version 2.0: May 3, 2012 Page 24 of 28

25 Figure 4: No Ruler. Figure 4 represents an unacceptable photograph since no ruler is included. This is particularly important in this example because this is necessary to assess the size of the tumor. Figure 4. Unacceptable due to lack of ruler The quality of the photograph is also important in order that the necessary information can be extracted. Figure 5: Ruler Unreadable. Figure 5 shows a photograph that has glare to the point of rendering the ruler unreadable. Figure 5. Unacceptable due to glare washing out markings on the ruler Other markings also may interfere with interpretation. In the model slice used for these explanations, there is an obvious tumor and it is not necessary to point out where the lesion is. In other cases, the lesion may be subtle, or there may be more than one lesion, or there may be something found on imaging that is not obvious in the liver itself that needs to be sampled and documented photographically, and pointing out the nodule or area of interest is necessary. Again, common sense should prevail, providing the information without compromising overall interpretation. Version 2.0: May 3, 2012 Page 25 of 28

26 Figures 6 and 7 use arrows to point out the tumor. It is best to highlight the tumor without masking any of the background liver. This is possible only when the tumor is near the edge of the specimen. Figure 6: Acceptable, Lesion Is Visible. Figure 6 is acceptable since minimal masking occurs. Figure 6. Acceptable use of pointer. Minimal masking of background. Figure 7: Slice Obscured. Though is exaggerated for effect, Figure 7 is meant to show that signpost information such as arrows should not obscure the overall assessment of the liver slice. Figure 7. Unacceptable use of pointer. Obscures significant amount of background liver. Version 2.0: May 3, 2012 Page 26 of 28

27 Labeling of Tumor Photograph In addition to sequential slice photographs with appropriate labels, closer images of each tumor are requested. As noted above, this liver slice represents hypothetical ACRIN Case #23, slice #12, with a single tumor nodule in segment 6 of the right lobe. A photographic image of each lesion identified on trial imaging, each suspicious area that the pathologist chooses to sample, and each area of treatment (post-ablation or TACE), such as the following, is submitted in addition to the liver slices photographs. (Note that in this model, the image quality of necessity is not optimal due to digital enlargement. Original close-up photographs of the tumors are requested in study cases). Figure 8. Close up photograph of lesion, labeled appropriately as: Case#.Slice#.Segment#.NoduleID-Running# Note the label in this case. This tumor photograph label conveys the information that this is case #23, from slice 12, segment 6, tumor nodule ID running #1. The tumor number should correspond with the Pathology: Nodule ID Running # from the PA Form (Local Pathology: Macroscopic Pathology Form) below (Figure 9, arrow) and may or may not correspond to the Radiology Lesion ID Nodule # (Figure 9, checkmark). Figure 4. PA Form. Local Pathology: Macroscopic Analysis Form. Radiology provides nodule numbers data under Lesion ID (checkmark). At explant examination, Pathology provides Pathology: Nodule Information, including Nodule ID Running #s (arrow) which typically, but may not always, correspond to Radiology numbers. Version 2.0: May 3, 2012 Page 27 of 28