8.5 Commerical Agent: N/A 9. BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES 9.1 Biomarker Studies 9.1.1 Collection of Specimens 9.1.1.1 Fine-Needle Aspiration (FNA) Biopsy of Tumor FNA biopsy will be prepared as detailed below and stored frozen in liquid nitrogen. 9.1.1.2 Tumor Biopsies Biopsy specimens must include at least 100 mm 3 of tumor tissue and ideally 1 cm3 or more. The specimens may be obtained by ex cisional biopsy, incisional biopsy, or core biopsy. Each surgical specimen or set of core biopsies will be di vided into 5 portions: 1/4th will be placed in RPMI and will be processed to yield cryopreserved viable cells for flow cytometric analysis, 1/4th will be fixed in formalin and then embedded in paraffin for histology, 1/6th will be flash frozen in OCT for histology, 1/6th will be flash frozen without medium for Kyn/Trp ratio and 1/6th will be flash frozen in RNA Later for gene expression. (See Laboratory Manual for additional information). If an archived pre-treatmen t biopsy is obtained from a fo rmalin-fixed paraffin-embedded (FFPE) tissue block, there must be at least 20 slides available for study. 9.1.1.3 Blood Blood will be collected for serum and PBMC isolation as designated below. 9.1.2 Processing and Shipping of Specimen(s) Tumor specimens will be collected as described above, at the bedside when tumor is biopsied. They will then be processed and shipped as specified below. 9.1.2.1 Portion for single-cell suspensions This will be submitted to mechanical disso ciation then, as appropriate, enzymatic digestion (typically collagenase, DNAase, and hyaluronidase). The resulting cells will be collected by centrifugatio n and cryopreserved viably in 10% dimethyl sulfoxide (DMSO) and either 12.5% HSA (Human serum albumin or 12.5% FCS (Fetal calf serum) using a NCI Protocol #: CITN-04 Version Date: 27 Jan 2016 56 controlled-rate freezer. Sample s will be stored at -80°C and then batch-shipped on dry ice to the CITN Central Laboratory. 9.1.2.2 Portion for Formalin Fixation These will be placed in formalin, and shippe d (ambient) overnight to the CITN Central Laboratory. After adequate fixa tion, samples will be embedded in paraffin and formalin- fixed paraffin embedded (FFPE) blocks stored at room temperature. 9.1.2.3 Portion for Quick-Freezing in OCT This will be placed in OCT at the patient bedside and placed immedi ately onto dry-ice for hardening. Samples will be stored at -80°C and then batch-shipped on dry ice to the CITN Central Laboratory. 9.1.2.4 Portion for Quick-Fr eezing Without Media This will be placed in a cryovial and placed immediately into liquid nitrogen for flash freezing. Samples will be stored at -80°C or in liquid nitrogen and then batch-shipped on dry ice to the CITN Central Laboratory. 9.1.2.5 Blood Samples A) Blood in red-top (or comparable) tubes fo r serum isolation and green-top tubes for plasma isolation, and CPT tubes (for R NA isolation) will be processed locally, aliquoted into cryovials, and frozen at - 80°C. They will be batch-shipped on dry ice to the CITN Central Laboratory. B) Blood in green-top (or comparable) t ubes for PBMC isolation and CellSave Preservation tubes will be sh ipped at ambient temperature in insulated containers to the CITN central laboratory at the University of Washington. 9.1.3 Shipping of Specimens Fresh blood collected for PBMC isolation will be shipped at ambient temperature overnight in insulated containers to the CITN central laboratory. Biopsy specimens in formalin will be shipped with the green t op tubes to the CITN Central Laboratory for paraffin embedding as described above. (FFPE ) tumor biopsies will be stored at room temperature, then shipped in batch periodical ly to the University of Virginia (Slingluff laboratory) for analysis. FNA biopsies will be stored in liquid nitrogen or at -80 oC, then shipped in batch to the CITN Central Laboratory for later batc h shipment to the laboratory of Drs. Marincola and Wang for analyses. Fro zen serum, plasma and viably frozen tumor cells and frozen tumor tissue will be shipped in batch to the CITN Central Laboratory on dry ice for same day or overnight delivery. 9.2 Laboratory Correlative Studies NCI Protocol #: CITN-04 Version Date: 27 Jan 2016 57 9.2.1 Immunohistochemical Evaluation of Tumor Biopsies - Laboratory Correlative Study #1 Tumors will be assessed by IH C for pattern of T-cell distribu tion and infiltration of cells expressing a variety of antigens incl uding CD3, CD4, CD8, CD56, CD34, CD45, CD20, CD138, CD163, DC-LAMP, FoxP3, PD-1, and IDO1, as well as PD-L1 on tumors. The primary endpoint of the trial will be a change in the number of CD8+ cells infiltrating tumor (per mm2) comparing pre- and post-treatment biopsies for each patient. We will use the assessment of total CD3+ T cells and total CD45+ lymphocytes as confirmatory assays. IHC staining will also be performed to detect other key parameters including IDO1 expression, and presence and patterns of CD4+ T cell and FoxP3+ Treg infiltration. These antibodies all work in FFPE tissue sections. Tissue sections will be processed with heat-induced antigen retrieval and stained as described [Erdag 2012], using antibodies to CD4, CD8, NKp46, CD31, CD45, CD20, CD3, CD138, CD163, DC-LAMP, FoxP3, PD -1, and IDO1. EnVision™ System (enzyme-conjugated polymer backbone c oupled to secondary antibodies) and 3,30- diaminobenzidine chromogen (Dako North Am erica, Inc., Carpin teria, CA) will be applied to develop the staining. Positive c ontrols for lymphocyte antibodies will include lymph node and for CD34 antibody, placenta. Ne gative control slides will use phosphate buffered saline instead of primary antibody, with other conditions held constant. If the tumor specimen is too small for evaluation of all antigens, the priority will be CD8 > CD3 > IDO > CD31 > CD45 > FoxP3 > remainder. Stained sections will be reviewed for type, density, and location of tumor-infiltrating immune cells, tumor cell type, necrosis, hemorrhage, and melanin pigmen t. At least 5 high-power ed fields will be examined. Fields with no tumor tissue, more than 50% necrosis, or hemorrhage will be excluded. Stained cells will be enumerated by a single pathologist on each core in a high-power microscope field, excluding cells with in blood vessels or in hemorrhagic or necrotic areas. Cell counts will be normalized to per mm 2. Alternatively, when automated image analysis software is available and va lidated, it may be used for quantification of the staining. Change in the number of tumor-infiltrating lymphocytes will be analyzed using standard descript ive statistical analyses described below. Immunotypes will be defined by staining for immune cells (CD45) and for intratumoral blood vessels (CD31) as described [ Erdag 2012]. Additional histologic studies may be perf ormed as appropriate to elucidate the immunologic changes in the tumor specimens. If IHC studies are need ed using antibodies that do not work well in FFPE sections, or for immunofluorescence studies, one of the quick-frozen tissue samples will be used for those purposes. 9.2.1.1 Site(s) Performing Correlative Study The primary endpoints (IHC for CD8 and CD 3) will be evaluated on FFPE slides of tumor tissue at Phenopath Laboratories, Seattle, WA. All other IHC will be performed at the University of Virginia. NCI Protocol #: CITN-04 Version Date: 27 Jan 2016 58 9.2.2 Gene Expression Analyses of Tumor Biopsi es, Fine Needle Aspirates of Metastases, and Peripheral Blood—Laboratory Correlative Study #2 Tumor biopsies will be obtaine d as described above for IHC analyses. FNA of melanoma metastases will be performed at each biopsy date, before the excisional/incisional/core biopsy; both tissues will be evaluated for ge ne signatures. Gene signatures will also be performed on PBMCs obtained before ther apy and on days 21 and 42, the same day of the tumor biopsies. PBMC gene signatures may be compared to data of tumor biopsy, immunologic response, and clinical response. 9.2.2.1 Collection of Specimen(s) For FNA a 22- or 23-gauge hypodermic needle will be inserted into the tumor and multiple passes with changing angles of inse rtion will be made through all quadrants of the tumor, while under suction. Tissue from the needle will be suspended in 10 mL of cold RPMI. For PBMC, a CPT tube will be drawn and processed as described below. 9.2.2.2 Handling of Specimen(s) Specimens will be collected at the clinical sites as described above and centrifuged at 1500 rpm for 5 minutes at 4°C. The cell pellet will then be harvested, red blood cells lysed as per the technique described by Weiss et al [ Weiss 2011 ]. Cells will be isolated by centrifugation and resuspended in Qiazol to obtain total RNA. Samples will be stored at -80°C and then batch-sh ipped as described below. 9.2.2.3 Shipping of Specimen(s) All RNA samples will be batched and shipped on dry ice using FedEx ® overnight delivery to the CITN Central Laborator y. The CITN Central Laboratory will hold specimens for shipment to the laboratory of Dr. Marincola and Wa ng for gene expression array studies. Any leftover sample will be returned to the CITN Central Laboratory for storage and future ancillary studies. 9.2.2.4 Site(s) Performing Correlative Study Gene expression studies, including analys es of the data, will be conducted in the laboratory of Dr. Francesco Marincola, Chie f Research Officer of Sidra Medical and Research Center, Qatar. 9.2.3 CD8+ T-cell IFN-gamma Responses to the 12-peptide Mixture, and CD4+ T-cell Responses to Tetanus Peptide—Laboratory Correlative Study #3 PBMC (and lymphocytes isolated from tumor bi opsies, when available) will be analyzed by IFN-gamma ELISpot assay for responses to the 12-peptide mixture. Assessment of immunologic response will be base d on a fold-increase measure from baseline as well as using a positivity threshold. Since 11 of the 12 melanoma-associated immunizing peptides are 9- and 10-mers, it is safely presumed that IFN-ga mma ELISpot responses among PBMC will derive from CD8 + T cells. No depletion of CD4+ T cells is planned for the ELISpot testing. The frequency of CD8+ T cells specific to 8 of the 12 immunizing peptides will be confirmed using tetramers and flow cytometric analysis. CD8+ T cell NCI Protocol #: CITN-04 Version Date: 27 Jan 2016 59 responses will be assayed directly ex vivo (using viably cryopreserved specimens), after stimulation with the 12-peptide mixture (10 μg/mL each) at baseline (i.e., before initiating INCB024360 treatment), at week 3 (before the first immunization with MELITAC12.1), at weeks 6, and optionally at week 16 (2 weeks after the last dose of MELITAC12.1). Similarly, these assays will include the tetanus peptide and multiple controls (CEF peptide, negative controls, etc.). Responses to the tetanus peptide wi ll be interpreted as helper (CD4+) T-cell responses. 9.2.3.1 Collection of Specimen(s) Whole blood will be collected into heparinized (green top) tubes. 9.2.3.2 Handling of Specimen(s) Samples will be collected at room temperature and shipped to the CITN Central Laboratory the same day as the blood draw, so that the sample is received at the central laboratory within 24 hours of blood draw. PBMC will be isolated and cryopreserved before testing. 9.2.3.3 Shipping of Specimen(s) Samples will be shipped at ambient temper ature for overnight Fe dEx delivery. Clinical research sites will utilize a web-based sp ecimen tracking system (BSI-II) to communicate with the CITN central laboratory. 9.2.3.4 Site(s) Performing Correlative Study The IFN-gamma ELISpot assay will be conducte d at the CITN Central Laboratory at the University of Washington Immunologic M onitoring Laboratory in Seattle, in collaboration with the laboratory of Dr. Slingluff at the Un iversity of Virginia. Flow cytometry for tetramer-positive cells will be co nducted in the laboratory of Dr. Slingluff. 9.2.4 Quantification of PBMC and T-cell Subsets—Laboratory Correlative Study #4 The effect of treatment on T lymphocyte number and phenotype (i.e., CD8+, effector memory CD8+, central memory CD8+, CD4+ , CD4+ Treg, naïve subsets) will be assessed. The frequency and pe rcentage of PBMC subsets will be determined using multiparameter flow cytometric analysis of whole blood. The markers include CD3, CD8, CD4, CD39, CD56, Foxp3, CD127, CD45RA, CD45RO, CCR7, CD28, CD27, CD25, CD122, CD86, CD14, CD16, CD19, CD123, and CD11c . Changes in myeloid-derived suppressor cell populations will also be assessed by flow cytometry. The effect of INCB024360 and MELITAC 12.1 on lymphocyte number and phenotype will be assessed at baseline (i.e., before initiating INCB024360 treatment), week 3 (before first immunization with MELITAC12.1), week 6 (before the second set of MELITAC12.1 vaccines), week 14 (just before the last dose of MELITAC12.1), and, if possible, week 16 (2 weeks after the last dose of MELITAC12.1). NCI Protocol #: CITN-04 Version Date: 27 Jan 2016 60 9.2.4.1 Collection of Specimen(s) Whole blood will be collected into heparinized tubes. 9.2.4.2 Handling of Specimen(s) Samples will be collected at room temperature and shipped to the CITN Central Laboratory the same day as the blood draw, so that the sample is received at the central laboratory within 24 hours of blood draw. 9.2.4.3 Shipping of Specimen(s) Samples will be shipped at ambient temper ature for overnight Fe dEx delivery. Clinical research sites will utilize a web-based sp ecimen tracking system (BSI-II) to communicate with the CITN central laboratory. 9.2.4.4 Sites Performing Correlative Study The immunophenotyping assays will be conduc ted at the CITN Central Laboratory. 9.2.5 Intratumor and Whole Blood Kyn/Trp Ra tios—Laboratory Correlative Study #5 Kyn/Trp ratios will be assayed in patients receiving a regimen of INCB024360 plus MELITAC 12.1 vaccine as measured in peripheral blood and in intra-tumoral specimens at baseline and on specimens obtained at subsequent tumor biopsy timepoints. Evaluations will occur at baseline (week 0) and after each vaccination. 9.2.5.1 Collection of Specimens Whole blood will be collected into heparinized tubes. Biopsy specimens will be collected as described in Section 9.1.1 9.2.5.2 Handling of Specimen(s) Blood samples will be processed locally, and froz en aliquots shipped to the CITN Central Laboratory. The Central Laboratory will distribu te aliquots to Incyte where testing will be done in a blinded fashion. Biopsy specimens will be collected and stored at -80°C (if frozen) at the collection site. 9.2.5.3 Shipping of Specimens Frozen plasma and flash frozen biopsy specimens will be batch shipped to the CITN Central Laboratory. The CITN Central Laboratory will ship specimens to Incyte Corporation by FedEx ove rnight delivery. 9.2.5.4 Site(s) Performing Correlative Study The Kyn/Trp analysis will be conducted at Incyte Corporation. NCI Protocol #: CITN-04 Version Date: 27 Jan 2016 61 9.2.6 Other Immunologic Assessments—Laboratory Correlative Study #6 9.2.6.1 Collection of Specimens At baseline, week 3, week 6 and optionally at week 16, 10 mL of serum will be collected in red-top tubes for serum storage, and 10 mL will be collected in green-top tubes for plasma storage. 9.2.6.2 Handling of Specimen(s) Samples will be collected at room temperat ure. Blood for serum, plasma, and the cell pellet into RNA will be processed at the Local Lab within 4 hours at the collection site, aliquoted according to protoc ol, and frozen at -80°C. 9.2.6.3 Shipping of Specimen(s) Serum, plasma and cell pellet into RNA will be processed at each clinical site and batch-shipped on dry ice overnight to the CITN centr al laboratory. Clinical research sites will utilize a web-based specimen tracking system (BSI-II) to communicate with the CITN central laboratory. Frozen samples will be batch-shipped to the CITN central laboratory/repository on dry ice. 9.2.6.4 Site(s) Performing Correlative Study It is anticipated that a number of CITN investigators with laboratories may propose ancillary studies to be conduc ted on CITN repository specimens, but the specific sites have yet to be determined. 9.3 Special Studies: Cytokine and INCB024360 Blood Levels 9.3.1 Collection of Specimens 10 mL of serum will be collect ed in red-top tubes for de termining the INCB024360 level, and cytokine levels. 9.3.2 Handling of Specimen(s) Samples will be collected at room temperat ure and processed in the local laboratory within 4 hours of blood draw, aliquoted accord ing to protocol, and frozen at -80°C. 9.3.3 Shipping of Specimen(s) Frozen aliquots of serum and plasma will be batch-shipped to the CITN Central Laboratory. The Central Laboratory will distribute aliquots to Incyte. Clinical research sites will utilize a web-based specimen trac king system (BSI-II) to communicate with the CITN Central Laboratory. 9.3.4 Site(s) Performing Correlative Study The INCB024360 drug levels will be c onducted at Incyte Corporation. NCI Protocol #: CITN-04 Version Date: 27 Jan 2016 62 10. STUDY CALENDAR All baseline evaluations are to be conducte d within 1 week before start of protocol therapy. Scans and x-rays must be done ≤4 weeks before the start of therapy. In the event that the patient’s condition is de teriorating, laboratory evaluations should be repeated within 48 hours before initi ating the next cycle of therapy. Pre- Study -30 day Pre- Study -7 day Wk 0 Day 0 Wk 1 Day 7 +/- 2 d Wk 2 Day 14 +/- 2d Wk 3 Day 21 +/- 2d Wk 4 Day 28 +/- 2dWk 5 Day 35 +/- 2d Wk 6 Day 42 +/- 2d Wk 8 Day 56 +/- 2d Wk 11 Day 77 +/- 2d Wk 14 Day 98 +/-2d Wk 16 Day 112 +/-2d Repeat b Cycles Wks c +/-2d 10 Wk Post-Tx F/U‡ F/U +/-14d 36 Wk Post- Tx FU F/U +/-30d Off Study INCB024360 daily X X X X X X X X X X X MELITAC 12.1 vaccine** X X X X X X I n f o r m e d c o n s e n t X D e m o g r a p h i c s X M e d i c a l h i s t o r y X Concomitant medication X X X X X X X X X X X X X X X X Physical exam X X X X X X X X X X X X Vital signs X X X X X X X X X X X X X X X X H e i g h t X X Weight X X X X X X X X X X X X X X X X Performance status X X X X X X X X X X X X X X X X Tumor Biopsy1,2 * Xa X X3 X3 X1 FNA4,5 Xa X X X3 X1 CBC w/diff, plts (local lab) X X X X X X X X X X X X X X X X Serum chemistry6 (local lab) X X X X X X X X X X X X X X X X B-HCG 7 (local lab) X7 PBMC5 and T-cell subsets X X X X X8 IFN-gamma ELISpot*** X X X X X Confirmatory Tetramer staining X X X X NCI Protocol #: CITN-04 Version Date: 27 Jan 2016 63 Pre- Study -30 day Pre- Study -7 day Wk 0 Day 0 Wk 1 Day 7 +/- 2 d Wk 2 Day 14 +/- 2d Wk 3 Day 21 +/- 2d Wk 4 Day 28 +/- 2dWk 5 Day 35 +/- 2d Wk 6 Day 42 +/- 2d Wk 8 Day 56 +/- 2d Wk 11 Day 77 +/- 2d Wk 14 Day 98 +/-2d Wk 16 Day 112 +/-2d Repeat b Cycles Wks c +/-2d 10 Wk Post-Tx F/U‡ F/U +/-14d 36 Wk Post- Tx FU F/U +/-30d Off Study K y n / T r p r a t i o X X X X * RNA Cell Pellet for gene expression analysis X X X X S e r u m C y t o k i n e s X X X X INCB024360 level X X X C i r c u l a t i n g t u m o r c e l l s X X PBMC storage*** X X X X8 X Serum storage X X X X8 EKG (pre-study and as indicated) X Adverse event evaluation X X X X X X X X X X X X X X X X Tumor measurements X Tumor measurements are repeated every 12 weeks or per institutional standard of care. Documentation (radiologic) must be provided for patients removed from study for progressive disease.