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Amplification of 7p12 Is Associated with Pathologic Nonresponse to Neoadjuvant Chemotherapy in Muscle-Invasive Bladder Cancer

Open AccessPublished:December 13, 2019DOI:https://doi.org/10.1016/j.ajpath.2019.10.018
      Pathologic downstaging (pDS) to neoadjuvant chemotherapy (NAC) is one of the most important predictors of survival in muscle-invasive bladder cancer (MIBC). The use of NAC is limited as pDS is only achieved in 30% to 40% of cases and predictive biomarkers are still lacking. We performed a comprehensive immunomolecular biomarker analysis to characterize the role of immune cells and inhibitory checkpoints, genome-wide frequencies of copy number alterations, mutational signatures in whole exome, and tumor mutational burden in predicting NAC response. Our retrospective study included 23 primary MIBC patients who underwent NAC, followed by radical cystectomy. pDS to NAC was a significant prognostic factor for better recurrence-free survival (P < 0.001), with a median time to recurrence of 41.2 versus 5.5 months in nonresponders. DNA damage repair alterations were noticed in 38.1% (n = 8), confirming a positive correlation with high tumor mutational burden (P = 0.007). Chromosomal 7p12 amplification, including the genes HUS1, EGFR, ABCA13, and IKZF1, predicted nonresponse in patients with a sensitivity, a negative predictive value, and a specificity of 71.4%, 87.5%, and 100%, respectively. Total count of CD3+ T cells/mm2 tumor was a significant predictor of NAC response. In conclusion, 7p12 amplification may predict nonresponse to NAC and worse survival in MIBC. Multicenter, prospective trials with sufficient statistical power may further fortify these findings.
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      Materials and Methods

      Patients and Cohorts

      Consecutive medical records of patients with centrally reviewed primary MIBC (cT2 to cT4a, N0 to N3, M0) diagnosed by transurethral resection from a single-center institution who received neoadjuvant cisplatin-based chemotherapy (gemcitabine, 1000 mg/m2, on days 1,8, and 15; and cisplatin, 70 mg/m2, on day 2, every 28 days) followed by radical cystectomy (RC) with extended bilateral pelvic lymphadenectomy were evaluated. The samples were collected consecutively. Repeated imaging was performed in all patients after completed NAC to exclude distant metastases or locally advanced tumor spread. pDS was defined as ypT0 to T1N0 stage, and pathologic nonresponse (pNR) was defined as ≥ypT2N+ on RC specimens. In all patients, the same follow-up investigations and visits after RC were scheduled, according to our institutional practice, as described previously.
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      Gender-related outcome in bladder cancer patients undergoing radical cystectomy.
      Patients who were followed up elsewhere postoperatively and patients with evidence of distant metastasis on standard imaging after NAC were excluded. This retrospective observational study was approved by the local Ethics Committee of the Medical University Innsbruck (Innsbruck, Austria; study number 1006/2017).

      Whole Exome Sequencing and Calculation of Tumor Mutation Burden

      DNA isolation was performed after macrodissection of the tumor with the formalin-fixed, paraffin-embedded (FFPE) tissue extraction kit (Qiagen, Hilden, Germany) on the automated Qiasymphony workstation. After shearing 100 ng genomic DNA with the Covaris M220 sonificator (Covaris Ltd, Brighton, UK), whole exome sequencing libraries were generated by the use of the TruSeqDNA Exome kit (Illumina Inc., San Diego, CA) combined with target enrichment by xGen Lockdown Probes (IDT, Coralville, IA) with an automated workflow on the Hamilton NGS-Star (Hamilton Robotics, Reno, NV); whole exome sequencing libraries were quantified with the Qubit 3.0 (Thermo Fisher Scientific, Waltham, MA) and sequenced on the NextSeq500 (Illumina Inc.) with a mean on-target coverage of 150×. Generation of FASTQ files and demultiplexing were performed on the BaseSpace Server (Illumina Inc.). The Enrichment App 3.1.0 of the BaseSpace Server was used for rapid alignment and variant detection tool. In fact, reads were aligned against the reference genome using IsaacCall (https://github.com/sequencing/isaac_variant_caller, last accessed February 10, 2019), and structural variants were called using Manta (https://github.com/Illumina/manta, last accessed February 10, 2019). Small germline variants (single-nucleotide variants and insertions-deletions) were called using Starling (https://github.com/sequencing/isaac_variant_caller, last accessed March 12, 2019), or somatic variants were called using Pisces (https://github.com/Illumina/Pisces, last accessed March 12, 2019). The Variant Calling Assessment Tool VCAT 3.0.0 (Illumina Inc.) was used for the comparison of variant call sets using VCF files as input and the Platinum Genomes version 2016-1.0 (Illumina Inc.) and dbSNP build 147 (http://www.ncbi.nlm.nih.gov/SNP, last accessed January 13, 2019) as reference databases. Genomic VCF files were filtered for 10% mutation frequency, EXAC allele frequency <1%, and nonsynonymous or frame-shift mutations to calculate the respective tumor mutational burden (TMB) for each MIBC specimen.

      Analysis of Chromosomal Aberrations in Tumor Cell DNA Using Array-Based Comparative Genomic Hybridization

      The OncoScan CNV FFPE Assay Kit (Thermo Fisher Scientific) working with 220,000 markers distributed with a resolution of 10 Mb all over the genome was used to monitor copy number variations and loss of heterozygosity. In addition, it covers approximately 900 oncogenes and tumor suppressors with a higher resolution of 50 to 125 kb. Genomic DNA (100 ng) was extracted from macrodissected tumor with the FFPE tissue extraction kit on the automated Qiasymphony Workstation. Chips were hybridized in the Gene Chip Hybridization Oven 645, processed in the Gene Chip Fluidics Station 450, and then scanned in the Gene Chip Scanner 7 G (Affymetrix, Santa Clara, CA; Thermo Fisher Scientific). Scanned data were imported in the Chromosome Analysis Suite software version 4.0 (ChAS 4.0; Thermo Fisher Scientific) and, thereafter, analyzed for DNA gain or loss (ie, changes in ≥25 SNP markers for defining a region with copy number variations). Regions with loss of heterozygosity were defined to be >5 Mb. Copy number gains or losses were detected for small chromosome arms, regions, and whole chromosomes; and they were analyzed in respective cohort analysis (Chromosome Analysis Suite software 3.1). Small genomic regions showing high-level amplifications (defined as log2 ratio >1), as well as regions indicating homozygous deletions (defined as log2 ratio <−1), could also be identified. Supplemental Figure S1 shows representative examples of the high-resolution analysis of two MIBC samples (one patient with pDS and one patient with pNR).

      Multispectral Analysis of Tumor-Infiltrating Lymphocytes and PD-L1 Expression by Digital Image Analysis

      The Opal 7 color immunohistochemistry kit (Perkin Elmer, Waltham, MA) was used for simultaneous detection of multiple biomarkers in FFPE tissue, specifically CD3, CD8, programmed death-ligand 1 (PD-L1), forkhead box P3 (FoxP3), and cytokeratin (CK) 7/20, together with a nuclear counterstain (DAPI) on tumor FFPE sections. Tissue staining was performed with an automated protocol for the respective antibodies in the automated stainer Bond RX (Leica, Vienna, Austria) with the respective Leica antigen retrieval solution. Thereafter, slides were mounted and scanned in the Mantra quantitative pathology workstation (Perkin Elmer) with the Mantra Snap software version 1.0.3 for image acquisition and the inForm Advanced Image Analysis Software version 2.3.0 (Akoya Biosciences Inc., Menlo Park, CA) for trainable image analysis and automatization of multispectral imaging. The immunoscoring was performed on areas with no necrosis and high lymphocyte infiltration on whole slide sections of the transurethral resection specimens. Tumor-infiltrating lymphocytes were counted in the tumor area and the respective tumor stroma and calculated in percentage of positive cells/slide. CK7/20-positive tumor cells were scanned for coexpression of PD-L1 and, thereafter, percentage PD-L1 positivity in tumor cells was calculated. Only tumor cells with membranous staining were taken into consideration. Digital pathology with quantification of tumor-infiltrating lymphocytes and PD-L1 is presented in Figures 1 and 2.
      Figure thumbnail gr1
      Figure 1Immunoscoring of programmed death-ligand 1 (PD-L1) quantification of transurethral resection specimens by multicolor-immunofluorescence and digital image analysis on the Mantra system. PD-L1–negative (A) and PD-L1–positive (B) muscle-invasive bladder cancer. PD-L1 expression is marked in green, and cytokeratin 7/20 is marked in red. Original magnification, ×200.
      Figure thumbnail gr2
      Figure 2Immunoscoring of tumor-infiltrating lymphocyte (TIL) expression of transurethral resection specimens by multicolor-immunofluorescence and digital image analysis on the Mantra system. Representative stains and quantification showing a cold tumor (few TILs; A) and a hot tumor (high TILs; B). CD3, CD8, forkhead box P3 (FOXP3), and cytokeratin 7/20 expression is labeled with yellow, pink, blue, and green, respectively. Original magnification, ×200.

      Molecular Subtyping by Immunohistochemistry

      Intrinsic molecular subtyping, referred to herein as luminal and basal, was performed using mouse monoclonal antibody against human GATA3 (luminal marker) and keratin 5/6 (KRT5/6; basal marker), as described previously.
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      Paraffin-embedded tissue sections were deparaffinized and hydrated in xylene and graded alcohol series. Thereafter, antigen retrieval was performed by microwave treatment in citrate buffer (10 mmol/L; pH 6.0) and endogenous peroxidase activity was blocked with 3% H2O2/methanol. Sections were incubated in blocking solution containing 10% bovine calf serum (Dako Cytomation, Glostrup, Denmark) for 45 minutes and then stained for 1 hour with primary antiserum [rabbit monoclonal anti-human CK5 and CK6 Cocktail (Epitomics-Abcan, Cambridge, MA) clone CK5 (EP24) and CK6 (EP67); dilution 1:200]. Moreover, serial sections were incubated with a monoclonal mouse anti-GATA3 (Roche, Unterhaching, Germany; clone L50-823; dilution 1:100). Primary antiserum was detected after incubation with the respective biotinylated secondary antibody (biotinylated horse anti-rabbit IgG or biotinylated rabbit anti-mouse IgG; Vector Laboratories, Burlingame, CA) using the Vectastain Elite ABC Kit (Vector Laboratories) and the FAST DAB Tablet Set (Sigma, Vienna, Austria). Sections were counterstained with Meyer's haemalaun and mounted with Pertex (Histolab, Gothenburg, Sweden). KRT5/6 and GATA3 were semiquantitatively assessed and scored as follows: − indicates negative staining; +, 10% to 25% (weak expression); ++, >25% to 50% (moderate expression); and +++, >50% (strong expression) positive tumor cells. An overview of all used antibodies is summarized in Table 1.
      Table 1Overview of All Antibodies, Providers, Clone Number Concentrations, and Antigen Retrieval Applied for Molecular Subtyping, Multispectral Analysis of Tumor-Infiltrating Lymphocytes, and PD-L1 Expression by Digital Image Analysis
      AntibodyOpal, nmCompanyCloneDilutionpH
      CD3650Dako AgilentCode: A05421:2509
      CD8570Dako AgilentC8/144B1:2009
      CK7690Dako AgilentOV-TL 12/301:1509
      CK20690Dako AgilentKS 20.81:1509
      FoxP3620Abcam236A/E71:1506
      PD-L1 (CD274)520Dako Agilent22C31:1509
      DAPI450Perkin Elmer1:206
      GATA3Roche DiagnosticsL50–8231:1006
      KRT5/6EpitomicsCK5 (EP24) and CK6 (EP67)1:2006
      CK, cytokeratin; FoxP, forkhead box P; KRT, keratin; PD-L1, programmed death-ligand 1.

      Statistical Analysis

      Statistical analyses were performed using SPSS software version 22 (IBM Corp., Armonk, NY) with two-sided P < 0.05 considered as statistically significant. The sensitivity, specificity, positive predictive value, and negative predictive value for 7p12 amplification in predicting NAC response were calculated. Correlations between parameters were assessed with Spearman's ρ correlation coefficient (rs). Patient immune and molecular characteristics were compared between the two groups (pDS versus pNR) by the U-test. OS and recurrence-free survival (RFS) were defined as the time period from the date of primary tumor diagnosis to death of any cause and the detection of local recurrence and distant lymphatic or hematogenous metastases, respectively. The median value of each marker was used as the cutoff point to dichotomize patients into two groups for Kaplan-Meier survival analysis and comparison by the log-rank test. Graphic diagrams were produced with GraphPad PrismTM6 version 8 (GraphPad Software Inc., La Jolla, CA). All values were presented as means ± SEM.

      Results

      Demographic and Clinicopathologic Characteristics and Molecular Subtyping

      At diagnosis of primary MIBC, the mean (±SD, range) age was 66.5 (±6.8, 48 to 76) years, including five females (21.7%) and 18 males (78.3%). Seven patients (30.4%) were defined as pNR, and 16 patients were classified as pDS. Supplemental Table S1 summarizes patient characteristics and response to NAC by clinical tumor-node stage. Transurethral resection before NAC confirmed at least pT2a urothelial carcinoma of the bladder in all patients.
      Median follow-up was 18 months (range, 6 to 89 months). There was a significant RFS benefit associated with pDS to cisplatin-based NAC (P < 0.001). Concerning pNR, six of seven patients (85.7%) developed tumor recurrence, whereas only two patients with pDS (12.5%) showed recurrent disease (both patients had ypT1 disease). Median time to recurrence was 5.5 months in the pNR group and 41.2 months in the pDS (ypT0 to ypT1) population. Focusing on patients with pDS after NAC, eight patients (50%) showed ypT0 and another eight patients showed ypT1 disease. The median RFS was 64.3 months in the ypT0 group compared with 18 months in patients with ypT1 tumor.
      Of 21 MIBCs, 14 (66.7%) were classified as luminal on the basis of positive GATA3 expression and negative KRT5/6 expression: six of seven patients (85.7%) were pNR, and 8 of 14 (57.1%) were pDS patients. However, a clear overlap with coexpression of GATA3 and KRT5/6 was observed in 6 of 21 cases (28.6%; pNR: n = 1; pDS: n = 5); of them, no KRT5/6-positive case had strong coexpression of GATA3 (staining in >50% of tumor cells). An example of GATA3/KRT5/6 coexpression is illustrated in Supplemental Figure S2. Double-negative tumors for GATA3 and KRT5/6 accounted for 1 of the 21 cases (4.7%) (Supplemental Figure S3).

      TMB Shows No Significant Differences concerning Pathologic Response to NAC and Survival Outcomes

      The landscape of the 16 most coding somatic mutations related to nucleotide excision repair genes, chromatin-modifying genes, DNA replication, DNA repair, and driver genes is reported in Figure 3, with TP53 (45%), ARID1A/B (40%), and KMT2B/C/D/E (35%) as the three most frequently mutated genes. Of 21 patients, 8 (38.1%) confirmed alterations in DDR genes (ATM, ATRX, BRCA1, CHEK2, ERCC2/4/5, FANCA, FANCD2, FANCG, MLH1, PALB2, PMS1, and FANCC). DDR alterations were significantly associated with a higher TMB (means ± SD: 15.2 ± 7.7 versus 9.1 ± 1.4 mutations/Mb; P = 0.007) (Supplemental Figure S4). The three patients with the highest TMB load had the highest frequency of DDR alterations [Patient 9: TMB 30.14 with seven DDR alterations (ATM, ATRX, ERCC2/4, FANCD2, PMS1, and FANCC); Patient 1: TMB 23.96 with three DDR alterations (ATRX, ERCC5, and FANCA); and Patient 2: TMB 13.51 with two DDR alterations (BRCA1 and PALB2)]. No significant differences in DDR alterations concerning response to NAC (pNR: 25% versus 27.3%) and RFS (P = 0.271 by log-rank test) were noticed. However, a trend toward better RFS was observed for patients with DDR alterations compared with DDR-unaltered tumors (Supplemental Figure S4).
      Figure thumbnail gr3
      Figure 3Whole exome sequencing on formalin-fixed, paraffin-embedded DNA of transurethral resection bladder cancer specimens to identify nonsynonymous mutations/exome. The landscape of the 16 most common coding driver mutations related to the RTK/RAS/phosphatidylinositol 3-kinase, T53/cell cycle, DNA repair, and oxidative damage pathways in our cohort. Samples were subdivided into nonresponders (left section of the graph) and pathologic downstaging (right section of the graph) and sorted by the total number of all mutations in descending order.
      Among 21 MIBC patients undergoing NAC, TMB was higher in the pDS group compared with nonresponders, but without statistical significance (median: 10.33 versus 9.41 mutations/Mb; 334.5 versus 317 mutations/exome; P = 0.201), with a broad range (6.67 to 30.14 mutations/Mb; 240 to 1052 mutations/exome) (Figure 4A). Evaluating by median TMB, there was no statistically significant difference between patients with high TMB (≥10 mutations/Mb) and low TMB (<10 mutations/Mb) concerning RFS (P = 0.071) and OS (P = 0.122), respectively (Figure 4B).
      Figure thumbnail gr4
      Figure 4Determination of the tumor mutational burden (TMB) by whole exome sequencing. A: Association between TMB (mutations/exome) and pathologic response to neoadjuvant chemotherapy. P values determined by U-test. B: Kaplan-Meier survival curves with recurrence-free survival (RFS) and overall survival (OS) in days according to TMB stratified by predefined cutoff points (<10 mutations/Mb indicates low TMB; ≥10 mutations/Mb, high TMB). P = 0.07 (log-rank test). Data are expressed as means ± SEM (A).

      High CD3+ T-Cell Infiltration Is Associated with Improved Response to NAC

      Digital pathology with quantification of tumor-infiltrating lymphocytes and PD-L1 is presented in Figures 1 and 2. Significant differences in mean expression levels of total counts (cells/mm2 tumor) based on NAC response were confirmed only for CD3+ T cells (pDS versus pNR: 733.3 versus 208.6; P < 0.001). In contrast, the levels of CD8+ T cells (P = 0.090), FOXP3+ regulatory T cells (Tregs; P = 0.103), CD3/FoxP3 ratio (P = 0.659), CD8/FoxP3 ratio (P = 0.495), and CD3/CD8 ratio (P = 0.129) did not differ significantly in regard to response to NAC, although there was a trend toward higher CD8+ T-cell infiltration in patients with NAC response (Figure 5, A–C ).
      Figure thumbnail gr5
      Figure 5Multispectral analysis of tumor-infiltrating lymphocytes and programmed death-ligand 1 (PD-L1) expression of transurethral resection bladder cancer specimens by digital image analysis. Total expression levels (cells/mm2 tumor) of immune cell subsets [CD3, CD8, and forkhead box P3 (FoxP3)] and tumoral PD-L1 expression (AC) based on response to neoadjuvant chemotherapy (NAC; D). Data are expressed as means ± SEM (AD). ***P < 0.001 (U-test).
      Differences in the localization pattern (stromal and tumoral) of immune cell percentage (CD3, CD8, and FoxP3) are shown in detail in Supplemental Figure S5. Moreover, mean (range) pretreatment tumor PD-L1 expression was not statistically different in patients with pDS to NAC (42.3%; 0% to 93%) compared with nonresponders (39.4%; 0% to 84%) (Figure 5D). Stratifying tumoral PD-L1 expression in three groups (0%, 1% to 50%, and >50%), pDS to NAC was seen in 31.2% (n = 5), 18.8% (n = 3), and 50% (n = 8), respectively; and in nonresponders in 20% (n = 1), 40% (n = 2), and 40% (n = 2), respectively. There was no significant association between tumoral PD-L1 expression and TMB (rs = −0.136; 95% CI, −0.566 to 0.350; P = 0.577). The median TMB was 13.5, 9.5, and 8.7 mutations/Mb in the PD-L1 expression group of 0%, 1% to 50%, and >50%, respectively (Supplemental Figure S6).

      Amplification of 7p12 Is Associated with Nonresponse to NAC

      Virtual karyotype analysis confirmed a loss of 17p13, encoding for TP53, in five of seven nonresponders (71.4%) and only in 2 of 14 pDS patients with loss of heterozygosity of TP53 (14.3%) and in eight pDS patients (57.1%). Interestingly, in five of seven samples of nonresponders (71.4%), an amplification of the chromosomal region 7p12 was observed. Focusing on the sequence of interest, 7p12.3-p11.2 (55 315 232- 47 323 074; a 7 990 000-bp fragment), this chromosomal region encompasses several genes that are implicated in the acquisition of resistance to cisplatin, such as HUS1, ABCA13, epidermal growth factor receptor (EGFR), FIGNL1, and IKZF1. All five MIBC samples of patients with HUS1/ABCA13/IKZF1/EGFR amplification showed a strong expression of GATA3 and were negative for basal cytokeratins 5 and 6 on immunohistochemistry analysis (Supplemental Figure S3). The combined up to 8× amplification of HUS1/ABCA13/IKZF1/EGFR was confirmed in all five of seven nonresponders, whereas none of the 14 pDS patients (0%) had an amplification in one or more of these genes (P < 0.001) and 2 of 14 pDS patients showed loss of heterozygosity of HUS1/ABCA13/IKZF1/EGFR (Supplemental Figure S7).
      The decision rule for nonresponse to NAC in case of HUS1/ABCA13/IKZF1/EGFR amplification results in a sensitivity, a specificity, a positive predictive value, and a negative predictive value of 71.4%, 100%, 100%, and 87.5%, respectively.
      Concerning survival outcomes, median time to recurrence after RC was three times shorter in the HUS1/ABCA13/IKZF1/EGFR amplification group compared with HUS1/ABCA13/IKZF1/EGFR wild-type patients (6.8 versus 18.1 months; P < 0.001). The HUS1/ABCA13/IKZF1/EGFR amplification was also predictive of poor RFS (hazard ratio, 4.0; 95% CI, 0.16–100.9; P < 0.001) and a trend toward poor OS (hazard ratio, 2.57; 95% CI, 0.12–55.2; P = 0.394) (Figure 6).
      Figure thumbnail gr6
      Figure 6Recurrence-free survival (RFS) and overall survival (OS) by HUS1/ABCA13/IKZF1/EGFR amplification status. Amplification in HUS1/ABCA13/IKZF1/EGFR predicts statistically significant poorer RFS (A), also with a trend for OS (B), but without statistical significance [P = 0.394 (log-rank test)]. ****P < 0.0001 (log-rank test).

      Discussion

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      confirming also a GATA3 and KRT5/6 coexpression in 48.35% of MIBC samples, although GATA3 and KRT5/6 demonstrated a significant negative correlation. Moreover, on the basis of the coexpression of immunohistochemical luminal and basal markers, 28% of basal tumors were coclustered with luminal tumors.
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      These preliminary findings must be interpreted with caution because of several major limitations. First, this was a retrospective study with limitation of statistical power and, thus, results must be reevaluated in further multicenter, prospective trials with sufficient statistical power before drawing any final conclusion. Nevertheless, our study population was homogeneous as all included patients underwent the identical cisplatin-based NAC protocol and number of chemotherapy cycles with follow-up visits at the same institution with equal time intervals. Second, the median follow-up was only 18 months, resulting in limitation of statistical power concerning OS analysis. Third, given the limited cohort size, the significant differences on digital karyotype analysis between the two groups of NAC response should be considered as hypothesis generating for now and further analyses are needed: To corroborate our preliminary results, the validation of a correlation of the expression level of the genes implicated in the 7p12 amplification would be necessary. Unfortunately, the gene expression of these genes was not analyzed as such analysis would need good-quality RNA from tissue blocks and RNA sequencing or real-time PCR analysis. Perhaps, many tissue blocks displayed already strong degradation of RNA, as determined by a quality check on the Agilent Bioanalyzer (Agilent, Santa Clara, CA). In addition, fluorescence in situ hybridization analysis that uses a fluorescently labeled, locus-specific indicator DNA probe to assess cells for the identified 7p12 amplification may reconfirm our preliminary results.
      In conclusion, DDR gene alterations were significantly associated with a high TMB. Nevertheless, no significant correlation was confirmed between DDR alterations or TMB and response to NAC, although there was a trend toward better survival rates after NAC. More important, chromosomal amplification of the region 7p12.3-p11.2, including the genes HUS1, EGFR, ABCA13, and IKZF1, predicted nonresponse to cisplatin-based NAC and worse RFS and OS outcomes after NAC in MIBC compared with patients with wild type. Thus, the detection of an amplified HUS1/EGFR/ABCA13/IKZF1 region resulted in a sensitivity, a specificity, a positive predictive value, and a negative predictive value of 71.4%, 100%, 100%, and 87.5% for nonresponse to NAC. Given the limited patient number, these significant differences on virtual karyotype analysis must be considered hypothesis generating at the moment and must be validated in further larger independent prospective data sets with further analysis (fluorescence in situ hybridization and validation of gene expression levels implicated in the 7p12 amplification).

      Supplemental Data

      • Supplemental Figure S1

        Whole genome profiles and genome-wide frequency of copy number alterations from chromosome 1 to 22. The frequency of copy number gains (blue), losses (red), and loss of heterozygosity (black) throughout the genome for pathologic nonresponse Patient 4 (A) and pathologic downstaging Patient 15 (B).

      • Supplemental Figure S2

        Immunohistochemical analysis of basal cytokeratins 5 and 6 and luminal zinc-finger transcription factor GATA3 in muscle-invasive bladder cancer. Tumor with coexpression of basal cytokeratins (A) and luminal GATA3 (B) in a patient with pathologic downstaging. Scale bars = 50 μm (A and B). Original magnification, ×200 (A and B).

      • Supplemental Figure S3

        Overview of semiquantitative immunohistochemical scoring for basal keratin 5/6 (KRT5/6) and the luminal marker GATA3 in muscle-invasive bladder cancer samples. GATA3+ and KRT5/6 cases are marked with orange; double-negative cases are marked in blue; and GATA3 and KRT5/6 coexpression is marked in yellow. +, 10% to 25% positive tumor cells; ++, >25% to 50% positive tumor cells; +++, >50% positive tumor cells; −, negative staining.

      • Supplemental Figure S4

        A: Tumor mutational burden (TMB) by the presence or absence of a DNA damage repair (DDR) gene mutation. Each data point reflects a different tumor sample. Black lines represent the data. P values were calculated by U-test. B: Recurrence-free survival (RFS) in days, according to the DDR alteration status. P values were calculated by log-rank test. Data are expressed as means ± SEM (A). **P < 0.01.

      • Supplemental Figure S5

        Multispectral analysis of tumor-infiltrating lymphocytes by digital image analysis. Percentage of immune cell expression levels based on localization pattern. Intratumoral (A) and stromal (B) CD3, CD8, and forkhead box P3 (FoxP3), stratified by response to neoadjuvant chemotherapy. Data are expressed as means ± SEM (A and B). *P < 0.05 (U-test).

      • Supplemental Figure S6

        A: Correlation analysis between baseline tumoral PD-L1 staining and tumor mutational burden (TMB). Dashed line represents linear regression line. B: No significant difference in baseline tumor PD-L1 expression (0% versus 1% to 50% versus >50%) was observed when comparing with levels of TMB. Data of TMB (mutations/Mb) are expressed as means ± SEM (B).

      • Supplemental Figure S7

        Virtual karyotype analysis by the Affymetrix Oncoscan (Affymetrix, Santa Clara, CA). A: Amplification of the chromosomal region 7p12 and loss of 17p13, encoding for TP53, is confirmed only in the pathologic nonresponse (pNR) group compared with the pathologic downstaging (pDS) cohort. B: Focusing on the 7p12 sequence, this region encompasses several genes that implicate in the acquisition of resistance to cisplatin, such as HUS1, EGFR, IKZF1, and ABCA13. C: Five of seven patients from the pNR group (71.4%) show up to 8× amplification of HUS1/ABCA13/EGFR/IKZF1 and a loss of TP53 (17p13), whereas in none of the pDS patients is HUS1/ABCA13/EGFR/IKZF1 amplified.

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