Loss Of 6p Research Articles

Tumor evolution metrics predict recurrence beyond 10 years in locally advanced prostate cancer.

Cancer evolution lays the groundwork for predictive oncology. Testing evolutionary metrics requires quantitative measurements in controlled clinical trials. We mapped genomic intratumor heterogeneity in locally advanced prostate cancer using 642 samples from 114 individuals enrolled in clinical trials with a 12-year median follow-up. We concomitantly assessed morphological heterogeneity using deep learning in 1,923 histological sections from 250 individuals. Genetic and morphological (Gleason) diversity were independent predictors of recurrence (hazard ratio (HR) = 3.12 and 95% confidence interval (95% CI) = 1.34-7.3; HR = 2.24 and 95% CI = 1.28-3.92). Combined, they identified a group with half the median time to recurrence. Spatial segregation of clones was also an independent marker of recurrence (HR = 2.3 and 95% CI = 1.11-4.8). We identified copy number changes associated with Gleason grade and found that chromosome 6p loss correlated with reduced immune infiltration. Matched profiling of relapse, decades after diagnosis, confirmed that genomic instability is a driving force in prostate cancer progression. This study shows that combining genomics with artificial intelligence-aided histopathology leads to the identification of clinical biomarkers of evolution.

Malignant Proliferating Pilar Tumor: Clinicopathologic, Immunohistochemical, and Molecular Study of 17 Cases.

Proliferating pilar tumors are rare neoplasms that differentiate toward the outer sheath near the isthmus and can rarely undergo malignant transformation. We performed histopathologic evaluation on 26 benign proliferating pilar tumor (BPPT) and 17 malignant proliferating pilar tumor (MPPT). Ki-67 and p53 immunostains were performed on 13 BPPT and 10 MPPT. Six MPPT cases were successfully analyzed by a next-generation sequencing platform which surveyed exonic DNA sequences of 447 cancer genes and 191 regions across 60 genes for rearrangement detection. Patient demographics and clinical characteristics were similar between the BPPT and MPPT groups. Follow-up data of 16 of 17 MPPT (median, 25mo) showed metastasis in 1 MPPT. The histologic features associated with MPPT include size >2.5cm, adjacent desmoplastic stroma, small nests or cords of atypical epithelium in surrounding stroma, irregular infiltration or borders, abnormal keratinization, large hyperchromatic nuclei, prominent nucleoli, severe cytologic atypia, nuclear pleomorphism, necrosis, and increased mitotic figures. MPPT harbors copy number gains of 15q and losses of 6p and 6q, findings previously reported in BPPT. However, MPPT harbors frequent TP53 mutations as molecular markers of progression. Different from cutaneous squamous cell carcinoma, MPPT more frequently demonstrates low tumor mutational burden and typically lacks a UV signature, suggestive of a different etiologic pathway than squamous cell carcinoma. In summary, with a median follow-up of 25 months, this study shows that MPPT is a biologically indolent carcinoma with rare metastasis. Molecular analyses suggest a non-UV-related pathogenesis with frequent TP53 aberration.

Next-Generation Sequencing of Newly-Diagnosed Primary CNS Lymphoma Reveals Alterations in BTG1, ETV6, and 6p Are Associated with Chemoresistance and Inferior Progression-Free Survival

Introduction: There is a significant unmet need for the development of reliable genetic biomarkers that identify patients with newly-diagnosed primary CNS lymphoma (PCNSL) who are at high risk of progression, both during standard methotrexate-based induction as well as delayed progression after receipt of dose-intensive consolidation. Currently, little is known about the genetic subtypes of PCNSL and the basis for differences in chemo-immunotherapeutic response. We hypothesized that an integrated clinical-genomic analysis may reveal new determinants of tumor progression and may ultimately guide a precision medicine-based approach to the selection of effective treatment regimens. Methods: We performed targeted next-generation sequencing of 529 pan-cancer genes of pre-treatment, formalin-fixed, paraffin-embedded tumors from 52 patients with CNS diffuse large B-cell lymphoma. We identified structural variants including single nucleotide variants, small insertions and deletions, and copy-number alterations. Clinical records were retrospectively reviewed to determine patients' baseline characteristics, IELSG index, and progression-free survival (PFS). 44 EBV-negative PCNSL patients treated with methotrexate, temozolomide, rituximab (MTR) induction with adequate genomic samples were available for complete analysis. Statistical testing was performed using R. Results: We identified three candidate genomic alterations that were significantly associated with chemotherapy resistance in PCNSL: (1) Alterations at 6p21.3, including both copy neutral loss of heterozygosity (CN LOH) at the HLA Class I (HLA-A, B, C) and Class II loci (HLA-DR, DP, DQ, TAP1) as well as homozygous deletion of HLA Class II; (2) Mutations of tumor suppressor genes BTG-1 and ETV6 (Fisher's exact, p < 0.01). Overall, these genomic alterations were detected in 18 out of 19 progression events in the cohort (Fisher's exact, p < 0.001). 7 of 9 tumors with 6p CN LOH also contained mutations at BTG-1 and/or ETV6. Kaplan-Meier survival analysis shows three subgroups stratified by alteration type with distinct PFS outcomes (Figure 1). The subgroup with either CN LOH or homozygous deletion at 6p had the earliest progression, followed by the subgroup with mutations at either BTG-1/ETV6 and no 6p CN LOH or homozygous deletion, compared to no alterations at these 3 loci (Log-rank, p < 0.01). Patients with hemizygous loss at 6p exhibited a trend towards worse outcome compared to no 6p loss. Furthermore, multivariate Cox proportional hazard analysis demonstrated a statistically significant 2.7-fold increased risk of progression with 6p CN LOH/homozygous deletion or mutations involving BTG-1/ETV6 (Table 1). While etoposide-cytarabine consolidation and maintenance lenalidomide were each significantly associated with a decreased risk of progression, age at diagnosis, Karnofsky performance status, intraocular disease status, and IELSG index were not significantly associated with PFS. Conclusions: Through harmonizing clinical and next-generation sequencing data, in this retrospective analysis we identify pathogenic alterations involving BTG-1 and ETV6, as well as major alterations at 6p21.3, that correlated with accelerated disease progression and an early tumor resistance phenotype in PCNSL. While 6p loss has been identified as a mechanism of tumor immune escape in PCNSL, our results suggest that 6p CN LOH may be a novel mechanism associated with early progression. Of note, in a parallel investigation using patient-derived CNS lymphoma tumor cells engrafted in a murine orthotopic xenograft model, we have demonstrated that lenalidomide significantly induces transcription of 6p21.3 genes involved in antigen presentation and immunomodulation, including HLA-A, TAP-1, and lymphotoxins. Taken together, 6p21.3 copy number loss, in particular CN LOH and homozygous deletion, as well as mutations in BTG-1 and ETV6 may be a promising biomarker set, potentially useful in risk stratification in clinical trials as well as to identify high risk patients more likely to derive benefit from lenalidomide. Prospective validation studies are planned to confirm these results. Supported by NCI. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

HLA Class I Allele-Specific Pathology Defines Clinical Manifestations of Immune Aplastic Anemia

Leukocytes that have lost the cell surface expression of an HLA class I allele due to somatic HLA gene mutations or 6p loss of heterozygosity (LOH) are often present in patients with immune aplastic anemia (AA), consistent with T cell-mediated bone marrow cell destruction. A recent NIH study suggested that the clinical significance and underlying pathology of HLA loss may differ according to the type of HLA allele affected (Zaimoku et al. Blood 2021) because (1) the significant correlation of HLA loss with clonal evolution was primarily attributed to patients who lost HLA-B*14:02 and A*02:01; (2) all patients who lost B*40:02 and A*02:06 achieved a hematologic response to immunosuppressive therapy (IST), though HLA loss in general was not correlated with the IST response; (3) loss of B*07:02 and B*40:01 was associated with older age of onset; (4) patients who retained B*14:02, B*07:02, and B*40:01 also showed clinical features of patients who lost respective HLA alleles. We aimed to confirm the HLA class I allele-related clinical manifestations in Japanese AA patients. HLA loss was assessed by SNP array, HLA flow cytometry, digital PCR, or deep nucleotide sequencing. An HLA allele was classified as "affected by loss" in individual patients when the single allele was lost or inactivated by somatic mutations or when both HLA-A and HLA-B alleles in a haplotype were lost due to 6p LOH with no evidence of loss or inactivation of a single HLA allele. Clinical characteristics were compared according to the affected HLA alleles. In 496 Japanese patients with AA (severe, n = 368; non-severe, n = 128; median age, 59 [range, 1-89] years), there were 146 (29%) patients with HLA loss. HLA-B*40:02, A*02:06, A*31:01, and B*54:01 were more frequently affected in our cohort than in the NIH cohort, where B*14:02, A*02:01, B*08:01, and B*07:02 held the majority of lost alleles (Figure 1). The median age of patients with B*40:01 loss (75 years) was highest among the groups of patients with HLA allele loss, consistent with the NIH study. The prevalence of ≥0.003% paroxysmal nocturnal hemoglobinuria (PNH) clones was significantly reduced in patients with B*40:02 loss but not in others. Sex and severity of AA were not markedly different across the groups of patients with HLA allele loss. Outcomes after anti-thymocyte globulin (ATG)-based IST were evaluable in 263 of the 496 patients. Although HLA loss was correlated with the IST response at 6 months (as reported by our group), loss of HLA-A*02:01 did not contribute to the high response, consistent with the NIH study. Loss of frequently affected HLA alleles in Japanese patients (B*40:02, A*02:06, B*54:01, etc.) was generally correlated with the IST-response. Further, patients who carried 5 HLA-B alleles (B*40:01, B*40:02, B*44:03, B*54:01, and B*56:01) without loss still displayed higher response rates than other patients. In contrast, the favorable effect of A*02:06 loss was not seen in patients who carried A*02:06 without loss. A*02:06 loss, the presence of the 5 HLA-B alleles (irrespective of loss), and previously reported HLA class II alleles (DRB1*13 and DRB1*15) were independently correlated with the IST response. The incidence of clonal evolution to myeloid neoplasms after ATG-based IST was significantly higher in patients with HLA-A*02:01 loss than other patients (Figure 2), as observed in the NIH study; A*02:01 was affected in 4 of 7 patients showing both HLA loss and clonal evolution in our cohort; B*40:02, B*44:02, and B*52:01 were affected in other 3 patients. In summary, our study in Japanese AA patients broadly confirmed the associations of loss of specific HLA alleles with clinical manifestations observed in American patients, including older age of onset in patients with HLA-B*40:01 loss, high IST response rates in patients who lost B*40:02 and A*02:06 but not in patients with A*02:01 loss, and frequent clonal evolution in patients with A*02:01 loss. We also revealed the correlation of 5 HLA-B alleles with IST response, irrespective of loss, similar to the relationship of B*14:02 to clonal evolution in the United States, and the reduced prevalence of PNH clones in patients with B*40:02 loss. These findings strongly suggest that the heterogeneous pathology of AA can be classified by affected HLA alleles. This should be useful for studies to unravel the pathophysiology and improve the outcomes of patients with immune AA. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Genetic Determinants of Isolated and Systemic Testicular Diffuse Large B-Cell Lymphoma Highlight a Disease Spectrum

Introduction Primary (diffuse) large B-cell lymphoma of the testis (PTL) is an aggressive extranodal B-cell lymphoma characterized by frequent relapses in the central nervous system (CNS) and other extranodal sites. While PTL has now been recognized as a distinct lymphoma in the latest classifications (International Consensus Classification and WHO HAEM5), however, the allowable extent of involvement outside of the testes is unclear from these publications. PTL are largely assigned to the MCD genetic subtype of diffuse large B-cell lymphoma (DLBCL) by LymphGen with frequent MYD88L265P mutations and have 6p loss associated with immune evasion. BCL6 rearrangements (BCL6-R), a feature of BN2, have been associated with an increased risk of CNS relapse (Twa et al., Blood 2021), though the mechanisms enabling tumor establishment in the CNS remain unclear. Here, we provide a characterization of the genomic landscape of DLBCL involving the testes, aiming to understand whether systemic disease is distinct from disease isolated to the testes and whether there are genetic features associated with patient outcomes. Methods Patients with DLBCL involving the testis, treated with R-CHOP immunochemotherapy, were identified in the BC Cancer Lymphoid Cancer database, yielding archival formalin-fixed paraffin-embedded orchidectomy samples from 65 patients. BCL6-R were identified by break-apart fluorescent in situ hybridization (FISH). High confidence simple somatic mutations (SSMs) from whole exome sequencing were selected using a voting approach of four variant callers (Strelka2, Lofreq, Mutect2, SAGE). Candidate copy number alterations (CNAs) were identified using CNVkit and PureCN and high confidence CNAs were determined using GISTIC2.0. LymphGen was run with SSMs to annotate genetic subgroups. Clinical endpoints included time to progression (TTP), disease-specific survival (DSS) and CNS relapse cumulative risk. Results The study cohort had a median age of 72 years (range 26-93) and median follow-up of living patients was 5.5 years. In 30 patients lymphoma was isolated to the testes (Stage IE), 27 had concurrent involvement outside of the testes (Stage II/E-IV), and eight had DLBCL isolated to the testes with low grade lymphoma in the bone marrow (herein labelled "BM" - excluded from isolated vs systemic comparisons). BCL6-R were detected in 29% of the tumors. The majority of the tumors (69%) were assigned to the MCD subgroup by LymphGen with 73% and 66% of tumors harboring MYD88L265P and PIM1 mutations, respectively, though 40% of BCL6-R tumors were assigned to BN2 or Other. To address whether there are biological differences between disease isolated to the testes versus those that also have systemic involvement, we compared tumors from patients with stage I vs II-IV disease. There were no significant differences in the proportions of BCL6-R or LymphGen subgroups. Similarly, there was no difference in CNAs and the mutational landscapes were largely the same, with the exception of two significant differences. There was a significant enrichment for CD58 truncating mutations in stage II-IV (31%; q < 0.1), with a missense mutation observed in one stage IE tumor. Secondly, BTG2 mutations were more frequently observed in stage I disease (37% vs 8%; q < 0.1). We observed significantly superior TTP (Hazard ratio (HR) = 0.46, P < 0.05) and DSS (HR = 0.34, P < 0.01) in patients with stage I vs stage II-IV diseases. In stage II-IV disease, the MCD (n=18) (vs non-MCD (n=9)) subgroup was associated with significantly superior TTP (2-year 72% vs 22% TTP; HR = 0.36, P < 0.05) and DSS (2-year 72% vs 22% DSS; HR = 0.37, P < 0.05) and lower frequency of CNS relapse (13% vs 55% cumulative relapse at 2 years; HR = 0.23, P < 0.05). In contrast, LymphGen subgroup was not prognostic in stage I disease. Conclusion Here, we show that there are very few genetic differences comparing testicular DLBCL isolated to the testes to those with stage II-IV disease. The novel observation of truncating mutations in CD58 exclusively in patients with systemic involvement suggests that these mutations contribute to immune evasion thereby promoting disease outside of the immune privileged site of the testes. Genetic subgroups appear to be associated with outcomes in patients with systemic involvement - an observation that requires validation in a larger cohort. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

EPEN-16. Epithelial Progenitor Cell Abundance and Copy Number Variant Gains and Losses Impact the Biology of Recurrent Ependymoma

Ependymoma (EPN) is a common pediatric brain tumor that is fatal in approximately 50% of cases. Posterior fossa A (PFA) EPN has the highest rate of recurrence and the worst prognosis of all EPN subtypes. At relapse, it is typically incurable even with re-resection and re-irradiation. The biology of recurrent ependymoma remains largely unknown, which hinders clinical advances. In this study, we use paired samples of primary and recurrent disease from the same patient to investigate the drivers of recurrence. DNA methylation studies reveal frequent copy number variants at recurrence that were not present at primary presentation. We report a frequent gain of chromosome 1q and loss of 6p at recurrence, which has not been previously reported and may be a driver of recurrent disease. We have previously shown that PFA EPN is comprised of 4 main neoplastic cell populations, two well-differentiated populations termed ciliated and transportive ependymal cells, a mesenchymal cell population, and an undifferentiated population. Using spatial transcriptomics (Visium) integrated with single-nuclei RNA-seq (Chromium), we discovered that a highly proliferative EPN progenitor population of epithelial lineage is significantly upregulated at recurrence which we hypothesize drives refractory disease. Accordingly, we found higher expression of EPN progenitor gene signatures in bulk RNA transcriptomes of primary tumors that later recurred compared to tumors that never recurred. Together, these findings highlight the biologic differences between primary and recurrent disease and add to our understanding of treatment resistance in childhood ependymoma.

Abstract IA44: ctDNA in indolent lymphoid cancers treated with targeted therapies

Abstract The use of circulating tumor DNA (ctDNA) in diagnosing and monitoring treatment responses in hematologic malignancies has multiple advantages: (1) ctDNA is noninvasive, allowing for frequent, serial, real-time tracking of multiple tumor clones; (2) ctDNA is highly sensitive for clone-specific genomic aberrations, and may rival traditional minimal residual disease (MRD) methodologies in detecting low-volume disease; and (3) ctDNA “integrates” the tumor genome across multiple body compartments, allowing the detection of emerging resistant clones that would otherwise be missed by single-site biopsies. Since 2014, our group has evaluated the use of ctDNA in patients with hematologic cancers initially as an investigational tool, and more recently as an accredited (ISO15189) hybridization-based NGS panel that can perform sequence variant detection, genome-wide copy number calling, and structural variant detection from ctDNA. In this talk, we will discuss different clinical scenarios and the evolution of ctDNA at our center. Our initial proof-of-concept study was conducted in patients receiving BTK inhibitor (BTKi) and/or BCL2 inhibitor (BCL2i) therapy for chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL). Clinical challenges in this population include response assessment being confounded by BTKi redistribution lymphocytosis, differential compartmental responses to different classes of novel agents, and emergence of resistant clones in disparate disease sites. In our study of CLL, serial samples from 32 patients treated with either ibrutinib (BTKi, n=22) or venetoclax (BCL2i, n=10) were analyzed, including 3 patients who developed RS. Using a bespoke TS panel of 7 commonly mutated genes in CLL (SF3B1, NOTCH1, ATM, TP53, MYD88, KRAS, and BIRC3), somatic mutations comprising 0.1% to 90% of total ctDNA were detected in 25 of 32 (78%) patients. Serial monitoring of MAF was concordant with disease response as determined by CT scans and, importantly, ctDNA was able to “look beyond” the artificial rise in circulating tumor cells caused by BTKi, showing an early and consistent fall in MAF. ctDNA was sensitive to the detection of disease clones in occult body compartments—of 88 serial timepoints assessed by both ctDNA and MNL DNA after the initiation of novel therapy, mutations were found to be detectable only in ctDNA in 18 (20%) timepoints. In 30 timepoints where MRD testing was performed by both ctDNA and high-resolution flow cytometry, there was 100% concordance in results. In a second study, serial ctDNA analyses were performed in a cohort of 24 patients with MCL receiving combination ibrutinib and venetoclax therapy. Using TS and a panel of 42 genes known to be recurrently mutated in MCL, trackable ctDNA mutations were detected in 71% of patients, with baseline MAF ranging from 0.4-60%. Similar to our experience with CLL, dynamic changes in ctDNA closely mirrored tumor responses as determined using traditional staging methods of CT, PET, endoscopy, and bone marrow examination. In one patient, ctDNA started rising several weeks before relapse was evident by traditional MRD methods. A further patient had progression in an occult site (pleura), with acquisition of new CNAs on chromosome 6, including 6p loss. In this patient, these new CNAs were readily identified by LC-WGS of plasma. Finally, in a patient with primary refractory disease, an acquired loss of chromosome 9p was identified in plasma 8 weeks before disease progression was evident on imaging. We have since optimized and validated ctDNA testing for routine use into our diagnostic laboratory NGS workflow and we will discuss recent cases of BTK and BCL2 mutations being readily detectable in patients in early stages of novel agent resistance, facilitating early transition to next-line therapy. In aggressive lymphomas, we will demonstrate the value of ctDNA in detecting CD274(PD-L1)/PDCD1LG2 (PD-L2) copy number abnormalities and structural variants in patients with lymphomas in inaccessible tissue sites, guiding the rational use of checkpoint inhibitors. Other important structural variants that are able to be detected in ctDNA through this methodology are IGH-MYC and IGH-BCL2 translocations, thus allowing rapid categorization of patients with diffuse large B-cell lymphoma as “double-hit” lymphoma. Taken together, these studies underscore the value of ctDNA as a noninvasive, highly sensitive tool for assessment of genomic landscape and tracking clonal dynamics across multiple body compartments in patients with hematologic malignancies. Citation Format: Constantine S. Tam, Piers Blombery. ctDNA in indolent lymphoid cancers treated with targeted therapies [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr IA44.

Spectrum and Clinical Significance of HLA Class I Alleles and Their Somatic Mutations in Immune Aplastic Anemia

Clonal hematopoiesis associated with loss of HLA class I alleles due to somatic mutations and/or 6p loss of heterozygosity (LOH) is frequent in immune aplastic anemia (AA). HLA-B*40:02 is more likely to be involved in HLA loss in Japanese AA patients, suggesting a role for this allele in immune pathophysiology (Zaimoku Y et al, Blood 2017). Mutations in non-B*40:02 HLA class I alleles have been reported in a limited number of patients from the United States (Babushok D et al, Blood Adv 2017) and Japan (Mizumaki H et al, 60th ASH meeting), but their prevalence and clinical significance are not well characterized. We investigated somatic mutations of HLA class I alleles, HLA allele frequencies, and their correlations with outcomes of therapy in a total of 532 AA patients, aged 2 years or older, treated on various Hematology Branch protocols (clinicaltrials.gov NCTs 00001964, 00061360, 00195624, 00260689, 00944749, 01193283, and 01623167). HLA allele-lacking (HLA-) monocytes from cryopreserved peripheral blood mononuclear cells were screened by flow cytometry after staining with allele-specific monoclonal antibodies for HLA-A and/or HLA-B (HLA-flow) in 172 AA patients. HLA- monocytes accounting for 0.5% to 100% (median 9.5%) of total monocytes were detected in 49 (28%) of the 172 patients and in 59 (15%) of 382 alleles analyzed (Figure 1). Loss of cell surface expression was frequent for HLA-B14 (46%), B27 (33%), B49 (33%), A68 (26%), A2 (23%), B40 (21%), and B8 (21%). One percent to 60% (median, 8.9%) of glycosylphosphatidylinositol-linked protein-negative (GPI-) monocytes were also present in 43% (21 of 49) of the patients with HLA- monocytes, but GPI- clones had normal HLA cell surface expression. Deep sequencing of HLA-A, HLA-B and HLA-C on sorted HLA- and HLA+GPI+ monocytes was performed in 42 of the 48 patients from whom adequate cells were available. Somatic mutations and/or LOH corresponding to the lacking alleles were detected in all 42 cases (Figure 1): 9 had both somatic mutations and LOH, 20 had somatic mutations only, and 13 had LOH only. Among the 13 patients who showed only LOH in the absent allele, 6 had somatic mutations in other alleles of HLA+ monocytes that was not analyzable of HLA expression, and 2 had a breakpoint of LOH between HLA-A and HLA-C, leading to loss of a single HLA-A allele. Somatic mutations or LOH involving only one allele were present in 37 patients among 6 HLA-A alleles (in 02:01 [7 patients], 02:05 [1], 02:06 [3], 02:11 [1], 68:01 [2], 68:02 [2]) and 10 HLA-B alleles (07:02 [1], 08:01 [4], 14:01 [1], 14:02 [7], 27:05 [1], 35:02 [1], 35:05 [1], 40:01 [1], 40:02 [3], 45:01 [1]), but were not found in HLA-C alleles. HLA allele frequencies in AA patients, including 271 white Americans, 120 African-Americans, and 99 Hispanics and Latinos, were compared with ethnicity-matched individuals in bone marrow donor datasets of the National Marrow Donor Program, and underwent random-effects meta-analyses. HLA-B*07, B*14, and B*40 were overrepresented in AA, while A*02, A*68, and B*08 frequencies were similar to those of healthy donors (Figure 2). In 164 severe AA patients who were initially treated with horse antithymocyte globulin (hATG), cyclosporine, and eltrombopag between 2012 and 2018, 36 and 79 were positive and negative for HLA- monocytes, respectively, and 49 were not tested by HLA-flow. There was no significant difference in overall and complete response rates at six months among the three groups (Figure 3). Clonal evolution, defined as acquisition of abnormal bone marrow cytogenetics or morphology, especially high-risk evolution to chromosome 7 abnormalities, complex cytogenetics, or morphological MDS/AML, tended to be more frequent in patients with HLA- monocytes, compared to the other two groups, but the difference did not reach statistical significance. Clinical outcomes were also assessed according to the presence of specific HLA alleles in 400 severe AA patients who were treated with hATG-based initial immunosuppressive therapy from 2000 to 2018: there was no significant differences in probabilities of response and clonal evolution according to the alleles associated with somatic mutations. Our study revealed that somatic mutations in HLA genes in AA are broadly distributed, but some alleles are preferentially affected. Inconsistent with previous studies, we found that outcomes of therapy did not significantly correlate with HLA gene mutations or with distinct HLA alleles. Disclosures No relevant conflicts of interest to declare.

Seminoma component of mixed testicular germ cell tumor shows a higher incidence of loss of heterozygosity than pure-type seminoma

Using analysis of allelic loss (loss of heterozygosity [LOH]), we previously reported a putative progression pathway from germ cell neoplasia in situ (GCNIS) to seminoma and then to embryonal carcinoma in mixed-type testicular germ cell tumors. To identify the genetic backgrounds related to the progression of nonseminomatous germ cell tumor, patterns of LOH were studied in seminoma components in mixed tumors (18 cases), pure seminomas (20 cases), and coexisting GCNIS lesions. Each tumor was assessed for LOH at 22 polymorphic loci located on 12 chromosomal arms: 3q, 5q, 6p, 9p, 10q, 11p, 12p, 12q, 13q, 17p, 17q, and 18q. For all informative loci, the frequency of LOH in seminoma components in mixed tumors was significantly higher than that in pure seminomas (32% [96/302 loci] versus 19% [60/323 loci], P < .0001). The frequency of LOH in GCNIS lesions was not significantly different between the 2 tumor groups. The frequencies of LOH at chromosomes 6p and 10q were significantly higher in seminoma components in mixed tumors than in pure seminomas (P = .020 and P = .0041, respectively). Immunohistochemical analysis demonstrated a close association between the allelic status of the 10q23 locus and levels of phosphatase and tensin homolog deleted from chromosome 10 protein expression in seminoma (P = .00051). These data indicate that the seminoma, which has a potential to progress to nonseminomatous germ cell tumor, already exhibits several genetic changes including allelic losses of 6p and 10q, unlike pure seminoma.

Subtype-specific signaling pathways and genomic aberrations associated with prognosis of glioblastoma.

A high heterogeneity and activation of multiple oncogenic pathways have been implicated in failure of targeted therapies in glioblastoma (GBM). Using The Cancer Genome Atlas data, we identified subtype-specific prognostic core genes by a combined approach of genome-wide Cox regression and Gene Set Enrichment Analysis. The results were validated with 8 combined public datasets containing 608 GBMs. We further examined prognostic chromosome aberrations and mutations. In classical and mesenchymal subtypes, 2 receptor tyrosine kinases (RTKs) (MET and IGF1R), and the genes in RTK downstream pathways such as phosphatidylinositol-3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR), and nuclear factor-kappaB (NF-kB), were commonly detected as prognostic core genes. Classical subtype-specific prognostic core genes included those in cell cycle, DNA repair, and the Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathway. Immune-related genes were enriched in the prognostic genes showing negative promoter cytosine-phosphate-guanine (CpG) methylation/expression correlations. Mesenchymal subtype-specific prognostic genes were those related to mesenchymal cell movement, PI3K/Akt, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), Wnt/β-catenin, and Wnt/Ca2+ pathways. In copy number alterations and mutations, 6p loss and TP53 mutation were associated with poor and good survival, respectively, in the classical subtype. In the mesenchymal subtype, patients with PIK3R1 or PCLO mutations showed poor prognosis. In the glioma CpG island methylator phenotype (G-CIMP) subtype, patients harboring 10q loss, 12p gain, or 14q loss exhibited poor survival. Furthermore, 10q loss was significantly associated with the recently recognized G-CIMP subclass showing relatively low CpG methylation and poor prognosis. These subtype-specific alterations have promising potentials as new prognostic biomarkers and therapeutic targets combined with surrogate markers of GBM subtypes. However, considering the small number of events, the results of copy number alterations and mutations require further validations.

P169 Is it really homozygous? Identifying 6P loss in the HLA typing

Aim A peripheral blood sample for HLA typing was received in the lab for a prospective HCT patient. As with all HCT patients, SeCore™ Sequence-Based Typing and One Lambda LABType™ SSOP were performed. Sequence analysis performed using uTYPE results were A∗02:01, A∗02:01; B∗08:01, B∗08:01, C∗07:01, C∗07:01; DQB1∗03:02, DQB1∗03:02, DRB1∗04:04, 04:04. DPB1 at first glance was determined to be DPB1∗04:01, DPB1∗04:01. However, on closer inspection, the Z38 GSSP primer typed as DPB1∗10:01, and the Z39 GSSP primer typed as DPB1∗04:01. The LABType SSOP typing was A∗02, A∗03; B∗08, 08; C∗07, 07; DPB1∗04:01, 04:01; DQB1∗03, 03; and DRB1∗04:04, 04:04. The heterozygous A locus typing gave a clear indication that our sequencing had not picked up the complete typing. The B and C and DPB1 locus typings appeared homozygous, but there were a few false positive and high negative beads present, indicating the possible presence of B∗27, C∗02 and DPB1∗10:01. The DQB1 and DRB1 were concordant with the homozygous sequencing results, with no cut off adjustments necessary to obtain the typings. Methods SeCore™ Sequence-Based Typing and One Lambda LABType™ SSOP were performed. High Resolution typing was performed using LABType SSOP. Results Reexamining to the sequencing data, and inserting B∗27:05 for the B locus and C∗02:02 for the C locus into the allele comparator on uTYPE, very small double peaks could be seen at all of the points of mismatch for the B∗08:01, 27:05 typing, and C∗02:02, 07:01 typing. At this point, cytogenetics analysis performed on this patient, and if 6p loss had been detected. The laboratory confirmed that the patient’s leukemia cells exhibited partial 6p loss. Buccal swab sample was requested by the HLA lab to perform HLA typing. The buccal swab typing was A∗02:01p, 03:01; B∗08:01, 27:05p; C∗02:02p, 07:01p; DPB1∗04:01, 10:01; DRB1∗04:04, 04:04; DQB1∗03:02, 03:02. Conclusions This example confirms the necessity of performing two typing methods. It also demonstrates the key benefit of communication from the Cytogenetics Laboratory whenever 6p loss is detected in prospective HCT patients. In this case, DNA from a buccal swab was absolutely required for assurance of an accurate typing due to the homozygous DRB1 and DQB1 present in the patient.

Cytogenetic Abnormalities in Uveal Melanoma Based on Tumor Features and Size in 1059 Patients: The 2016 W. Richard Green Lecture

To determine the risks for altered cytogenetic profile based on melanoma features and size. Retrospective case series. A total of 1059 patients with uveal melanoma. Fine-needle aspiration biopsy (FNAB) of tumor for DNA amplification and whole genome array-based assay. Risk for cytogenetic abnormalities based on features and size: small (≤3 mm thickness), medium (>3-<8 mm), and large (≥8 mm). Of 1059 patients with uveal melanoma sampled for status of chromosomes 3, 6, and 8, comparison (normal [disomy] chromosomes 3, 6, and 8 vs. any 3, 6, or 8 abnormality) revealed differences in mean age (55 vs. 58 years, P= 0.018), ocular melanocytosis (1% vs. 5%, P= 0.027), mean visual acuity (VA) (20/30 vs. 20/50, P=0.011), poor VA (≤20/200) (9% vs. 15%, P= 0.041), ciliary body location (5% vs. 11%, P < 0.001), extramacular location (73% vs. 87%, P < 0.001), increased mean distance to optic disc (3.3 vs. 5.0 mm, P < 0.001) and foveola (3.1 vs. 4.7 mm, P < 0.001), and increased mean basal diameter (9.8 vs. 12.6 mm, P < 0.001) and thickness (3.8 vs. 5.9 mm, P < 0.001). Tumors classified as small, medium, and large showed abnormalities with loss of disomy of chromosomes 3 (35%/52%/65%), 6 (15%/34%/51%), and 8 (19%/41%/69%), respectively. By comparison (medium/large vs. small melanoma), the odds ratio (OR) included complete monosomy 3 (3.09, P < 0.001), partial monosomy 3 (1.44, P= 0.053), 6p gain (3.78, P < 0.001), 6q gain (1.37, P= 0.537), 6p loss (2.52, P= 0.410), 6q loss (12.61, P < 0.001), 8p gain (6.16, P < 0.001), 8p loss (6.04, P < 0.001), and 8q gain (4.87, P < 0.001). For chromosome 3 monosomy, the OR was highest for ciliary body location (8.17, P < 0.001), tumor thickness ≥8 mm (2.70, P < 0.001), tumor base ≥10 mm (2.59, P < 0.001), and age ≥60 years (1.83, P < 0.001). For chromosome 8p loss, the OR was highest for ciliary body location (53.91, P= 0.008), ocular melanocytosis (3.95, P= 0.038), and thickness ≥8 mm (5.14, P < 0.001), whereas for 8q gain, the OR was highest for ciliary body location (102.87, P= 0.001), thickness >8 mm (4.44, P < 0.001), and ocular melanocytosis (2.75, P= 0.049). Increasing melanoma size demonstrates greater cytogenetic alterations. Alterations in chromosome 8 show unique correlation with melanocytosis. This suggests that prompt management of small melanoma might reduce chromosomal instability and could improve overall patient survival.

Microarray Analysis of Formalin-Fixed Paraffin-Embedded (FFPET) and Frozen (FZT) Uveal Melanoma Detects Clinically Relevant Abnormalities, Including Chromosome 3 Loss of Heterozygosity

Molecular cytogenetic alterations, specifically monosomy 3, are strongly correlated with metastases and death in uveal melanoma (UVM). Although FISH can be used for the identification of monosomy 3, a subset of UVM exhibit only partial loss of chromosome 3 potentially missed with chromosome enumeration probes. Moreover, often limited material available can compromise detection of additional alterations with potential clinical relevance. Microarray analysis is an alternative method for the analysis of such specimens affording whole genome interrogation. In the current study we performed microarray to detect genomic changes in DNA from FFPET and FZT UVM samples (clinical trial NCT00952939). Of the available cases, 23.5% yielded DNA of sufficient quantity and quality to obtain interpretable microarray results, representing 28 patients. All but one case demonstrated significant abnormalities. Gains of 8q, consistent with an apparent i(8)(q10) karyotype, were the most common finding, seen in 21 patients. Monosomy 3 was detected by microarray in 15 cases. A single case showed partial loss of 3 (3q11.2q25.31), the only clinically significant abnormality detected in that case. All cases lacking chromosome 3 abnormalities showed a copy gain of 6p sharing a small 28.18 Mb region of overlap at 6p25.2p21.33. Additional findings included TERT and NEDD9 amplifications, and CDKN2A/B and LUM gene deletions. SNP analysis revealed three cases with unique regions of copy neutral LOH involving 5p15.33q35.3, 15q11.2q21.1 and one case with whole chromosome LOH for chromosomes 3, 4 and 6. The latter was detected in a FZT specimen, while the paired FFPET sample lacked evidence of LOH and showed monosomy 3, trisomy 4 and whole arm gains and losses of 6p and 6q, respectively, suggesting the presence of tumor heterogeneity. Microarray analysis has identified new recurrent abnormalities associated with UVM with potential relevance to UVM biology, diagnosis and prognosis. Conflict of Interest: Roger Schultz is an employee of Perkin Elmer.

Neurofibroma-Like Spindle Cell Melanoma

We present a case that highlights the use of two maneuvers useful in the diagnosis of spindle cell melanoma. A shave biopsy from the cheek of a 58-year-old man demonstrated a thin invasive melanoma of 0.3 mm thickness with a less than 2-mm-wide intra-epidermal component. Below this melanocytic lesion, but not contiguous with it, there was a transected S-100-positive and Melan-A-negative spindle cell proliferation. Upon re-excision, no residuum of conventional melanoma was identified, but a residual spindle cell neoplasm that was 4 mm in diameter, nodular, well-circumscribed, cytologically bland, and S-100 positive was noted. At our consensus conference, our group favored neurofibroma but agreed that spindle cell melanoma could not be excluded based on histopathologic features alone. To further address the differential diagnosis, we performed CD34 staining that demonstrated lack of a CD34 fingerprint. We also completed array-based comparative genomic hybridization, which demonstrated gain of chromosome 6p, loss of 6p and gain of 7. These two methods of analysis support a diagnosis of spindle cell melanoma.

Genetic alterations in a primary medullary thyroid carcinoma and its lymph node metastasis in a patient with 15 years follow-up

BackgroundAssociation between DNA alterations and clinical parameters as recurrence, survival or prognosis has been found in a variety of tumors. A clear association between Medullary Thyroid Carcinoma (MTC) and RET oncogene mutation has been accepted. Specifically M918T RET mutation represents the main genetic event in most cases of sporadic MTC (SMTC) and limited chromosomal alterations analyses have been performed.MethodsIn the present work, a comparative genomic hybridization (CGH) study was performed using DNA from a primary tumor in a M918T RET mutation-positive SMTC patient and from its lymph node metastasis to investigate additional genetic alterations. We studied a patient with 15 years of follow-up and persistence of disease, confirmed by periodical elevated serum calcitonin (CT) levels.ResultsOnly 3 chromosomal imbalances were identified in the primary tumor, gain of 18p, and loss of 6p and 16p region, whereas 25 chromosomal imbalances were identified in the metastasis (9 gains and 16 losses).ConclusionThe chromosomal changes 6p-, 16p-, 18p + could determine in part the oncogenic phenotype in the primary M918T RET positive tumor and probably related to persistence of high serum CT levels in this patient. The additional chromosomal changes observed could be related to the metastasis phenotype. We suggest that some genes mapped at 6p, 16p and 18p chromosomal regions, could act as genes associated to cancer and could be related to persistent SMTC and good prognosis.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1720753793691097

Oncogenetic tree modeling of human hepatocarcinogenesis

Classical comparative genomic hybridization (CGH) has been used to identify recurrent genomic alterations in human HCC. As hepatocarcinogenesis is considered as a stepwise process, we applied oncogenetic tree modeling on all available classical CGH data to determine occurrence of genetic alterations over time. Nine losses (1p, 4q, 6q, 8p, 9p, 13q, 16p, 16q and 17p) and ten gains (1q, 5p, 6p, 7p, 7q, 8q, 17q, 20p, 20q and Xq) of genomic information were used to build the oncogenetic tree model. Whereas gains of 1q and 8q together with losses of 8p formed a cluster that represents early etiology-independent alterations, the associations of gains at 6q and 17q as well as losses of 6p and 9p were observed during tumor progression. HBV-induced HCCs had significantly more chromosomal aberrations compared to HBV-negative tumors. Losses of 1p, 4q and 13q were associated with HBV-induced HCCs, whereas virus-negative HCCs showed an association of gains at 5p, 7, 20q and Xq. Using five aberrations that were significantly associated with tumor dedifferentiation a robust progression model of stepwise human hepatocarcinogensis (gain 1q → gain 8q → loss 4q → loss 16q → loss 13q) was developed. In silico analysis revealed that protumorigenic candidate genes have been identified for each recurrently altered hotspot. Thus, oncogenic candidate genes that are coded on chromosome arms 1q and 8q are promising targets for the prevention of malignant transformation and the development of biomarkers for the early diagnosis of human HCC that may significantly improve the treatment options and thus prognosis of HCC patients.

Oncogenetic tree modeling of human hepatocarcinogenesis

Classical Comparative Genomic Hybridization (CGH) has been used to identify recurrent genomic alterations in human HCC. Since hepatocarcinogenesis is considered a step-wise process, we applied oncogenetic tree modeling on all currently available classical CGH data to determine occurrence of genetic alterations over time. Nine losses (1p, 4q, 6q, 8p, 9p, 13q, 16p, 16q, 17p) and ten gains (1q, 5p, 6p, 7p, 7q, 8q, 17q, 20p, 20q, Xq) of genomic information were detected in more than 10% of cases and were selected to build the oncogenetic tree model. Whereas gains of 1q and 8q together with losses of 8p formed a cluster that represents early etiology-independent alterations, the associations of gains at 6q and 17q as well as losses of 6p and 9p were observed later during tumor progression. HBV-induced HCCs had significantly more chromosomal aberrations compared to HBV-negative tumors. Losses of 1p, 4q, and 13q were typically associated in HBV-induced HCCs, while virus-negative HCCs showed an association of gains at 5p, 7, 20q, and Xq. Using five aberrations that were significantly associated with tumor dedifferentiation a robust progression model of step-wise human hepatocarcinogensis (gain 1q → gain 8q → loss 4q → loss 16q → loss 13q) was developed. In silico analysis revealed that protumorigenic candidate genes have been identified for each recurrently altered hotspot. Thus, oncogenic candidate genes that are coded on chromosome arms 1q and 8q are promising targets for the prevention of malignant transformation and the development of biomarkers for the early diagnosis of human HCC that may significantly improve the treatment options and thus prognosis of HCC patients.

An Investigation into a Panel of Isogenic Taxane Resistant Breast Cancer Cell Lines Using Transcriptomic and Genomic Methods.

Abstract Background: Taxanes are a powerful chemotherapeutics used to treat breast cancer. Taxane resistance has become increasingly more prevalent in the clinic; This problem is exacerbated by a lack of understanding of mechanisms underlying taxane resistance.Materials and Methods: 3 isogenic breast cancer cell-line panels: paclitaxel resistant (PACR) MDA-MB-231s/ZR75-1s and docetaxel resistant (DOCR) ZR75-1s were derived by culturing cells in incrementally escalating doses of taxanes over time. MDA-MB-231: native, 5nM, 25nM and 100nM PACR cells, ZR75-1: native, and 5nM, 25nM, and 50nM PACR and ZR75-1: native, and 5nM, 25nM and 50nM DOCR cells were analysed. RNA from native and PACR MDA-MB-231 cell lines was analysed on the Illumina® human ref.8V2 chip. Data were analysed using IlluminaGui. For aCGH, DNA was extracted from MDA-MB-231 PACR, ZR75-1 PACR and DOCR and respective parental lines, labelled and hybridised to a tiling path BAC microarray (resolution ∼50kb). Each cell-line was tested against reference samples of DNA from pooled female blood. Taxane resistant cells were tested against their parental cell line.Results: Illumina data demonstrated that resistance to increasing doses of paclitaxel is associated with sequential increases in mRNA dysregulation in MDA-MB-231 cells.A number of comparisons between the illumina data of native and the PACR cells were made using a p value of P≤0.001. The first comparison made using the illumina data, was between the 5nM PACR cells and the native cell lines: 25 significantly up or downregulated genes were identified. When the 25nM PACR cells were compared to the native cell lines, 225 genes were found to be significantly up or downregulated. Finally, when 100nM PACR cells were compared to native cells, 425 genes were found to be significantly up or downregulated. A group of genes identified that were common to multiple paclitaxel resistant groups included candidates such as YY1, AURKA and CCND2. aCGH has identified significant differences in patterns of copy number loss, gain and amplification in the taxane resistant cell lines, including chromosome loss of 6p and gain of 2p. Illumina and aCGH data were overlaid using specially designed algorithms.Discussion: Both aCGH and expression arrays highlight a progressive acquisition of molecular and transcriptomic alterations in isogenic breast cancer cell lines resistant to increasing doses of taxanes. Further mapping of transcriptomic and genomic profiles will enable identification of key molecular targets for restoration of taxane sensitivity. Genes upregulated in taxane resistant clones identified in the illumina experiment, in particular those whose upregulation is underpinned by DNA copy number gains in resistant cells, may represent genes which may be targeted with inhibitors to test potential candidates for reversing taxane resistance. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1134.

Characterization of PacMetUT1, a recently isolated human prostate cancer cell line

Existing prostate cancer cell lines have limitations. Cells were characterized using Western blotting, immunohistochemistry, invasion into Matrigel, and by studying xenograft tumors. We describe a cell line (PacMetUT1) isolated from a lymph node of a 57-year-old male with prostate cancer. Compared to existing prostate cancer cell lines, the growth rate of PacMetUT1 xenograft tumors is slower with tumors occurring at injection sites and with metastases to lung and liver. Androgen receptor (AR) was detected in vivo by Western blotting and the cells responded to methyltrienolone (R1881). PacMetUT1 cells are more invasive in Matrigel than DU-145, PC-3, and LNCaP cells, and showed greater anchorage-independent growth in soft agar. The cells do not express prostate specific antigen (PSA) in vitro or in xenografts. However, the green fluorescent protein (GFP) gene was introduced and stably expressed in PacMetUT1 cells, allowing tumor imaging in vivo. Xenograft tumors show epithelial features and are positive for keratin, epithelial membrane antigen, EGF receptor, and E cadherin. In contrast, fibroblast markers vimentin, desmin, and Factor VIII, were negative. Karyotyping showed losses of 6p, 7q, 8p, 18q, and 22q, and gains of 8q and 9q; additional genetic material was observed at 2q and 12p. The PacMetUT1 cell line allows metastases to be assessed using a single animal model. Because of its slower growth, PacMetUT1 more closely mimics the human disease. Studies of tumor progression or metastasis can be conducted over a longer period of time.

Genomic copy number alterations in non-small cell lung cancers identified using CBS and MSA

7695 Background: Lung cancer is the leading cause of cancer death in both men and women with a 5-yr survival rate of 15.5%. Previous studies have begun to characterized genomic copy number alterations of non-small-cell lung CA using array CGH and SNP arrays. We have used aCGH using our 1MB BAC arrays and two algorthims for making copy number alteration (CNA) determinations. We have also pursued the exact copy number gains and losses of several genes using Q-PCR. Methods: Genomic DNA from fresh frozen tumors of 27 patients with NSCLC. We performed aCGH using 1MB Arrays. We used CBS, and MSA to identify regions of CNA. We further pursued several genes of interest (including HRAS, CRK, and CDC42) identified using Q-PCR. Unsupervised hierarchical clustering was performed to look for distinct subgroups. Significant Analysis of Microarrays (SAM) was applied to identify the association between CNAs clinical parameters including tumor subtype, gender, lymph node involvement, tumor stage, and overall survival. Results: 240 regions of amplification and 181 regions of deletions were found, and included all previously published regions implicated in lung cancer. CNAs in &gt; 70% of tumors included amplifications in 1q, 3q, 5p, 6p, 11p, 16q, 20q, and Xq, and deletions in 1p, 8p and 13q. We verified CNAs of HRAS, CRK and CDC42 using Q-PCR. Hierarchical clustering revealed 2 subgroups: one with amplifications in 2q, 4p, 4q, 8q, 21q, 15q, and 16p, and the other with amplifications in 3q, and 5q. These were confirmed by supervised SAM analysis. Using SAM we found that gain of 2q, 4p and 10q, and loss of 16p and 19q were significantly present in adenocarcinomas. (q = 0, FDR = 0%). Gain of 10q, and loss of 6p and 14q were associated with female gender. (q = 0, FDR = 0%). Conclusions: We used aCGH to identify CNAs that characterize non-small cell lung CA tumors with the aim of finding key regions which may harbor important oncogenes and tumor suppressors. Several regions of CNA have been identified, several of which have been associated with clinical parameters. Because much heterogeneity exists in non-small-cell lung tumors, we have demonstrated that clustering analysis is useful in identifying subtypes which may possess prognostic and therapeutic significance. No significant financial relationships to disclose.