Molecularly Heterogeneous Research Articles

Morphologic and Molecular Heterogeneity of High-grade Serous Carcinoma Precursor Lesions.

Serous tubal intraepithelial carcinoma (STIC) is the fallopian tube precursor lesion for most cases of pelvic high-grade serous carcinoma (HGSC). To date, the morphologic, molecular, and clinical heterogeneity of STIC and a less atypical putative precursor lesion, termed serous tubal intraepithelial lesion, has not been well characterized. Better understanding of precursor heterogeneity could impact the clinical management of women with incidental STICs (without concurrent carcinoma) identified in cases of prophylactic or opportunistic salpingectomy. This study analyzed morphologic and molecular features of 171 STICs and 21 serous tubal intraepithelial lesions. We assessed their histologic features, Ki-67 and p53 staining patterns, and genome-wide DNA copy number alterations. We classified all precursor lesions into 2 morphologic subtypes, one with a flat surface (Flat) and the other characterized by budding, loosely adherent, or detached (BLAD) morphology. On the basis of pathology review by a panel of 8 gynecologic pathologists, we found 87 BLAD, 96 Flat, and 9 indeterminate lesions. As compared with Flat lesions, BLAD lesions were more frequently diagnostic of STIC ( P <0.0001) and were found concurrently with HGSC ( P <0.0001). BLAD morphology was also characterized by higher Ki-67 proliferation index ( P <0.0001), presence of epithelial stratification ( P <0.0001), and increased lymphocyte density ( P <0.0001). BLAD lesions also exhibited more frequent DNA copy number gain/amplification at the CCNE1 or CMYC loci canonical to HGSCs ( P <0.0001). Both BLAD morphology and STIC diagnoses are independent risk factors for an elevated Ki-67 proliferation index. No correlation was observed between BLAD and Flat lesions with respect to patient age, presence of germline BRCA1/2 mutation, or p53 staining pattern. These findings suggest that tubal precursor lesions are morphologically and molecularly heterogeneous, laying the foundation for further studies on the pathogenesis of HGSC initiation and identifying histologic features predictive of poor patient outcomes.

Chimeric Antigen Receptor-T Cell and Oncolytic Viral Therapies for Gastric Cancer and Peritoneal Carcinomatosis of Gastric Origin: Path to Improving Combination Strategies

Precision immune oncology capitalizes on identifying and targeting tumor-specific antigens to enhance anti-tumor immunity and improve the treatment outcomes of solid tumors. Gastric cancer (GC) is a molecularly heterogeneous disease where monoclonal antibodies against human epidermal growth factor receptor 2 (HER2), vascular endothelial growth factor (VEGF), and programmed cell death 1 (PD-1) combined with systemic chemotherapy have improved survival in patients with unresectable or metastatic GC. However, intratumoral molecular heterogeneity, variable molecular target expression, and loss of target expression have limited antibody use and the durability of response. Often immunogenically “cold” and diffusely spread throughout the peritoneum, GC peritoneal carcinomatosis (PC) is a particularly challenging, treatment-refractory entity for current systemic strategies. More adaptable immunotherapeutic approaches, such as oncolytic viruses (OVs) and chimeric antigen receptor (CAR) T cells, have emerged as promising GC and GCPC treatments that circumvent these challenges. In this study, we provide an up-to-date review of the pre-clinical and clinical efficacy of CAR T cell therapy for key primary antigen targets and provide a translational overview of the types, modifications, and mechanisms for OVs used against GC and GCPC. Finally, we present a novel, summary-based discussion on the potential synergistic interplay between OVs and CAR T cells to treat GCPC.

Integrative genetic and genomic networks identify microRNA associated with COPD and ILD

Chronic obstructive pulmonary disease (COPD) and interstitial lung disease (ILD) are clinically and molecularly heterogeneous diseases. We utilized clustering and integrative network analyses to elucidate roles for microRNAs (miRNAs) and miRNA isoforms (isomiRs) in COPD and ILD pathogenesis. Short RNA sequencing was performed on 351 lung tissue samples of COPD (n = 145), ILD (n = 144) and controls (n = 64). Five distinct subclusters of samples were identified including 1 COPD-predominant cluster and 2 ILD-predominant clusters which associated with different clinical measurements of disease severity. Utilizing 262 samples with gene expression and SNP microarrays, we built disease-specific genetic and expression networks to predict key miRNA regulators of gene expression. Members of miR-449/34 family, known to promote airway differentiation by repressing the Notch pathway, were among the top connected miRNAs in both COPD and ILD networks. Genes associated with miR-449/34 members in the disease networks were enriched among genes that increase in expression with airway differentiation at an air–liquid interface. A highly expressed isomiR containing a novel seed sequence was identified at the miR-34c-5p locus. 47% of the anticorrelated predicted targets for this isomiR were distinct from the canonical seed sequence for miR-34c-5p. Overexpression of the canonical miR-34c-5p and the miR-34c-5p isomiR with an alternative seed sequence down-regulated NOTCH1 and NOTCH4. However, only overexpression of the isomiR down-regulated genes involved in Ras signaling such as CRKL and GRB2. Overall, these findings elucidate molecular heterogeneity inherent across COPD and ILD patients and further suggest roles for miR-34c in regulating disease-associated gene-expression.

Integrated molecular and pharmacological characterization of patient-derived xenografts from bladder and ureteral cancers identifies new potential therapies.

BackgroundMuscle-invasive bladder cancer (MIBC) and upper urinary tract urothelial carcinoma (UTUC) are molecularly heterogeneous. Despite chemotherapies, immunotherapies, or anti-fibroblast growth factor receptor (FGFR) treatments, these tumors are still of a poor outcome. Our objective was to develop a bank of patient-derived xenografts (PDXs) recapitulating the molecular heterogeneity of MIBC and UTUC, to facilitate the preclinical identification of therapies.MethodsFresh tumors were obtained from patients and subcutaneously engrafted into immune-compromised mice. Patient tumors and matched PDXs were compared regarding histopathology, transcriptomic (microarrays), and genomic profiles [targeted Next-Generation Sequencing (NGS)]. Several PDXs were treated with chemotherapy (cisplatin/gemcitabine) or targeted therapies [FGFR and epidermal growth factor (EGFR) inhibitors].ResultsA total of 31 PDXs were established from 1 non-MIBC, 25 MIBC, and 5 upper urinary tract tumors, including 28 urothelial (UC) and 3 squamous cell carcinomas (SCCs). Integrated genomic and transcriptomic profiling identified the PDXs of three different consensus molecular subtypes [basal/squamous (Ba/Sq), luminal papillary, and luminal unstable] and included FGFR3-mutated PDXs. High histological and genomic concordance was found between matched patient tumor/PDX. Discordance in molecular subtypes, such as a Ba/Sq patient tumor giving rise to a luminal papillary PDX, was observed (n=5) at molecular and histological levels. Ten models were treated with cisplatin-based chemotherapy, and we did not observe any association between subtypes and the response. Of the three Ba/Sq models treated with anti-EGFR therapy, two models were sensitive, and one model, of the sarcomatoid variant, was resistant. The treatment of three FGFR3-mutant PDXs with combined FGFR/EGFR inhibitors was more efficient than anti-FGFR3 treatment alone.ConclusionsWe developed preclinical PDX models that recapitulate the molecular heterogeneity of MIBCs and UTUC, including actionable mutations, which will represent an essential tool in therapy development. The pharmacological characterization of the PDXs suggested that the upper urinary tract and MIBCs, not only UC but also SCC, with similar molecular characteristics could benefit from the same treatments including anti-FGFR for FGFR3-mutated tumors and anti-EGFR for basal ones and showed a benefit for combined FGFR/EGFR inhibition in FGFR3-mutant PDXs, compared to FGFR inhibition alone.

DIPG-35. Personalized treatment for molecularly heterogeneous Diffuse midline glioma, H3 k27-altered Paediatric case

Abstract Diffuse midline glioma, H3 k27-altered (DMG) is a type of Paediatric- type diffuse high grade gliomas according to the 2021 WHO CNS tumors Classification. Diffuse intrinsic pontine glioma (DIPG) is another acceptable related term when it located in the pons with fatal prognosis. The combination of H3K27M with BRAF V600 mutations rarely reported in DMG although more commonly in Paediatric-type low grade gliomas (Diffuse low-grade glioma, MAPK pathway-altered). We present a twenty-month-old boy, previously healthy, presented with 2 weeks history of unsteady gait, drooling, cranial nerves palsy MRI imaging showed diffuse pontine mass with classic radiological features of DIPG. 2.6 x 1.6 x 3.2 (AP x TV x CC) with no evidence for spinal metastases. Patient underwent right retro sigmoid approach and open biopsy of lesion he received focal Radiation Therapy 54GY/30fx as stander of care of DIPG with mild neurogical improvement Pathological &amp; molecular Diagnosis was Diffuse midline glioma, H3 k27-altered with Co-occurrence BRAF V600E mutation. Two months after end of radiation, he presented with vomiting, and neurological deterioration with new right-side hemiplegia. Imaging studies showed interval increase in the pontine lesion with increased edema causing narrowing of the fourth ventricle, no active hydrocephalus. He was started on combination therapy BRAF inhibitor Dabrafenib and MEK Inhibitor Trametinib as maintenance therapy the patient gradually showed Marked neurological and clinical improvement. A 6-month MRI after start of targeted therapy showed favorable treatment response with complete resolution of the previous diffusion restriction, reduced tumor volume on MR perfusion ,reduced perilesional edema otherwise almost stabilization of nonenhancing pontine lesion. The poor prognosis of recurrent DIPG is well known but our patient is clinically and radiologically stable with excellent quality of life and well tolerating the therapy . Our case show that personalized treatment approach that address molecular heterogeneity of H3K27M glioma are safe and feasible

Molecular classification of aggressive lymphomas-past, present, future.

Aggressive large B-cell lymphomas (LBCLs) represent a frequent but clinically and molecularly heterogeneous group of tumors. Technological advances over the last decades prompted the development of different classification schemas to either sharpen diagnoses, dissect molecular heterogeneity, predict outcome, or identify rational treatment targets. Despite increased diagnostic precision and a noticeably improved molecular understanding of these lymphomas, clinical perspectives of patients largely remain unchanged. Recently, finished comprehensive genomic studies discovered genetically defined LBCL subtypes that predict outcome, provide insight into lymphomagenesis, and suggest rational therapies with the hope of generating patient-tailored treatments with increased perspective for patients in greatest need. Current and future efforts integrate multiomics studies and/or leverage single-cell technologies and will provide us with an even more fine-grained picture of LBCL biology. Here, we highlight examples of how high-throughput technologies aided in a better molecular understanding of LBCLs and provide examples of how to select rationally designed targeted treatment approaches that might personalize LBCL treatment and eventually improve patients' perspective in the near future.

Re-evaluating tumors of purported specialized prostatic stromal origin reveals molecular heterogeneity, including non-recurring gene fusions characteristic of uterine and soft tissue sarcoma subtypes.

Tumors of purported specialized prostatic stromal origin comprise prostatic stromal sarcomas (PSS) and stromal tumors of uncertain malignant potential (STUMP). Prior studies have described their clinicopathologic characteristics, but the molecular features remain incompletely understood. Moreover, these neoplasms are morphologically heterogeneous and the lack of specific adjunctive markers of prostatic stromal lineage make precise definition more difficult, leading some to question whether they represent a specific tumor type. In this study, we used next-generation DNA and RNA sequencing to profile 25 primary prostatic mesenchymal neoplasms of possible specialized prostatic stromal origin, including cases originally diagnosed as PSS (11) and STUMP (14). Morphologically, the series comprised 20 cases with solid architecture (11 PSS and 9 STUMP) and 5 cases with phyllodes-like growth pattern (all STUMP). Combined DNA and RNA sequencing results demonstrated that 19/22 (86%) cases that underwent successful sequencing (either DNA or RNA) harbored pathogenic somatic variants. Except for TP53 alterations (6 cases), ATRX mutations (2 cases), and a few copy number variants (-13q, -14q, -16q and +8/8p), the findings were largely nonrecurrent. Eight gene rearrangements were found, and 4 (NAB2-STAT6, JAZF1-SUZ12, TPM3-NTRK1 and BCOR-MAML3) were useful for reclassification of the cases as specific entities. The present study shows that mesenchymal neoplasms of the prostate are morphologically and molecularly heterogeneous and include neoplasms that harbor genetic aberrations seen in specific mesenchymal tumors arising in other anatomic sites, including soft tissue and the uterus. These data suggest that tumors of purported specialized prostatic stromal origin may perhaps not represent a single diagnostic entity or specific disease group and that alternative diagnoses should be carefully considered.

Variability in the Munc13-1 content of excitatory release sites.

The molecular mechanisms underlying the diversity of cortical glutamatergic synapses are still incompletely understood. Here, we tested the hypothesis that presynaptic active zones (AZs) are constructed from molecularly uniform, independent release sites (RSs), the number of which scales linearly with the AZ size. Paired recordings between hippocampal CA1 pyramidal cells and fast-spiking interneurons in acute slices from adult mice followed by quantal analysis demonstrate large variability in the number of RSs (N) at these connections. High-resolution molecular analysis of functionally characterized synapses reveals variability in the content of one of the key vesicle priming factors - Munc13-1 - in AZs that possess the same N. Replica immunolabeling also shows a threefold variability in the total Munc13-1 content of AZs of identical size and a fourfold variability in the size and density of Munc13-1 clusters within the AZs. Our results provide evidence for quantitative molecular heterogeneity of RSs and support a model in which the AZ is built up from variable numbers of molecularly heterogeneous, but independent RSs.

Abstract PS18-05: Using blueprint to elucidate the molecular heterogeneity of triple negative breast cancers

Abstract Background: Triple negative breast cancers (TNBC) are more aggressive, have a worse prognosis, and few targeted therapies compared to other BC subtypes. TNBC is molecularly heterogeneous, with at least 4 distinct subtypes: basal-like immune-activated (BLIA), basal-like immunosuppressed (BLIS), luminal androgen receptor (LAR), and mesenchymal (MES). The molecular subtyping gene signature, BluePrint (BP), classifies breast tumors into Luminal, HER2, or Basal subtype based on the assessment of downstream signaling pathways and independently of IHC expression. Compared to IHC-defined TNBC, a higher frequency of BLIS or BLIA subtypes and fewer LAR or MES tumors were reported in BP-defined Basal tumors. To advance our understanding of TNBC heterogeneity, we evaluated the relationship between gene expression signatures, TNBC subtype and BluePrint, in IHC-defined TNBC. Methods: The FLEX registry (NCT03053193) is an ongoing, prospective study evaluating primary tumors from stage I-III BC patients who receive the risk of distant recurrence gene signature, MammaPrint (MP), and BP testing and consent to clinically annotated full transcriptome data collection. This analysis includes 204 IHC-defined TNBC patients. TNBC subtypes BLIA, BLIS, LAR, and MES were derived using an adjusted version of the Burstein centroid signature. BP classified patient samples into Luminal, HER2, and Basal subtypes. A proportion of tumors may exhibit a secondary but less pronounced activated pathway or BP subtype. Therefore, each BP subtype was divided into single activated or mixed subtype based on BP indices. Results: Of 204 TNBC tumors, 84% were classified as Basal by BP, most of which were BLIS (65%), followed by BLIA (22%), with a low frequency of MES (8%) and LAR (5%) subtypes (Table). Approximately 14% of TNBCs were reclassified as Luminal by BP, most of which were LAR (76%), whereas 24% were MES. Clustering analysis revealed similar gene expression profiles between Basal-BLIS and Basal-BLIA tumors. Interestingly, the transcriptional profile of Basal-MES and Basal-LAR tumors were similar to Luminal-MES and Luminal-LAR tumors. BP Basal indices distinguished between different TNBC subtypes. The Basal pathway was predominantly activated in 90% of BP Basals (single activated tumors), most of which were either BLIS or BLIA (96%), whereas 10% of BP Basals were mixed subtype and more likely to classify as LAR (53%) or MES (35%). Approximately 25% of the BP Basal gene signature overlapped with the TNBC subtype gene signature. Expression of 18 and 12 genes out of 28 genes that make up the BP basal signature were significantly different in Basal-BLIA/BLIS compared to LAR or MES, respectively (P &amp;lt; 0.05). PRR15 and CAPN13 were significantly differentially expressed between LAR and MES within Basals. Conclusion: BP reclassified a subgroup of TNBC tumors to Luminal, explaining the discrepancy in the distribution of TNBC subtypes between IHC-defined TNBC and BP Basal tumors. Furthermore, BP indices distinguished between single activated and mixed subtypes, which correlated with different TNBC subtypes. These data suggest that molecular classification by BP adds further precision in classifying TNBC patients and sheds new light on the heterogeneity of these tumors. These findings have clinical implications in stratifying patients and identifying successful targeted treatment options. Future studies are warranted to investigate treatment response and prognosis in these molecular subgroups. BasalLuminalHER2TotalBLIA370037BLIS11100111LAR1022335MES147021Total172 (84.3%)29 (14.2%)3 (1.5%)204 (100%) Citation Format: Virginia G. Kaklamani, Cathy Graham, Karen L. Tedesco, Abirami Sivapiragasam, Jennifer A. Crozier, Apurva N. Shah, Yuan Yuan, Josien Haan, Andrea Menicucci, Michelle L. Bolner, Shiyu Wang, Lorenza Mittempergher, Erin Yoder, William Audeh, FLEX Investigators' Group. Using blueprint to elucidate the molecular heterogeneity of triple negative breast cancers [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-05.

Abstract IA07: Molecular classification of T-cell lymphomas

Abstract Peripheral T-cell lymphomas (PTCLs) represent a clinically, histologically, and molecularly heterogeneous group of non-Hodgkin lymphomas derived from mature post-thymic T cells. Among them, the most common in Western countries is PTCL, not otherwise specified (NOS), accounting for approximately 30% of all PTCLs. Combining whole-exome and deep targeted-capture sequencing of 133 cases, we delineated the entire picture of genetic alterations in PTCL, NOS. Of note is the identification of a previously undescribed molecular subtype characterized by TP53 and/or CDKN2A mutations and deletions in PTCL, NOS without showing a T follicular helper cell phenotype. This subtype exhibited different prognosis and unique genetic features, including extensive chromosomal instability, which preferentially affected molecules involved in immune escape and transcriptional regulation. Among PTCLs, the most common entity in Japan is adult T-cell leukemia/lymphoma (ATL), which is an aggressive peripheral T-cell lymphoma associated with human T-cell leukemia virus type-1 (HTLV-1) infection. We previously carried out an integrated molecular study, in which whole-exome, transcriptome, and targeted resequencing, as well as array-based copy number analysis, were performed. We found recurrent genetic alterations in T-cell receptor/NF-κB signaling, T-cell trafficking, and other T cell-related pathways as well as immunosurveillance. Although our previous study discovered many driver mutations and copy number alterations, the whole-genome landscape of ATL still remains elusive. To this end, we have recently performed whole-genome sequencing (WGS) of 155 ATL samples, with a median depth of 95-fold for tumor samples. WGS presented a substantially different overview of driver alterations compared with WES: most driver genes were affected by more than one classes of somatic alterations, including structural variations (SV) and noncoding mutations. We integrated these genetic drivers using non-negative factorization clustering and identified several molecular subsets with distinct clinical features, including a leukemic subtype characterized by PLCG1 and STAT3 mutations. In addition, we newly identified several putative driver SVs and noncoding mutations (such as NOTCH1 truncations and mutations involving NFKBIZ 3′-untraslated region). Several types of SVs were linked to distinct alterations, suggesting their etiology, including associations of TP53 disruption with inversions and translocations, and inactivation of immune-related molecules with deletions. Our WGS analysis not only identifies novel somatic alterations, but also extends the overview of ATL genome, which can lead to future improvement of patient management. Taken together, our findings provide novel insights into genetic and molecular heterogeneity in PTCLs, which should help to devise a novel molecular classification and to exploit a new therapeutic strategy for these malignancies. Citation Format: Keisuke Kataoka. Molecular classification of T-cell lymphomas [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 IA07.

Central nervous system high grade neuroepithelial tumor with BCOR immunopositivity: Is there a molecular heterogeneity?

Central nervous system high grade neuroepithelial tumor - BCOR altered is a newly defined entity which is characterised by internal tandem duplication (ITD) in exon 15 of BCOR. These tumors resemble high grade glioma histologically and exhibit BCOR immunopositivity. However, recently fusions of BCOR are also described in CNS lower grade gliomas, thus questioning the sensitivity and specificity of BCOR immunohistochemistry for identification of BCOR-ITD. We describe four cases of high grade neuroepithelial tumor with BCOR immunopositivity which were diagnosed over a period of one year at our institute. Amongst these, only one tumor revealed BCOR-ITD on sequencing. SATB2 immunopositivity which is a sensitive marker of BCOR-ITD, BCOR fusions and YWHAE fusions was noted in three out of four cases. Our study suggests that BCOR immunopositive CNS high grade tumors are molecularly heterogeneous and could harbour genetic alterations other than BCOR-ITD.

Stem-Mesenchymal Signature Cell Genes Detected in Heterogeneous Circulating Melanoma Cells Correlate With Disease Stage in Melanoma Patients.

During the process of metastasis, cancer cells dissociate from primary tumors, migrate to distal sites, and finally colonize, eventually leading to the formation of metastatic tumors. These cancer cells, defined circulating tumor cells (CTCs) spreading through the blood stream, may develop metastatic lesions or remain dormant. Some emerging clinical evidence supports that some tumor cells may possess metastatic properties already in the earlier stages of tumorigenesis. Because the initiation and progression of vertical growth in human melanoma is fundamental to the notion of tumor virulence and progression, we decided to immune-magnetic collect and molecularly characterize circulating melanoma cells (CMCs) from melanoma patients AJCC staged = pT1b (i.e., transition from radial to vertical phase). CMCs are phenotypically and molecularly heterogeneous, thus we performed a “home-made Liquid-Biopsy,” by targeting the melanoma-associated-antigen, MCAM/MUC18/CD146, and/or the melanoma-initiating marker, ABCB5. We assessed a biomarker qualitative expression panel, contemplating the angiogenic-potential, melanoma-initiating and melanoma-differentiation drivers, cell-cell adhesion molecules, matrix-metallo-proteinases, which was performed on three enriched subpopulations from a total of 61 blood-samples from 21 melanoma patients. At first, a significant differential expression of the specific transcripts was documented between and within the CMC fractions enriched with MCAM-, ABCB5-, and both MCAM/ABCB5-coated beads, when analyzing two distinct groups: early AJCC- (stage I–II) and advanced- staged patients (stage II–IV). Moreover, in the early-AJCC staged-group, we could distinguish “endothelial,” CD45–MCAM+ enriched-, “stem” S-CMCs, CD45–ABCB5+ enriched- and a third hybrid bi-phenotypic CD45–MCAM+/ABCB5+ enriched-fractions, due to three distinct gene-expression profiles. In particular, the endothelial-CMCs were characterized by positive expression of genes involved in migration and invasion, whilst the stem CMC-fraction only expressed stem and differentiation markers. The third subpopulation isolated based on concurrent MCAM and ABCB5 protein expression showed an invasive phenotype. All three distinct CMCs sub-populations, exhibited a primitive, “stem-mesenchymal” profile suggesting a highly aggressive and metastasizing phenotype. This study confirms the phenotypic and molecular heterogeneity observed in melanoma and highlights those putative genes involved in early melanoma spreading and disease progression.

Molecularly Heterogeneous Structure of a Nonionic Deep Eutectic Solvent Composed of N-Methylacetamide and Lauric Acid.

Deep eutectic solvents (DES) are a new class of designer solvents with a wide range of possible applications. DESs are typically made from an ionic compound and a hydrogen bond donor, but lately DESs made of nonionic compounds with interesting properties have started to appear. This study focuses on describing the structure, dynamics, and interactions of a nonionic DES composed of N-methylacetamide (NMA) and lauric acid (LA). Linear IR and time-resolved IR experiments show that LA-NMA DES is the result of hydrogen bond interaction between the two molecular components, though the interaction energy is only favored by ∼1 kJ/mol with respect to the pure components. The IR experiments also reveal the presence of molecular heterogeneities produced by the nanosegregation of polar and nonpolar domains formed by N-methylacetamide and lauric acid, respectively. In addition, it is observed that the hydrogen bond interaction between components occurs at the interface of the two distinct polarity domains. The proposed molecular picture is also supported by the observation of nanodomains with the approximate size of two lauric acids via small-angle X-ray scattering.

Single-Cell Mutational Profiling of Clonal Evolution in De Novo AML during Therapy and Relapse

Background: De novo acute myeloid leukemia (AML) is a molecularly heterogeneous disorder with clinically variable outcomes. Recent studies on the mutational landscape of AML have been informative in better stratifying risk of relapse. However, bulk sequencing techniques have been limited in their ability to delineate the true complexity of tumoral molecular heterogeneity and allow for efficient identification of drug resistant subclones. Here, we applied high-throughput single cell sequencing technique to identify patterns of clonal heterogeneity and evolution in longitudinal samples from patients with AML undergoing induction chemotherapy.Methods: Matched diagnosis, remission, and relapse samples were examined for 20 de novo AML cases including 15 relapsed and 5 non-relapsed controls. Mutational bulk sequencing was performed by NGS panel sequencing and exome sequencing was available in select cases. Single cell processing was performed using the Tapestri (Mission Bio) platform. Briefly, individual cells were isolated using a microfluidic approach, followed by barcoding and genomic DNA amplification for individual cancer cells confined to droplets. Barcodes were then used to reassemble the genetic profiles of cells from next generation sequencing data. We applied this approach to individual AML samples, genotyping the most clinically relevant loci across upwards of 10,000 individual cells.Results: Targeted single-cell sequencing was able to recapitulate bulk sequencing data from both peripheral blood and bone marrow aspirate samples. We observed high concordance between bulk VAFs and sample level VAFs derived from single cell sequencing data. Additionally, single cell analysis allowed for resolution of subclonal architecture and tumor phylogenetic evolution beyond what was predicted from bulk sequencing alone. Rare subclones associated with disease relapse, were identified in initial diagnostic samples that were frequently under the limit of detection of bulk NGS.Conclusions:Taken together, our results suggest a greater degree of heterogeneity in de novo AML samples than suggested with bulk sequencing methods alone and shows the utility of single-cell sequencing for longitudinal monitoring and identification of resistant clones prior to therapy initiation in select patients. We show here that this approach is a feasible and effective way to identify and track heterogeneous populations of cells in AML and may be valuable for MRD identification. DisclosuresAleshin:Mission Bio, Inc.: Consultancy; Natera, Inc.: Employment. Durruthy-Durruthy:Mission Bio, Inc.: Employment, Equity Ownership. Liedtke:Prothena: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech/Roche: Research Funding; Caelum: Membership on an entity's Board of Directors or advisory committees; Amgen/Onyx: Consultancy, Honoraria, Research Funding; BlueBirdBio: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; celgene: Research Funding. Medeiros:Celgene: Consultancy, Research Funding; Genentech: Employment. Eastburn:Mission Bio, Inc.: Employment, Equity Ownership.

Intratumoral Heterogeneity of Bladder Cancer by Molecular Subtypes and Histologic Variants

Molecular subtyping may inform on prognosis and treatment response in bladder cancer. However, intratumoral molecular heterogeneity is not well studied in this disease and could complicate efforts to use molecular subtyping to guide patient management. To investigate intratumoral heterogeneity in bladder cancer, we examined molecular subtypes in a consecutive, retrospective cystectomy series of histologic variant bladder cancers and conventional urothelial carcinomas co-occurring with them. Molecular subtypes were assigned as per the approach reported by Lund University, an approach that incorporates cell cycle alterations and markers of differentiation, to give the urothelial-like, genomically unstable, basal-squamous, mesenchymal-like, and neuroendocrine-like subtypes. The majority (93%) of tumors were classified as urothelial like, genomically unstable, or basal squamous. Among patients with more than one tumor histology, 39% demonstrated molecular heterogeneity among the different tumor histologies. This was greatest for the basal-squamous subtype, 78% of which co-occurred with either urothelial-like or genomically unstable carcinoma (among cases with multiple histologies). In contrast, there was no co-occurrence of urothelial-like and genomically unstable carcinoma in the same patient. The findings indicate that bladder cancer is often molecularly heterogeneous, particularly in the basal-squamous subtype. This raises the concern for sampling error in laboratory tests that guide therapy based on molecular subtyping.Patient summary: In this report, we investigated molecular diversity among different areas from the same tumor in patients with bladder cancer. We found that different areas from the same tumor are often molecularly different. We conclude that this biological diversity must be taken into account when interpreting clinical molecular tests performed on bladder cancer samples.

Impact of cholangiocarcinoma (CC) molecular heterogeneity on outcome during first-line chemotherapy and access to targeted therapies in early clinical trials (CT).

4091Background: CC has poor prognosis and limited therapeutic options beyond first-line therapy. It is molecularly heterogeneous with several gene alterations (alt) that can be matched to targeted ...

Application of NSG in Newly Diagnosed AML

Acute myeloid leukemia (AML) is a hematologic disease characterized by maturation arrest and proliferation of primitive clonal hematopoietic stem cells (blasts) resulting in bone marrow (BM) failure and eventually death if not timely treated. In 2017, approximately 20,000 new cases of AML and 10,000 disease-related deaths occurred in the United States alone. The median age at diagnosis is 67 years and the incidence of the disease increases with age. According to SEER/NCI database, survival in the US AML patients’ population is 26.6% for the last 5 years. Importantly, AML comprises several distinct entities that are cytogenetically and molecularly heterogeneous. Cytogenetic and molecular heterogeneity is clinically relevant, and it has been used for disease classification, prognostication and treatment selection. Despite cytogenetic/molecular risk-based approaches, however, only approximately 40% of younger (<60 year/old) and 10% of older (>60 years) patients achieve cure when treated with currently available chemotherapies. Even if allogeneic hematopoietic stem cells (alloHCT) may improve cure rate, the long-term success of this procedure may be somewhat limited by transplant-related toxicity and disease relapse. Thus, “one size fits all” treatments are insufficient to achieve complete remission (CR) and eventually long-term survival in the vast majority of the molecularly heterogeneous AML patients. Emerging new technologies for sequencing of the human genome have provided the tools for a deeper understanding of the mechanisms of malignant myeloid transformation, and have discovered previously unreported gene mutations and aberrantly silenced or expressed genes that participate in leukemogenesis and treatment resistance and, therefore may be use as therapeutic targets. These novel approaches however have also unveiled the magnitude of the biological complexity of the single AML blasts. Indeed, a myriad of genes have been found concurrently mutated, epigenetically modified or aberrantly expressed in unique molecular profiles of individual patients and contribute to clinical outcome. Thus, the challenge is how to match these complex profiles comprising concurrent mutations, epigenetic changes and aberrantly expressed genes with the most appropriate molecular targeting therapies in individual patients. Although the most pragmatic use of genomic data analyses is to apply a reductive approach and utilize only a relatively small number of genes, novel algorithms for molecular data prioritization and comprehensive analyses must be implemented to capture the entire biologic diversity among distinct AML cells and utilize this information for “personalized” clinical intervention. We will review how the development of molecular profiling using data from different omics platforms may be used for a comprehensive view of the disease biology and to match integrated profiles with the most appropriate cutting- edge treatment for individual AML patients.

T-cell Receptor Signaling Activates an ITK/NF-κB/GATA-3 axis in T-cell Lymphomas Facilitating Resistance to Chemotherapy

Purpose: T-cell lymphomas are a molecularly heterogeneous group of non-Hodgkin lymphomas (NHL) that account for a disproportionate number of NHL disease-related deaths due to their inherent and acquired resistance to standard multiagent chemotherapy regimens. Despite their molecular heterogeneity and frequent loss of various T cell-specific receptors, the T-cell antigen receptor is retained in the majority of these lymphomas. As T-cell receptor (TCR) engagement activates a number of signaling pathways and transcription factors that regulate T-cell growth and survival, we examined the TCR's role in mediating resistance to chemotherapy.Experimental Design: Genetic and pharmacologic strategies were utilized to determine the contribution of tyrosine kinases and transcription factors activated in conventional T cells following TCR engagement in acquired chemotherapy resistance in primary T-cell lymphoma cells and patient-derived cell lines.Results: Here, we report that TCR signaling activates a signaling axis that includes ITK, NF-κB, and GATA-3 and promotes chemotherapy resistance.Conclusions: These observations have significant therapeutic implications, as pharmacologic inhibition of ITK prevented the activation of this signaling axis and overcame chemotherapy resistance. Clin Cancer Res; 23(10); 2506-15. ©2016 AACR.

Molecular genetic heterogeneity in undifferentiated endometrial carcinomas

AbstractUndifferentiated and dedifferentiated endometrial carcinomas are rare and highly aggressive subtypes of uterine cancer, not well characterized at a molecular level. To investigate whether dedifferentiated carcinomas carry molecular genetic alterations similar to those of pure undifferentiated carcinomas, and to gain insight into the pathogenesis of these tumors, we selected a cohort of 18 undifferentiated endometrial carcinomas, 8 of them with a well-differentiated endometrioid carcinoma component (dedifferentiated endometrioid carcinomas), and studied them by immunohistochemistry and massive parallel and Sanger sequencing. Whole-exome sequencing of the endometrioid and undifferentiated components, as well as normal myometrium, was also carried out in one case. According to The Cancer Genome Atlas classification, we distributed 95% of the undifferentiated carcinomas in this series as follows: (a) hypermutated tumors with loss of any mismatch repair protein expression and microsatellite instability (eight cases, 45%); (b) ultramutated carcinomas carrying mutations in the exonuclease domain of POLE (two cases, 11%); (c) high copy number alterations (copy-number high) tumors group exhibiting only TP53 mutations and high number of alterations detected by FISH (two cases, 11%); and (d) low copy number alterations (copy-number low) tumors with molecular alterations typical of endometrioid endometrial carcinomas (five cases, 28%). Two of the latter cases, however, also had TP53 mutations and higher number of alterations detected by FISH and could have progressed to a copy-number high phenotype. Most dedifferentiated carcinomas belonged to the hypermutated group, whereas pure undifferentiated carcinomas shared molecular genetic alterations with copy-number low or copy-number high tumors. These results indicate that undifferentiated and dedifferentiated endometrial carcinomas are molecularly heterogeneous tumors, which may have prognostic value.

Clinical implications of molecular heterogeneity in triple negative breast cancer

Triple negative breast cancer (TNBC) is a molecularly heterogeneous disease lacking recurrent targetable alterations and thus therapeutic advances have been challenging. The absence of ER, PR and HER2 amplifications, leaves combination chemotherapy as the standard of care treatment option in the adjuvant, neoadjuvant and metastatic settings. Recently, multiple studies have shed some light on the heterogeneity of TNBC and identified distinct transcriptional subtypes with unique biologies. Herein we review the molecular heterogeneity and the impact on previous and future clinical trials.