Genomic dissection and mutation-specific target discovery for breast cancer PIK3CA hotspot mutations.

BACKGROUND: Recent advancements in high-throughput genomics and targeted therapies have provided tremendous potential to identify and therapeutically target distinct mutations associated with cancers. However, to date the majority of targeted therapies are used to treat all functional mutations within the same gene, regardless of affected codon or phenotype. RESULTS: In this study, we developed a functional genomic analysis workflow with a unique isogenic cell line panel bearing two distinct hotspot PIK3CA mutations, E545K and H1047R, to accurately identify targetable differences between mutations within the same gene. We performed RNA-seq and ATAC-seq and identified distinct transcriptomic and epigenomic differences associated with each PIK3CA hotspot mutation. We used this data to curate a select CRISPR knock out screen to identify mutation-specific gene pathway vulnerabilities. These data revealed AREG as a E545K-preferential target that was further validated through in vitro analysis and publicly available patient databases. CONCLUSIONS: Using our multi-modal genomics framework, we discover distinct differences in genomic regulation between PIK3CA hotspot mutations, suggesting the PIK3CA mutations have different regulatory effects on the function and downstream signaling of the PI3K complex. Our results demonstrate the potential to rapidly uncover mutation specific molecular targets, specifically AREG and a proximal gene regulatory region, that may provide clinically relevant therapeutic targets. The methods outlined provide investigators with an integrative strategy to identify mutation-specific targets for the treatment of other oncogenic mutations in an isogenic system.

Alternative Tissue Fixation Protocols Dramatically Reduce the Impact of DNA Artifacts, Unraveling the Interpretation of Clinical Comprehensive Genomic Profiling.

Formalin-fixed paraffin-embedded (FFPE) samples represent the cornerstone of tissue-based analysis in precision medicine. Targeted next-generation sequencing panels are routinely used to analyze a limited number of genes to guide treatment decision-making for advanced-stage patients. The number and complexity of genetic alterations to be investigated are rapidly growing; in several instances, a comprehensive genomic profiling analysis is needed. The poor quality of genetic material extracted from FFPE samples may impact the feasibility/reliability of sequencing data. We sampled 9 colorectal cancers to allow 4 parallel fixations: (1) neutral buffered formalin (NBF), (2) acid-deprived formalin fixation (ADF), (3) precooled ADF (coldADF), and (4) glyoxal acid free (GAF). DNA extraction, fragmentation analysis, and sequencing by 2 large next-generation sequencing panels (OCAv3 and TSO500) followed. We comprehensively analyzed library and sequencing quality controls and the quality of sequencing results. Libraries from coldADF samples showed significantly longer reads than the others with both panels. ADF-derived and coldADF-derived libraries showed the lowest level of noise and the highest levels of uniformity with the OCAv3 panel, followed by GAF and NBF samples. The data uniformity was confirmed by the TSO500 results, which also highlighted the best performance in terms of the total region sequenced for the ADF and coldADF samples. NBF samples had a significantly smaller region sequenced and displayed a significantly lower number of evaluable microsatellite loci and a significant increase in single-nucleotide variations compared with other protocols. Mutational signature 1 (aging and FFPE artifact related) showed the highest (37%) and lowest (17%) values in the NBF and coldADF samples, respectively. Most of the identified genetic alterations were shared by all samples in each lesion. Five genes showed a different mutational status across samples and/or panels: 4 discordant results involved NBF samples. In conclusion, acid-deprived fixatives (GAF and ADF) guarantee the highest DNA preservation/sequencing performance, thus allowing more complex molecular profiling of tissue samples.

Unique Patterns of Heterogeneous Mismatch Repair Protein Expression in Colorectal Cancer Unveil Different Degrees of Tumor Mutational Burden and Distinct Tumor Microenvironment Features.

Mismatch repair (MMR) protein expression in colorectal cancer (CRC) cells is usually homogeneously retained or lost. Rare lesions may show a heterogeneous pattern of MMR protein expression. We evaluated MMR protein expression (MLH1, MSH2, MSH6, and PMS2) in 200 CRCs, identifying 3 groups with proficient MMR protein expression (MMRp), deficient MMR protein expression (MMRd), and heterogeneous MMR protein expression (MMRh). MMRh tumors were microdissected on the basis of the expression of the heterogeneous marker. DNA was extracted and subjected to targeted sequencing. RNA was purified from bulk tumors of all MMRh cases and in a control series of 15 MMRp and 10 MMRd CRCs and analyzed using the PanCancer IO 360 Panel (NanoString Technologies). Twenty-nine of the 200 cases (14.5%) were MMRd. Nine cases (4.5%) showed a heterogeneous pattern of MMR expression, with 6 tumors harboring concomitant loss of one of the other MMR proteins, thus featuring areas with double loss at immunohistochemistry (IHC) testing (MMRh double-loss cases). Four of the 6 MMRh double-loss cases were suitable for a separate sequence variant analysis of IHC double-negative and IHC single-negative components of the tumor. In all lesions, both components exhibited a high tumor mutation burden (TMB). Nevertheless, a significant increase in TMB in the double-negative components was observed (mean TMB: negative, 70 mut/Mb vs positive, 59 mut/Mb) because of a higher number of subclonal variants compared with the other component. Comparative gene expression analyses among MMRd, MMRp, and MMRh CRCs highlighted differential gene expression patterns and an increased number of tumor-infiltrating lymphocytes in MMRh lesions, which is also characterized by a substantial population of exhausted CD8+ lymphocytes. We describe a unique subgroup of CRCs showing heterogeneous expression of MMR proteins in a background of concomitant loss of one of the other markers.

TROP2, androgen receptor, and PD-L1 status in histological subtypes of high-grade metaplastic breast carcinomas.

AIMS: High-grade metaplastic breast carcinoma (HG-MBC) is a rare subtype of invasive breast carcinoma, mostly triple-negative. Metaplastic carcinomas are less responsive to neoadjuvant chemotherapy and are associated with a worse outcome than invasive carcinomas of no special type. METHODS: Clinicopathological characteristics and immunophenotype were retrospectively assessed in a series of 65 patients diagnosed with HG-MBC between 2005 and 2017 at the Curie Institute (antibody panel: oestrogen receptor [ER], progesterone receptor [PR], androgen receptor [AR], human epidermal growth factor receptor 2 [HER2], programmed death ligand-1 [PD-L1], and trophoblast cell surface antigen 2 [TROP2]). RESULTS: The median age at diagnosis was 59.5 years. Six (9%) patients had metastatic disease at diagnosis. Among the nonmetastatic patients receiving neoadjuvant therapy, 26% (5/19) achieved pathological complete response. Most tumours were pT1/pT2 (77%) and 12% were pN+. Histological subtypes (mixed, squamous, mesenchymal, and spindle cell) were 40%, 35.5%, 15.5%, and 9%, respectively. Tumour-infiltrating lymphocytes were low or moderate except when squamous differentiation was present. Most tumours were triple-negative (92%). AR and TROP2 were positive in 34% and 85% of the cases, respectively. PD-L1 was positive in tumour cells in 18% (cutoff: 1% of positive tumour cells) of the cases and in tumour-infiltrating immune cells in 40% (cutoff: 1% of tumour area) of the cases. Notably, spindle cell and mesenchymal metaplastic breast carcinomas were mostly PDL1-negative. Lastly, 21 (32.3%) cases were HER2-low, all being HER2 1+, with no HER2 2+. CONCLUSION: Metaplastic breast carcinoma could benefit from tailored therapeutic strategies adapted to the phenotypic specificities of histological subtypes.

Tissue Fixation with a Formic Acid-Deprived Formalin Better Preserves DNA Integrity over Time.

INTRODUCTION: Optimization of pre-analytic procedures and tissue processing is a basic requirement for reliable and reproducible data to be obtained. Tissue fixation in formalin represents the extensively favored method for surgical tissue specimen processing in diagnostic pathology; however, formalin fixation exerts a blasting effect on DNA and RNA. METHODS: A formic acid-deprived formaldehyde solution was prepared by removing acids with an ion-exchange basic resin and the concentrated, acid-deprived formaldehyde (ADF) solution was employed to prepare a 4% ADF solution in 0.1 M phosphate buffer, pH 7.2-7.4. Human (n = 27) and mouse (n = 20) tissues were fixed in parallel and similar conditions in either ADF or neutral buffered formalin (NBF). DNAs and RNAs were extracted, and fragmentation analyses were performed. RESULTS: Besides no significant differences in terms of extraction yield and absorbance ratio, ADF fixation reduced DNA fragmentation, i.e., the largest fragments (>5,000 bp) were significantly more prevalent in the DNAs purified from ADF-fixed tissues (p < 0.001 in both cohorts). Moreover, we observed that DNA preservation is more stable in ADF-fixed tissue compared to NBF-fixed tissues. CONCLUSION: Although DNA fragmentation in FFPE tissues is a multifactor process, we showed that the removal of formic acid is responsible for a significant improvement in DNA preservation.

The Genes-Stemness-Secretome Interplay in Malignant Pleural Mesothelioma: Molecular Dynamics and Clinical Hints.

MPM has a uniquely poor somatic mutational landscape, mainly driven by environmental selective pressure. This feature has dramatically limited the development of effective treatment. However, genomic events are known to be associated with MPM progression, and specific genetic signatures emerge from the exceptional crosstalk between neoplastic cells and matrix components, among which one main area of focus is hypoxia. Here we discuss the novel therapeutic strategies focused on the exploitation of MPM genetic asset and its interconnection with the surrounding hypoxic microenvironment as well as transcript products and microvesicles representing both an insight into the pathogenesis and promising actionable targets.

Think "HER2" different: integrative diagnostic approaches for HER2-low breast cancer.

This work explores the complex field of HER2 testing in the HER2-low breast cancer era, with a focus on methodological aspects. We aim to propose clear positions to scientific societies, institutions, pathologists, and oncologists to guide and shape the appropriate diagnostic strategies for HER2-low breast cancer. The fundamental question at hand is whether the necessary tools to effectively translate our knowledge about HER2 into practical diagnostic schemes for the lower spectrum of expression are available. Our investigation is centered on the significance of distinguishing between an immunohistochemistry (IHC) score 0 and score 1+ in light of the clinical implications now apparent, as patients with HER2-low breast cancer become eligible for trastuzumab-deruxtecan treatment. Furthermore, we discuss the definition of HER2-low beyond its conventional boundaries and assess the reliability of established diagnostic procedures designed at a time when therapeutic perspectives were non-existent for these cases. In this regard, we examine potential complementary technologies, such as gene expression analysis and liquid biopsy. Ultimately, we consider the potential role of artificial intelligence (AI) in the field of digital pathology and its integration into HER2 testing, with a particular emphasis on its application in the context of HER2-low breast cancer.

Evolving concepts in HER2 evaluation in breast cancer: Heterogeneity, HER2-low carcinomas and beyond.

The human epidermal growth factor receptor 2 (HER2) is a well-known negative prognostic factor in breast cancer and a target of the monoclonal antibody trastuzumab as well as of other anti-HER2 compounds. Pioneering works on HER2-positive breast cancer in the 90s' launched a new era in clinical research and oncology practice that has reshaped the natural history of this disease. In diagnostic pathology the HER2 status is routinely assessed by using a combination of immunohistochemistry (IHC, to evaluate HER2 protein expression levels) and in situ hybridization (ISH, to assess HER2 gene status). For this purpose, international recommendations have been developed by a consensus of experts in the field, which have changed over the years according to new experimental and clinical data. In this review article we will document the changes that have contributed to a better evaluation of the HER2 status in clinical practice, furthermore we will discuss HER2 heterogeneity defined by IHC and ISH as well as by transcriptomic analysis and we will critically describe the complexity of HER2 equivocal results. Finally, we will introduce the clinical impact of HER2 mutations and we will define the upcoming category of HER2-low breast cancer with respect to emerging clinical data on the efficacy of specific anti-HER2 agents in subgroups of breast carcinomas lacking the classical oncogene addition dictated by HER2 amplification.

Unusual Patterns of HER2 Expression in Breast Cancer: Insights and Perspectives.

The biomarker human epidermal growth factor receptor-2 (HER2) has represented the best example of successful targeted therapy in breast cancer patients. Based on the concept of "oncogene addiction," we have learnt how to identify patients likely benefitting from anti-HER2 agents. Since HER2 gene amplification leads to marked overexpression of the HER2 receptors on the cell membrane, immunohistochemistry with clinically validated antibodies and scoring system based on intensity and completeness of the membranous expression constitute the screening method to separate negative (score 0/1+) and positive (score 3+) carcinomas and to identify those tumours with complete yet only moderate HER2 expression (score 2+, equivocal carcinomas), which need to be investigated further in terms of gene status to confirm the presence of a loop of oncogene addiction. This process has demanded quality controls and led to recommendations by Scientific Societies, which pathologists routinely need to follow to guarantee reproducibility. In this review, we will span from the description of classical HER2 evaluation to the discussion of those scenarios in which HER2 expression is unusual and/or difficult to define. We will dissect HER2 heterogeneity, HER2 conversion from primary to relapsed/metastatic breast cancer, and we will introduce the new category of HER2-low breast carcinomas.

Integrated Antitumor Activities of Cellular Immunotherapy with CIK Lymphocytes and Interferons against KIT/PDGFRA Wild Type GIST.

Gastrointestinal stromal tumors (GISTs) are rare, mesenchymal tumors of the gastrointestinal tract, characterized by either KIT or PDGFRA mutation in about 85% of cases. KIT/PDGFRA wild type gastrointestinal stromal tumors (wtGIST) account for the remaining 15% of GIST and represent an unmet medical need: their prevalence and potential medical vulnerabilities are not completely defined, and effective therapeutic strategies are still lacking. In this study we set a patient-derived preclinical model of wtGIST to investigate their phenotypic features, along with their susceptibility to cellular immunotherapy with cytokine-induced killer lymphocytes (CIK) and interferons (IFN). We generated 11 wtGIST primary cell lines (wtGISTc). The main CIK ligands (MIC A/B; ULBPs), along with PD-L1/2, were expressed by wtGISTc and the expression of HLA-I molecules was preserved. Patient-derived CIK were capable of intense killing in vitro against wtGISTc resistant to both imatinib and sunitinib. We found that CIK produce a high level of granzyme B, IFNα and IFNγ. CIK-conditioned supernatant was responsible for part of the observed tumoricidal effect, along with positive bystander modulatory activities enhancing the expression of PD-L1/2 and HLA-I molecules. IFNα, but not In, had direct antitumor effects on 50% (4/8) of TKI-resistant wtGISTc, positively correlated with the tumor expression of IFN receptors. wtGIST cells that survived IFNα were still sensitive to CIK immunotherapy. Our data support the exploration of CIK immunotherapy in clinical studies for TKI-resistant wtGIST, proposing reevaluation for IFNα within this challenging setting.