RB1 Genetic Alterations in Estrogen Receptor-Positive Breast Carcinomas: Correlation With Neuroendocrine Differentiation.

Genetic alterations in the retinoblastoma susceptibility gene (RB1) are present in up to 40% of triple-negative breast cancers (BCs) and frequent in tumors with neuroendocrine differentiation, including small cell neuroendocrine carcinoma. Data on RB1 genetic alterations in estrogen receptor (ER)-positive BCs are scarce. In this study, we sought to define the morphologic, immunohistochemical, and genetic features of ER-positive BCs harboring somatic alterations in RB1, with emphasis on neuroendocrine differentiation. ER-positive BCs with pathogenic RB1 genetic alterations were identified in <1% of cases (N = 55) from a cohort of 6026 BCs previously subjected to targeted next-generation sequencing, including 23 primary BCs (pBCs) and 32 recurrent/metastatic BCs (mBCs). In cases where loss of heterozygosity of the wild-type RB1 allele could be assessed (93%, 51/55), most pBCs (82%, 18/22) and mBCs (90%, 26/29) exhibited biallelic RB1 inactivation, primarily through loss-of-function mutation and loss of heterozygosity (98%, 43/44). Upon histologic review, a subset of RB1-altered tumors exhibited neuroendocrine morphology (13%, 7/55), which correlated with expression of neuroendocrine markers (39%, 9/23) in both pBCs (27%, 3/11) and mBCs (50%, 6/12). Loss of Rb protein expression was observed in BCs with biallelic RB1 loss only, with similar frequency in pBCs (82%, 9/11) and mBCs (75%, 9/12). All cases with neuroendocrine marker expression (n = 9) and/or neuroendocrine morphology (n = 7) harbored biallelic genetic inactivation of RB1 and exhibited Rb loss of expression. TP53 (53%, 29/55) and PIK3CA (45%, 25/55) were the most frequently comutated genes across the cohort. Overall, these findings suggest that ER-positive BCs with biallelic RB1 genetic alterations frequently exhibit Rb protein loss, which correlates with neuroendocrine differentiation in select BCs. This study provides insights into the molecular and phenotypic heterogeneity of BCs with RB1 genetic inactivation, underscoring the need for further research into the potential clinical implications associated with these tumors.

APOBEC3 mutagenesis drives therapy resistance in breast cancer.

Acquired genetic alterations commonly drive resistance to endocrine and targeted therapies in metastatic breast cancer 1-7 , however the underlying processes engendering these diverse alterations are largely uncharacterized. To identify the mutational processes operant in breast cancer and their impact on clinical outcomes, we utilized a well-annotated cohort of 3,880 patient samples with paired tumor-normal sequencing data. The mutational signatures associated with apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) enzymes were highly prevalent and enriched in post-treatment compared to treatment-naïve hormone receptor-positive (HR+) cancers. APOBEC3 mutational signatures were independently associated with shorter progression-free survival on antiestrogen plus CDK4/6 inhibitor combination therapy in patients with HR+ metastatic breast cancer. Whole genome sequencing (WGS) of breast cancer models and selected paired primary-metastatic samples demonstrated that active APOBEC3 mutagenesis promoted resistance to both endocrine and targeted therapies through characteristic alterations such as RB1 loss-of-function mutations. Evidence of APOBEC3 activity in pre-treatment samples illustrated a pervasive role for this mutational process in breast cancer evolution. The study reveals APOBEC3 mutagenesis to be a frequent mediator of therapy resistance in breast cancer and highlights its potential as a biomarker and target for overcoming resistance.

Converging and evolving immuno-genomic routes toward immune escape in breast cancer.

The interactions between tumor and immune cells along the course of breast cancer progression remain largely unknown. Here, we extensively characterize multiple sequential and parallel multiregion tumor and blood specimens of an index patient and a cohort of metastatic triple-negative breast cancers. We demonstrate that a continuous increase in tumor genomic heterogeneity and distinct molecular clocks correlated with resistance to treatment, eventually allowing tumors to escape from immune control. TCR repertoire loses diversity over time, leading to convergent evolution as breast cancer progresses. Although mixed populations of effector memory and cytotoxic single T cells coexist in the peripheral blood, defects in the antigen presentation machinery coupled with subdued T cell recruitment into metastases are observed, indicating a potent immune avoidance microenvironment not compatible with an effective antitumor response in lethal metastatic disease. Our results demonstrate that the immune responses against cancer are not static, but rather follow dynamic processes that match cancer genomic progression, illustrating the complex nature of tumor and immune cell interactions.

Genomic and epigenomic basis of breast invasive lobular carcinomas lacking CDH1 genetic alterations.

CDH1 (E-cadherin) bi-allelic inactivation is the hallmark alteration of breast invasive lobular carcinoma (ILC), resulting in its discohesive phenotype. A subset of ILCs, however, lack CDH1 genetic/epigenetic inactivation, and their genetic underpinning is unknown. Through clinical targeted sequencing data reanalysis of 364 primary ILCs, we identified 25 ILCs lacking CDH1 bi-allelic genetic alterations. CDH1 promoter methylation was frequent (63%) in these cases. Targeted sequencing reanalysis revealed 3 ILCs harboring AXIN2 deleterious fusions (n = 2) or loss-of-function mutation (n = 1). Whole-genome sequencing of 3 cases lacking bi-allelic CDH1 genetic/epigenetic inactivation confirmed the AXIN2 mutation and no other cell-cell adhesion genetic alterations but revealed a new CTNND1 (p120) deleterious fusion. AXIN2 knock-out in MCF7 cells resulted in lobular-like features, including increased cellular migration and resistance to anoikis. Taken together, ILCs lacking CDH1 genetic/epigenetic alterations are driven by inactivating alterations in other cell adhesion genes (CTNND1 or AXIN2), endorsing a convergent phenotype in ILC.

Nonlobular Invasive Breast Carcinomas with Biallelic Pathogenic CDH1 Somatic Alterations: A Histologic, Immunophenotypic, and Genomic Characterization.

CDH1 encodes for E-cadherin, and its loss of function is the hallmark of invasive lobular carcinoma (ILC). Albeit vanishingly rare, biallelic CDH1 alterations may be found in nonlobular breast carcinomas (NL-BCs). We sought to determine the clinicopathologic characteristics and repertoire of genetic alterations of NL-BCs harboring CDH1 biallelic genetic alterations. Analysis of 5842 breast cancers (BCs) subjected to clinical tumor-normal sequencing with an FDA-cleared multigene panel was conducted to identify BCs with biallelic CDH1 pathogenic/likely pathogenic somatic mutations lacking lobular features. The genomic profiles of NL-BCs with CDH1 biallelic genetic alterations were compared with those of ILCs and invasive ductal carcinomas (IDCs), matched by clinicopathologic characteristics. Of the 896 CDH1-altered BCs, 889 samples were excluded based on the diagnosis of invasive mixed ductal/lobular carcinoma or ILC or the detection of monoallelic CDH1 alterations. Only 7 of the 5842 (0.11%) BCs harbored biallelic CDH1 alterations and lacked lobular features. Of these, 4/7 (57%) cases were ER-positive/HER2-negative, 1/7 (14%) was ER-positive/HER2-positive, and 2/7 (29%) were ER-negative/HER2-negative. In total, 5/7 (71%) were of Nottingham grade 2, and 2/7 (29%) were of grade 3. The NL-BCs with CDH1 biallelic genetic alterations included a mucinous carcinoma (n = 1), IDCs with focal nested growth (n = 2), IDC with solid papillary (n = 1) or apocrine (n = 2) features, and an IDC of no special type (NST; n = 1). E-cadherin expression, as detected by immunohistochemistry, was absent (3/5) or aberrant (discontinuous membranous/cytoplasmic/granular; 2/5). However, NL-BCs with CDH1 biallelic genetic alterations displayed recurrent genetic alterations, including TP53, PIK3CA (57%, 4/7; each), FGFR1, and NCOR1 (28%, 2/7, each) alterations. Compared with CDH1 wild-type IDC-NSTs, NL-BCs less frequently harbored GATA3 mutations (0% vs 47%, P = .03), but no significant differences were detected when compared with matched ILCs. Therefore, NL-BCs with CDH1 biallelic genetic alterations are vanishingly rare, predominantly comprise IDCs with special histologic features, and have genomic features akin to luminal B ER-positive BCs.

Genomic profiling of primary and metastatic thyroid cancers.

The genetic repertoire of primary thyroid cancers (TCs) is well documented, but there is a considerable lack of molecular profiling in metastatic TCs. Here, we retrieved and analyzed the molecular and clinical features of 475 primary and metastatic TCs subjected to targeted DNA sequencing, from the cBioPortal database. The cohort included primary and metastatic samples from 276 papillary thyroid carcinomas (PTCs), 5 follicular thyroid carcinomas, 22 Hürthle cell carcinomas (HCCs), 127 poorly differentiated thyroid carcinomas (PDTCs), 30 anaplastic thyroid carcinomas (ATCs) and 15 medullary thyroid carcinomas. The ATCs had the highest tumor mutational burden and the HCCs the highest fraction of the genome altered. Compared to primary PTCs, the metastases had a significantly higher frequency of genetic alterations affecting TERT (51% vs 77%, P < 0.001), CDKN2A (2% vs 10%, P < 0.01), RET (2% vs 7%, P < 0.05), CDKN2B (1% vs 6%, P < 0.05) and BCOR (0% vs 4%, P < 0.05). The distant metastases had a significantly lower frequency of BRAF (64% vs 85%, P < 0.01) and a significantly higher frequency of NRAS (13% vs 3%, P < 0.05) hotspot mutations than the lymph node metastases. Metastases from HCCs and PDTCs were found to be enriched for NF1 (29%) and TP53 (18%) biallelic alterations, respectively. The frequency of subclonal mutations in ATCs was significantly higher than in PTCs (43% vs 25%, P < 0.01) and PDTCs (43% vs 22%, P < 0.01). Metastatic TCs are enriched in clinically informative genetic alterations such as RET translocations, BRAF hotspot mutations and NF1 biallelic losses that may be explored therapeutically.

Interferon response and epigenetic modulation by SMARCA4 mutations drive ovarian tumor immunogenicity.

Cell-intrinsic mechanisms of immunogenicity in ovarian cancer (OC) are not well understood. The presence of damaging mutations in the SWI/SNF chromatin remodeling complex, such as the SMARCA4 (BRG1) catalytic subunit, has been associated with improved response to ICB, however the mechanism by which this occurs is unclear. The aim of this current study was to examine the alterations in tumor cell-intrinsic and extrinsic immune signaling caused by SMARCA4 loss. Using OC models with loss-of-function mutations in SMARCA4 , we found that SMARCA4 loss resulted in increased cancer cell-intrinsic immunogenicity, characterized by upregulation of long-terminal RNA repeats such as endogenous retroviruses, increased expression of interferon-stimulated genes, and upregulation of antigen presentation machinery. Notably, this response was dependent on IRF3 signaling, but was independent of the type I interferon receptor. Mice inoculated with cancer cells bearing SMARCA4 loss demonstrated increased activation of cytotoxic T cells and NK cells in the tumor microenvironment as well as increased infiltration with activated dendritic cells. These results were recapitulated when animals bearing SMARCA4- proficient tumors were treated with a BRG1 inhibitor, suggesting that modulation of chromatin remodeling through targeting SMARCA4 may serve as a strategy to reverse immune evasion in OC.

Genomic Profiling Reveals Germline Predisposition and Homologous Recombination Deficiency in Pancreatic Acinar Cell Carcinoma.

PURPOSE: To determine the genetic predisposition underlying pancreatic acinar cell carcinoma (PACC) and characterize its genomic features. METHODS: Both somatic and germline analyses were performed using an Food and Drug Administration-authorized matched tumor/normal sequencing assay on a clinical cohort of 28,780 patients with cancer, 49 of whom were diagnosed with PACC. For a subset of PACCs, whole-genome sequencing (WGS; n = 12) and RNA sequencing (n = 6) were performed. RESULTS: Eighteen of 49 (36.7%) PACCs harbored germline pathogenic variants in homologous recombination (HR) and DNA damage response (DDR) genes, including BRCA1 (n = 1), BRCA2 (n = 12), PALB2 (n = 2), ATM (n = 2), and CHEK2 (n = 1). Thirty-one PACCs displayed pure, and 18 PACCs harbored mixed acinar cell histology. Fifteen of 31 (48%) pure PACCs harbored a germline pathogenic variant affecting HR-/DDR-related genes. BRCA2 germline pathogenic variants (11 of 31, 35%) were significantly more frequent in pure PACCs than in pancreatic adenocarcinoma (86 of 2,739, 3.1%; P < .001), high-grade serous ovarian carcinoma (67 of 1,318, 5.1%; P < .001), prostate cancer (116 of 3,401, 3.4%; P < .001), and breast cancer (79 of 3,196, 2.5%; P < .001). Genomic features of HR deficiency (HRD) were detected in 7 of 12 PACCs undergoing WGS, including 100% (n = 6) of PACCs with germline HR-related pathogenic mutations and 1 of 6 PACCs lacking known pathogenic alterations in HR-related genes. Exploratory analyses revealed that in PACCs, the repertoire of somatic driver genetic alterations and the load of neoantigens with high binding affinity varied according to the presence of germline pathogenic alterations affecting HR-/DDR-related genes and/or HRD. CONCLUSION: In a large pan-cancer cohort, PACC was identified as the cancer type with the highest prevalence of both BRCA2 germline pathogenic variants and genomic features of HRD, suggesting that PACC should be considered as part of the spectrum of BRCA-related malignancies.

Clinicopathologic and genomic features of lobular like invasive mammary carcinoma: is it a distinct entity?

This study describes "lobular-like invasive mammary carcinomas" (LLIMCas), a group of low- to intermediate-grade invasive mammary carcinomas with discohesive, diffusely infiltrative cells showing retained circumferential membranous immunoreactivity for both E-cadherin and p120. We analyzed the clinical-pathologic features of 166 LLIMCas compared to 104 classical invasive lobular carcinomas (ILCs) and 100 grade 1 and 2 invasive ductal carcinomas (IDCs). Tumor size and pT stage of LLIMCas were intermediate between IDCs and ILCs, and yet often underestimated on imaging and showed frequent positive margins on the first resection. Despite histomorphologic similarities to classical ILC, the discohesion in LLIMCa was independent of E-cadherin/p120 immunophenotypic alteration. An exploratory, hypothesis-generating analysis of the genomic features of 14 randomly selected LLIMCas and classical ILCs (7 from each category) was performed utilizing an FDA-authorized targeted capture sequencing assay (MSK-IMPACT). None of the seven LLIMCas harbored CDH1 loss-of-function mutations, and none of the CDH1 alterations detected in two of the LLIMCas was pathogenic. In contrast, all seven ILCs harbored CDH1 loss-of-function mutations coupled with the loss of heterozygosity of the CDH1 wild-type allele. Four of the six evaluable LLIMCas were positive for CDH1 promoter methylation, which may partially explain the single-cell infiltrative morphology seen in LLIMCa. Further studies are warranted to better define the molecular basis of the discohesive cellular morphology in LLIMCa. Until more data becomes available, identifying LLIMCas and distinguishing them from typical IDCs and ILCs would be justified. In patients with LLIMCas, preoperative MRI should be entertained to guide surgical management.

KIT genetic alterations in breast cancer.

AIMS: Activating somatic mutations or gene amplification of KIT result in constitutive activation of its receptor tyrosine kinase, which is targetable in various solid tumours. Here, we sought to investigate the presence of KIT genetic alterations in breast cancer (BC) and characterise the histological and genomic features of these tumours. METHODS: A retrospective analysis of 5,575 BCs previously subjected to targeted sequencing using the FDA-authorised Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Targets (MSK-IMPACT) assay was performed to identify BCs with KIT alterations. A histological assessment of KIT-altered BCs was conducted, and their repertoire of genetic alterations was compared with that of BCs lacking KIT genetic alterations, matched for age, histological type, oestrogen receptor/HER2 status and sample type. RESULTS: We identified 18 BCs (0.32%), including 9 primary and 9 metastatic BCs, with oncogenic/likely oncogenic genetic alterations affecting KIT, including activating somatic mutations (n=4) or gene amplification (n=14). All KIT-altered BCs were of high histological grade, although no distinctive histological features were observed. When compared with BCs lacking KIT genetic alterations, no distinctive genetic features were identified. In two metastatic KIT-altered BCs in which the matched primary BC had also been analysed by MSK-IMPACT, the KIT mutations were found to be restricted to the metastatic samples, suggesting that they were late events in the evolution of these cancers. CONCLUSIONS: KIT genetic alterations are vanishingly rare in BC. KIT-altered BCs are of high grade but lack distinctive histological features. Genetic alterations in KIT might be late events in the evolution and/or progression of BC.

Morphologic and Genomic Characteristics of Breast Cancers Occurring in Individuals with Lynch Syndrome.

PURPOSE: Lynch syndrome is defined by germline pathogenic mutations involving DNA mismatch repair (MMR) genes and linked with the development of MMR-deficient colon and endometrial cancers. Whether breast cancers developing in the context of Lynch syndrome are causally related to MMR deficiency (MMRd), remains controversial. Thus, we explored the morphologic and genomic characteristics of breast cancers occurring in Lynch syndrome individuals. EXPERIMENTAL DESIGN: A retrospective analysis of 20,110 patients with cancer who underwent multigene panel genetic testing was performed to identify individuals with a likely pathogenic/pathogenic germline variant in MLH1, MSH2, MSH6, or PMS2 who developed breast cancers. The histologic characteristics and IHC assessment of breast cancers for MMR proteins and programmed death-ligand 1 (PD-L1) expression were assessed on cases with available materials. DNA samples from paired tumors and blood were sequenced with Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (≥468 key cancer genes). Microsatellite instability (MSI) status was assessed utilizing MSISensor. Mutational signatures were defined using SigMA. RESULTS: A total of 272 individuals with Lynch syndrome were identified, 13 (5%) of whom had primary breast cancers. The majority of breast cancers (92%) were hormone receptor-positive tumors. Five (42%) of 12 breast cancers displayed loss of MMR proteins by IHC. Four (36%) of 11 breast cancers subjected to tumor-normal sequencing showed dominant MSI mutational signatures, high tumor mutational burden, and indeterminate (27%) or high MSISensor scores (9%). One patient with metastatic MMRd breast cancer received anti-PD1 therapy and achieved a robust and durable response. CONCLUSIONS: A subset of breast cancers developing in individuals with Lynch syndrome are etiologically linked to MMRd and may benefit from anti-PD1/PD-L1 immunotherapy.

Cancer-Causative Mutations Occurring in Early Embryogenesis.

Mosaic mutations in normal tissues can occur early in embryogenesis and be associated with hereditary cancer syndromes when affecting cancer susceptibility genes (CSG). Their contribution to apparently sporadic cancers is currently unknown. Analysis of paired tumor/blood sequencing data of 35,310 patients with cancer revealed 36 pathogenic mosaic variants affecting CSGs, most of which were not detected by prior clinical genetic testing. These CSG mosaic variants were consistently detected at varying variant allelic fractions in microdissected normal tissues (n = 48) from distinct embryonic lineages in all individuals tested, indicating their early embryonic origin, likely prior to gastrulation, and likely asymmetrical propagation. Tumor-specific biallelic inactivation of the CSG affected by a mosaic variant was observed in 91.7% (33/36) of cases, and tumors displayed the hallmark pathologic and/or genomic features of inactivation of the respective CSGs, establishing a causal link between CSG mosaic variants arising in early embryogenesis and the development of apparently sporadic cancers. SIGNIFICANCE: Here, we demonstrate that mosaic variants in CSGs arising in early embryogenesis contribute to the oncogenesis of seemingly sporadic cancers. These variants can be systematically detected through the analysis of tumor/normal sequencing data, and their detection may affect therapeutic decisions as well as prophylactic measures for patients and their offspring. See related commentary by Liggett and Sankaran, p. 889. This article is highlighted in the In This Issue feature, p. 873.

TERT promoter hotspot mutations and gene amplification in metaplastic breast cancer.

Metaplastic breast cancers (MBCs) are characterized by complex genomes, which seem to vary according to their histologic subtype. TERT promoter hotspot mutations and gene amplification are rare in common forms of breast cancer, but present in a subset of phyllodes tumors. Here, we sought to determine the frequency of genetic alterations affecting TERT in a cohort of 60 MBCs with distinct predominant metaplastic components (squamous, 23%; spindle, 27%; osseous, 8%; chondroid, 42%), and to compare the repertoire of genetic alterations of MBCs according to the presence of TERT promoter hotspot mutations or gene amplification. Forty-four MBCs were subjected to: whole-exome sequencing (WES; n = 27) or targeted sequencing of 341-468 cancer-related genes (n = 17); 16 MBCs were subjected to Sanger sequencing of the TERT promoter, TP53 and selected exons of PIK3CA, HRAS, and BRAF. TERT promoter hotspot mutations (n = 9) and TERT gene amplification (n = 1) were found in 10 of the 60 MBCs analyzed, respectively. These TERT alterations were less frequently found in MBCs with predominant chondroid differentiation than in other MBC subtypes (p = 0.01, Fisher's exact test) and were mutually exclusive with TP53 mutations (p < 0.001, CoMEt). In addition, a comparative analysis of the MBCs subjected to WES or targeted cancer gene sequencing (n = 44) revealed that MBCs harboring TERT promoter hotspot mutations or gene amplification (n = 6) more frequently harbored PIK3CA than TERT wild-type MBCs (n = 38; p = 0.001; Fisher's exact test). In conclusion, TERT somatic genetic alterations are found in a subset of TP53 wild-type MBCs with squamous/spindle differentiation, highlighting the genetic diversity of these cancers.

Genetic characterisation of adult primary pleomorphic uterine rhabdomyosarcoma and comparison with uterine carcinosarcoma.

AIMS: To characterise the genetic alterations in adult primary uterine rhabdomyosarcomas (uRMSs) and to investigate whether these tumours are genetically distinct from uterine carcinosarcomas (UCSs). METHODS AND RESULTS: Three tumours originally diagnosed as primary adult pleomorphic uRMS were subjected to massively parallel sequencing targeting 468 cancer-related genes and RNA-sequencing. Mutational profiles were compared with those of UCSs (n = 57) obtained from The Cancer Genome Atlas. Sequencing data analyses were performed using validated bioinformatic approaches. Pathogenic TP53 mutations and high levels of genomic instability were detected in the three cases. uRMS1 harboured a likely pathogenic YTHDF2-FOXR1 fusion. uRMS2 harboured a PPP2R1A hotspot mutation and amplification of multiple genes, including WHSC1L1, FGFR1, MDM2, and CCNE1, whereas uRMS3 harboured an FBXW7 hotspot mutation and an ANKRD11 homozygous deletion. Hierarchical clustering of somatic mutations and copy number alterations revealed that these tumours initially diagnosed as pleomorphic uRMSs and UCSs were similar. Subsequent comprehensive pathological re-review of the three uRMSs revealed previously unidentified minute pan-cytokeratin-positive atypical glands in one case (uRMS3), favouring its reclassification as UCS with extensive rhabdomyosarcomatous overgrowth. CONCLUSIONS: Adult pleomorphic uRMSs harbour TP53 mutations and high levels of copy number alterations. Our findings underscore the challenge in discriminating between uRMS and UCS with rhabdomyosarcomatous differentiation.

Whole-exome sequencing analysis of juvenile papillomatosis and coexisting breast carcinoma.

Juvenile papillomatosis (JP) of the breast is a rare benign mass-forming lesion occurring in young women, which is histologically characterized by a constellation of proliferative changes and large cysts, giving it the gross appearance of Swiss cheese. A subset of patients with JP report a family history of breast carcinoma and/or coexisting or subsequent breast carcinoma. We performed whole-exome sequencing of the hyperplastic epithelial component of three JPs, including one with coexisting ductal carcinoma in situ (DCIS) and invasive ductal carcinoma of no special type (IDC-NST). JPs harbored clonal somatic PIK3CA hotspot mutations in two cases. In the JP with coexisting DCIS and IDC-NST, these lesions were clonally related to the associated JP, sharing a clonal PIK3CA E542K somatic hotspot mutation. JP showed a paucity of copy number alterations, whereas the associated DCIS and IDC-NST showed concurrent 1q gains/16q losses, hallmarks of estrogen receptor (ER)-positive breast cancers. We observed JP to harbor a dominant aging-related mutational signature, whereas coexisting DCIS and IDC-NST showed greater exposure to an APOBEC signature. Taken together, our findings suggest that, at least in a subset of cases, JP might constitute the substrate from which DCIS and invasive breast carcinomas develop.

Whole-Exome Sequencing Analysis of the Progression from Non-Low-Grade Ductal Carcinoma In Situ to Invasive Ductal Carcinoma.

PURPOSE: Ductal carcinoma in situ (DCIS) is a nonobligate precursor of invasive breast cancer. Here, we sought to investigate the level of intralesion genetic heterogeneity in DCIS and the patterns of clonal architecture changes in the progression from DCIS to invasive disease. EXPERIMENTAL DESIGN: Synchronous DCIS (n = 27) and invasive ductal carcinomas of no special type (IDC-NSTs; n = 26) from 25 patients, and pure DCIS (n = 7) from 7 patients were microdissected separately and subjected to high-depth whole-exome (n = 56) or massively parallel sequencing targeting ≥410 key cancer-related genes (n = 4). Somatic genetic alterations, mutational signatures, clonal composition, and phylogenetic analyses were defined using validated computational methods. RESULTS: DCIS revealed genetic alterations similar to those of synchronously diagnosed IDC-NSTs and of non-related IDC-NSTs from The Cancer Genome Atlas (TCGA), whereas pure DCIS lacked PIK3CA mutations. Clonal decomposition and phylogenetic analyses based on somatic mutations and copy number alterations revealed that the mechanisms of progression of DCIS to invasive carcinoma are diverse, and that clonal selection might have constituted the mechanism of progression from DCIS to invasive disease in 28% (7/25) of patients. DCIS displaying a pattern of clonal selection in the progression to invasive cancer harbored higher levels of intralesion genetic heterogeneity than DCIS where no clonal selection was observed. CONCLUSIONS: Intralesion genetic heterogeneity is a common feature in DCIS synchronously diagnosed with IDC-NST. DCIS is a nonobligate precursor of IDC-NST, whose mechanisms of progression to invasive breast cancer are diverse and vary from case to case.

Using game theory and decision decomposition to effectively discern and characterise bi-locus diseases.

In order to gain insight into oligogenic disorders, understanding those involving bi-locus variant combinations appears to be key. In prior work, we showed that features at multiple biological scales can already be used to discriminate among two types, i.e. disorders involving true digenic and modifier combinations. The current study expands this machine learning work towards dual molecular diagnosis cases, providing a classifier able to effectively distinguish between these three types. To reach this goal and gain an in-depth understanding of the decision process, game theory and tree decomposition techniques are applied to random forest predictors to investigate the relevance of feature combinations in the prediction. A machine learning model with high discrimination capabilities was developed, effectively differentiating the three classes in a biologically meaningful manner. Combining prediction interpretation and statistical analysis, we propose a biologically meaningful characterization of each class relying on specific feature strengths. Figuring out how biological characteristics shift samples towards one of three classes provides clinically relevant insight into the underlying biological processes as well as the disease itself.

Predicting disease-causing variant combinations.

Notwithstanding important advances in the context of single-variant pathogenicity identification, novel breakthroughs in discerning the origins of many rare diseases require methods able to identify more complex genetic models. We present here the Variant Combinations Pathogenicity Predictor (VarCoPP), a machine-learning approach that identifies pathogenic variant combinations in gene pairs (called digenic or bilocus variant combinations). We show that the results produced by this method are highly accurate and precise, an efficacy that is endorsed when validating the method on recently published independent disease-causing data. Confidence labels of 95% and 99% are identified, representing the probability of a bilocus combination being a true pathogenic result, providing geneticists with rational markers to evaluate the most relevant pathogenic combinations and limit the search space and time. Finally, the VarCoPP has been designed to act as an interpretable method that can provide explanations on why a bilocus combination is predicted as pathogenic and which biological information is important for that prediction. This work provides an important step toward the genetic understanding of rare diseases, paving the way to clinical knowledge and improved patient care.

Understanding mutational effects in digenic diseases.

To further our understanding of the complexity and genetic heterogeneity of rare diseases, it has become essential to shed light on how combinations of variants in different genes are responsible for a disease phenotype. With the appearance of a resource on digenic diseases, it has become possible to evaluate how digenic combinations differ in terms of the phenotypes they produce. All instances in this resource were assigned to two classes of digenic effects, annotated as true digenic and composite classes. Whereas in the true digenic class variants in both genes are required for developing the disease, in the composite class, a variant in one gene is sufficient to produce the phenotype, but an additional variant in a second gene impacts the disease phenotype or alters the age of onset. We show that a combination of variant, gene and higher-level features can differentiate between these two classes with high accuracy. Moreover, we show via the analysis of three digenic disorders that a digenic effect decision profile, extracted from the predictive model, motivates why an instance was assigned to either of the two classes. Together, our results show that digenic disease data generates novel insights, providing a glimpse into the oligogenic realm.

DEOGEN2: prediction and interactive visualization of single amino acid variant deleteriousness in human proteins.

High-throughput sequencing methods are generating enormous amounts of genomic data, giving unprecedented insights into human genetic variation and its relation to disease. An individual human genome contains millions of Single Nucleotide Variants: to discriminate the deleterious from the benign ones, a variety of methods have been developed that predict whether a protein-coding variant likely affects the carrier individual's health. We present such a method, DEOGEN2, which incorporates heterogeneous information about the molecular effects of the variants, the domains involved, the relevance of the gene and the interactions in which it participates. This extensive contextual information is non-linearly mapped into one single deleteriousness score for each variant. Since for the non-expert user it is sometimes still difficult to assess what this score means, how it relates to the encoded protein, and where it originates from, we developed an interactive online framework (http://deogen2.mutaframe.com/) to better present the DEOGEN2 deleteriousness predictions of all possible variants in all human proteins. The prediction is visualized so both expert and non-expert users can gain insights into the meaning, protein context and origins of each prediction.