Repertoire Of Somatic Genetic Alterations Research Articles

Abstract P2-23-15: Histologic, immunohistochemical and genomic comparison between classic Invasive lobular carcinomas and lobular-like invasive ductal carcinomas

Abstract Background: Invasive lobular carcinomas (ILCs) are the most frequent special histologic subtype of breast cancer, accounting for up to 15% of all breast cancer cases. ILCs are characterized by a distinctive discohesive growth pattern, with cells arranged in single cell infiltrative file and dispersed throughout the stroma, which stems from the loss of E-cadherin expression due to bi-allelic inactivation of the CDH1 gene. A subset of breast cancers display a similar single cell infiltrative growth pattern but, in contrast to classic ILC, display diffuse strong membranous E-cadherin reactivity and membranous p120 expression. We refer to such cases as “lobular-like invasive ductal carcinoma” (LLIDC), but it is unclear if this terminology is appropriate and if such cases show biallelic inactivation of CDH1, similarly to ILCs. Here, we sought to define whether LLIDCs would harbor bi-allelic alterations of CDH1 and to perform an exploratory, hypothesis generating analysis of the repertoire of somatic genetic alterations of LLIDCs and classic ILCs. Materials and methods: Representative H&Es, as well as sections subjected to E-cadherin and p120 immunohistochemistry from seven classic ILCs and seven bona fide “lobular-like invasive ductal carcinomas” were retrieved and independently reviewed by two pathologists with experience and expertise in breast pathology. DNA samples were extracted from representative sections from tumor and normal breast tissue from each patient and subjected to an FDA-approved targeted sequencing assay comprising the coding regions and selected regulatory elements of 515 genes. Somatic single nucleotide variants (SNVs) were detected with MuTect, indels with Strelka, Varscan2, Scalpel and Lancet. All mutations were manually inspected using the Integrative Genomics Viewer (IGV). The cancer cell fraction (CCF) of each mutation was inferred, as well as clonal probability, using ABSOLUTE. Copy number alterations and loss of heterozygosity were determined using FACETS. Mutational signatures were inferred using SigMA based on all synonymous and nonsynonymous somatic mutations. Results: Based on the histopathologic evaluation, of the 14 cases analyzed, seven were classified as ILC, and the other seven were classified as LLIDC. Sequencing analysis revealed that the classic ILCs harbored 16q LOH and CDH1 mutations (7/7), of which five were frameshift indel and two were splice site mutations consistently coupled with loss-of-heterozygosity (LOH) of the wild-type allele. Conversely, five of the seven LLIDCs did not harbor CDH1 mutations or genomic rearrangements. CDH1 mutations were identified in 2 LLIDCs: one harbored a subclonal CDH1 in-frame indel mutation coupled with LOH. This case displayed membranous E-cadherin and p120 expression with areas of aberrant expression. The other CDH1-mutated LLIDC harbored a complex in-frame indel with subclonal LOH. This case displayed membranous E-cadherin and p120 expression. The comparative analysis of the repertoire of somatic genetic alterations and mutational signatures present in LLIDCs and classic ILCs did not reveal any significant differences. Conclusion: Despite the histologic similarities, LLIDCs differ from classic lobular carcinomas based on the lack of CDH1 bi-allelic inactivation and the patterns of expression of E-cadherin and p120 catenin. Further whole-genome sequencing analyses are warranted to define the molecular basis of the discohesive cancer cells of LLIDC display. Citation Format: Edaise M. da Silva, Thais Basili, Jing Yu, Juan Blanco-Heredia, Pier Selenica, Qiqi Ye, Arnaud da Cruz Paula, Higinio Dopeso, Antonio Marra, Steffi Oesterreich, Jorge Reis-Filho, Rohit Bhargava. Histologic, immunohistochemical and genomic comparison between classic Invasive lobular carcinomas and lobular-like invasive ductal carcinomas [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-23-15.

Abstract HER2-07: HER2-07 Genomic Characterization of Primary and Metastatic HER2-low Breast Cancers

Abstract Background: Varying levels of HER2 expression without ERBB2 gene amplification can be detected by immunohistochemistry (IHC) in approximately 60% of all invasive breast cancers (BCs). HER2-low-expressing BCs have recently been shown to respond to novel anti-HER2 antibody drug conjugates. Several studies have demonstrated that HER2-low BCs do not seem to constitute a distinct clinical and transcriptomic subtype as compared to HER2-0 BCs. Here we sought to define the clinicopathologic features and repertoire of somatic genetic alterations in HER2-low BCs. Methods: We retrieved clinical, pathological, and genomic data of BCs that were subjected to targeted sequencing using the FDA-cleared MSK-IMPACT assay from April 2014 to December 2021. After removing cases where any available biopsy had HER2 3+ and/or positive ERBB2 fluorescence in situ hybridization (FISH), 3608 samples (primary=1347; post-treatment/metastatic=2261) were included. Tumors were classified as HER2-low if they had an HER2 IHC score of 1+ or 2+ with a non-amplified FISH assay and HER2-0 if they had an HER2 IHC score of 0. Somatic mutations and DNA copy number alterations from MSK-IMPACT were analyzed. Multiple testing correction using the Benjamini-Hochberg method was applied to control for the false discovery rate (q). Q values < 0.1 were considered significant. Results: Among 3608 HER2- BCs, 1460 (40%) and 2148 (60%) were HER2-0 and HER2-low, respectively. Hormone receptor (HR) expression was significantly higher in HER2-low than HER2-0 tumors in both primary (781 [68.3%] vs 362 [31.7%]; p< 0.001) and metastatic (1031 [60.5%] vs 673 [39.5%]; p< 0.001) samples. A higher proportion of HER2-low tumors was found in metastatic than primary samples (59% vs 41%; p< 0.001) in this cohort. No difference in histology subtype, tumor grade, disease stage (among primary tumors), mutational signatures, and tumor mutational burden was found overall and when cases were stratified by HR expression. In HR-positive BCs, HER2-0 BCs harbored higher frequency of TP53 (33% vs 25%; odds ratio [OR] 1.49, 95% confidence interval [CI] 1.25-1.78, q< 0.001) and CDKN1A (1% vs 0%; OR 17.47, 95% CI 2.48-756.37, q=0.02) alterations than HER2-low BCs. Similar findings were observed in metastatic but not in primary HR-positive BCs. No differences were detected in HR-negative BCs stratified into HER2-low and HER2-0. Given the potential misclassification that exists between IHC HER2-0 and HER2-1+, we then conducted an exploratory analysis splitting the HER2-low group into HER2 1+ and 2+. TP53 alterations remained significantly enriched in HER2-0 compared to HER2-1+ HR-positive tumors (33% vs 24%; OR 1.55, 95% CI 1.28-1.87, q< 0.001). In HR-positive BCs, HER2-2+ displayed a higher frequency of genetic alterations in genes encoding for transcription factors, such as MYC (14.2% vs 7.3%; OR 2.09, 95% CI 1.44-3.04, q=0.02) and YAP1 (2% vs 0.3%; OR 6.86, 95% CI 1.7-39.57, q=0.1), and DNA damage response, such as FAM175A (1.6% vs 0%; OR 18.23, 95% CI 2.43-807.73, q=0.03) and BRCA2 (4% vs 1%; OR 3.09, 95% CI 1.49-6.55, q=0.1), than HER2-0 tumors. In HR-negative HER2-2+ tumors, a higher frequency of PIK3CA mutations was observed in comparison to HER2-0 (36.9% vs 19.5%; OR 2.41, 1.4-4.1, q=0.1), overall and in the metastatic setting. Conclusions: HER2-low BCs seem not to represent a distinct pathologic subtype. At the genomic level, however, some differences were identified and these became more conspicuous upon subclassification of HER2 IHC expression into 1+ and 2+. Further investigation into methods that more accurately detect and quantify low levels of HER2 expression in BC samples as well as better characterize the biology behind the HER2-low/ultralow expression is warranted. Citation Format: Antonio Marra, Anton Safonov, Joshua Drago, Emanuela Ferraro, Pier Selenica, Andrea Gazzo, Giuseppe Curigliano, Shanu Modi, Pedram Razavi, Jorge Reis-Filho, Sarat Chandarlapaty. HER2-07 Genomic Characterization of Primary and Metastatic HER2-low Breast Cancers [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr HER2-07.

Clinical and pathologic features associated with PD-L1 (SP142) expression in stromal tumor-infiltrating immune cells of triple-negative breast carcinoma

The Ventana PD-L1 SP142 immunohistochemistry (IHC) assay is the FDA-approved companion diagnostic assay for atezolizumab therapy selection for patients with PD-L1-positive locally advanced or metastatic triple-negative breast carcinoma (TNBC). We aimed to elucidate clinical, pathologic, and molecular features associated with PD-L1 expression in TNBCs. Clinical, pathologic, and next-generation sequencing (NGS)-based molecular data for TNBCs tested with PD-L1 (SP142) IHC at our institution between 11/2018 and 12/2019 were retrieved and reviewed. PD-L1 positivity was defined as ≥1% IC staining. Patients with metastatic TNBC treated at first line with atezolizumab regimens were evaluated for treatment response and for time to treatment failure (TTF). Among 156 TNBCs, PD-L1 was positive in 47.4% of cases. Primary TNBCs were significantly more frequently PD-L1 positive, compared with recurrent/metastatic samples (p = 0.002). PD-L1-positive TNBCs had increased stromal IC, compared with PD-L1-negative samples (p < 0.001). The repertoire of somatic genetic alterations of PD-L1-positive and PD-L1-negative TNBCs was similar. Matched primary and recurrent/metastatic TNBC samples were available for eight patients, in whom four had discordant results. Thirty patients with metastatic TNBC were treated with atezolizumab regimens, with treatment failure occurring in 16 patients and a median TTF of 5.1 months in this early evaluation. The findings of this study show stromal ICs in primary TNBCs are more likely to show PD-L1 positivity than in recurrent or metastatic samples. This information should guide selection of samples suitable for testing. Further studies are needed to identify other features associated with PD-L1-positive breast carcinomas and clinical benefit of treatment.

The genomic landscape of metastatic histologic special types of invasive breast cancer

Histologic special types of breast cancer (BC) account for ~20% of BCs. Large sequencing studies of metastatic BC have focused on invasive ductal carcinomas of no special type (IDC-NSTs). We sought to define the repertoire of somatic genetic alterations of metastatic histologic special types of BC. We reanalyzed targeted capture sequencing data of 309 special types of BC, including metastatic and primary invasive lobular carcinomas (ILCs; n = 132 and n = 127, respectively), mixed mucinous (n = 5 metastatic and n = 14 primary), micropapillary (n = 12 metastatic and n = 8 primary), and metaplastic BCs (n = 6 metastatic and n = 5 primary), and compared metastatic histologic special types of BC to metastatic IDC-NSTs matched according to clinicopathologic characteristics and to primary special type BCs. The genomic profiles of metastatic and primary special types of BC were similar. Important differences, however, were noted: metastatic ILCs harbored a higher frequency of genetic alterations in TP53, ESR1, FAT1, RFWD2, and NF1 than primary ILCs, and in CDH1, PIK3CA, ERBB2, TBX3, NCOR1, and RFWD2 than metastatic IDC-NSTs. Metastatic ILCs displayed a higher mutational burden, and more frequently dominant APOBEC mutational signatures than primary ILCs and matched metastatic IDC-NSTs. ESR1 and NCOR mutations were frequently detected in metastatic mixed mucinous BCs, whereas PIK3CA and TP53 were the most frequently altered genes in metastatic micropapillary and metaplastic BCs, respectively. Taken together, primary and metastatic BCs histologic special types have remarkably similar repertoires of somatic genetic alterations. Metastatic ILCs more frequently harbor APOBEC mutational signatures than primary ILCs and metastatic IDC-NSTs.

Genomic Profiling Aids Classification of Diagnostically Challenging Uterine Mesenchymal Tumors With Myomelanocytic Differentiation.

Although diagnosis of high-grade uterine mesenchymal tumors (UMTs) exhibiting classic morphologic features is straightforward, diagnosis is more challenging in tumors in which prototypical features are poorly developed, focal, and/or coexist with features seen in other neoplasms. Here, we sought to define the repertoire of somatic genetic alterations in diagnostically challenging UMTs with myomelanocytic differentiation, including some reported as perivascular epithelioid cell tumors (PEComas). In 17 samples from 15 women, the tumors were histologically heterogenous. Immunohistochemical expression of at least 1 melanocytic marker (HMB45, Melan-A, or MiTF) was identified in all tumors, and of myogenic markers (desmin or smooth muscle actin) in most tumors. Targeted massively parallel sequencing revealed several genetic alterations, most commonly in TP53 (41% mutation, 12% deletion), TSC2 (29% mutation, 6% deletion), RB1 (18% deletion), ATRX (24% mutation), MED12 (12% mutation), BRCA2 (12% deletion), CDKN2A (6% deletion) as well as FGFR3, NTRK1, and ERBB3 amplification (each 6%). Gene rearrangements (JAZF1-SUZ12; DNAJB6-PLAG1; and SFPQ-TFE3) were identified in 3 tumors. Integrating histopathologic, immunohistochemical, and genetic findings, tumors from 4 patients were consistent with malignant PEComa (1 TFE3-rearranged); 6 were classified as leiomyosarcomas; 3 showed overlapping features of PEComa and other sarcoma types (leiomyosarcoma or low-grade endometrial stromal sarcoma); and 2 were classified as sarcoma, not otherwise specified. Our findings suggest that diagnostically challenging UMTs with myomelanocytic differentiation represent a heterogenous group of neoplasms which harbor a diverse repertoire of somatic genetic alterations; these genetic alterations can aid classification.

Abstract PD8-06: The genomic landscape of metastatic special histologic types of breast cancer

Abstract Introduction: Breast cancer (BC) is heterogeneous and comprises diverse entities with distinctive phenotypes, biology and clinical presentations. There are 21 special histologic types, accounting for approximately 20% of invasive BCs. Large-scale sequencing studies have focused preferentially on common histologic forms, and data on the genomic landscape of special histologic types, particularly in the metastatic setting, are scarce. Here, we sought to define the repertoire of somatic genetic alterations of metastatic special histologic types of BC. Materials and methods: We conducted a reanalysis of MSK-IMPACT targeted sequencing data of 346 special histologic types of BC from Razavi et al (Cancer Cell 2018), including invasive lobular (ILC; primary, n=129; metastasis, n=150), mucinous (primary n=11, metastasis n=5), micropapillary (primary n=8, metastasis n=13) and metaplastic carcinomas (primary, n=6; metastasis, n=11). Somatic single nucleotide variants (SNVs) and copy number alterations (CNAs) were inferred using a validated bioinformatics pipeline, and mutational signatures were assessed with SigMA. Genetic alterations of metastatic special histologic type BCs were compared to those of primary tumors and metastatic invasive ductal carcinomas of no special type (IDC-NSTs) matched by age, menopausal status and estrogen receptor (ER)/HER2 status at a 1:2 ratio for ILCs and at a 1:3 ratio for the remaining BCs. Results: The most frequently mutated genes in metastatic ILCs were CDH1 (71%), PIK3CA (49%), ESR1 (20%) and KMT2C (19%). ESR1 mutations were more frequent in metastatic than in primary ILCs (20% vs 3%; P&amp;lt;0.01). Metastatic ILCs, as compared to matched metastatic IDC-NSTs, had a higher frequency of mutations in CDH1 (71% vs 2%; P&amp;lt;0.001), KMT2C (19% vs 10%; P&amp;lt;0.05), TBX3 (16% vs 5%; P&amp;lt;0.001), ERBB2 (12% vs 3%; P&amp;lt;0.01) and FOXA1 (10% vs 2%; P&amp;lt;0.05), and a lower frequency of mutations in TP53 (16% vs 36%; P&amp;lt;0.001) and GATA3 (6% vs 16%; P&amp;lt;0.01). GATA3, NCOR1 and ESR1 (each, 40%) were the most frequently mutated genes in metastatic mucinous BCs. ESR1 and NCOR were numerically more frequent in metastatic than in primary mucinous BCs (40% vs 0%; P&amp;gt;0.05). Metastatic mucinous BCs had a numerically higher frequency of mutations in ESR1 (40% vs 6%; P&amp;gt;0.05) and GATA3 (40% vs 0%; P&amp;gt;0.05), and lower frequency of TP53 mutations (20% vs 53%; P&amp;gt;0.05) than matched metastatic IDC-NSTs. PIK3CA and TP53 were the most recurrently mutated genes in metastatic micropapillary BCs and were numerically more frequent in metastatic than in primary tumors (PIK3CA, 61% vs 25%; TP53, 46% vs 25%; P&amp;gt;0.05). PIK3CA was more frequently mutated in metastatic micropapillary BCs than IDC-NSTs (61% vs 23%; P&amp;lt;0.05). Metastatic metaplastic BCs most frequently harbored mutations in TP53 (72%) and PIK3CA (45%), with comparable frequencies in primary metaplastic BCs. ATM (27% vs 3%; P&amp;lt;0.05), MAP3K13 and YAP1 (27% vs 0%; P&amp;lt;0.05) were more frequently mutated in metastatic metaplastic BCs than in matched IDC-NSTs. No differences in the frequency of CNAs were seen between metastatic BCs of special histologic type and matched primary tumors or metastatic IDC-NSTs. Akin to metastatic IDC-NSTs, aging or APOBEC were the predominant mutational SNV signatures in metastatic ILCs, micropapillary and metaplastic BCs. Conclusions: The repertoire of somatic genetic alterations of primary and metastatic special histologic type BCs markedly overlaps. Nonetheless, there is an enrichment of mutations in ESR1 in metastatic BCs of typically ER-positive special histologic type. Metastatic BCs of special histologic type show an enrichment of similar genetic alterations as primary tumors, such as a higher frequency of mutations in CDH1, FOXA1 and TBX3 in ILCs, a lower frequency of PIK3CA and TP53 mutations in mucinous BCs and a higher frequency of PIK3CA and TP53 mutations in micropapillary and metaplastic BCs. Citation Format: Fresia Pareja, Simon SK Lee, Francisco Beca, Lorenzo Ferrando, Pier Selenica, David N Brown, Hannah Y Wen, Hong Zhang, Edi Brogi, Pedram Razavi, Sarat Chandarlapaty, Britta Weigelt, Jorge S Reis-Filho. The genomic landscape of metastatic special histologic types of breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD8-06.

Homologous recombination DNA repair defects in PALB2-associated breast cancers

Mono-allelic germline pathogenic variants in the Partner And Localizer of BRCA2 (PALB2) gene predispose to a high-risk of breast cancer development, consistent with the role of PALB2 in homologous recombination (HR) DNA repair. Here, we sought to define the repertoire of somatic genetic alterations in PALB2-associated breast cancers (BCs), and whether PALB2-associated BCs display bi-allelic inactivation of PALB2 and/or genomic features of HR-deficiency (HRD). Twenty-four breast cancer patients with pathogenic PALB2 germline mutations were analyzed by whole-exome sequencing (WES, n = 16) or targeted capture massively parallel sequencing (410 cancer genes, n = 8). Somatic genetic alterations, loss of heterozygosity (LOH) of the PALB2 wild-type allele, large-scale state transitions (LSTs) and mutational signatures were defined. PALB2-associated BCs were found to be heterogeneous at the genetic level, with PIK3CA (29%), PALB2 (21%), TP53 (21%), and NOTCH3 (17%) being the genes most frequently affected by somatic mutations. Bi-allelic PALB2 inactivation was found in 16 of the 24 cases (67%), either through LOH (n = 11) or second somatic mutations (n = 5) of the wild-type allele. High LST scores were found in all 12 PALB2-associated BCs with bi-allelic PALB2 inactivation sequenced by WES, of which eight displayed the HRD-related mutational signature 3. In addition, bi-allelic inactivation of PALB2 was significantly associated with high LST scores. Our findings suggest that the identification of bi-allelic PALB2 inactivation in PALB2-associated BCs is required for the personalization of HR-directed therapies, such as platinum salts and/or PARP inhibitors, as the vast majority of PALB2-associated BCs without PALB2 bi-allelic inactivation lack genomic features of HRD.

Genomic analysis of recurrences and high-grade forms of polymorphous adenocarcinoma.

Polymorphous adenocarcinoma (PAC) usually follows an indolent course, but some cases may show recurrences and high-grade features. The genetic events associated with recurrences and high-grade versions are yet to be defined. Our aim was to determine the genetic underpinning of recurrent PACs of the salivary gland and the repertoire of somatic genetic alterations in cases with high-grade histology. Four PACs from three patients, including one case with matching primary and recurrent tumours, one de-novo high-grade PAC, and a PAC that transformed to a high-grade tumour following multiple recurrences, were subjected to targeted sequencing (Memorial Sloan Kettering Mutation Profiling of Actionable Cancer Targets assay) or whole-exome sequencing. Both matching primary and recurrent tumours, and the de-novo high-grade PAC, harboured clonal PRKD1 E710D hotspot mutations, whereas the PAC that underwent high-grade transformation upon recurrence, which was wild-type for PRKD1, harboured a PRKD2 rearrangement. The PACs analysed here also harboured mutations targeting cancer genes such as PIK3CA, SETD2, ARID1A, and NOTCH2. A clonal decomposition analysis of the matching primary and recurrent PACs revealed that a minor subclone from the primary tumour became dominant in the recurrent tumour following a clonal selection evolutionary pattern. Our findings demonstrate that recurrent and high-grade PACs are underpinned by PRKD1 E710D hotspot mutations or PRKD2 rearrangements, and that recurrences of PACs may stem from the selection of pre-existing subclones in the primary tumour.

Massively parallel sequencing analysis of benign melanocytic naevi.

Melanocytic naevi are benign lesions of the skin or mucosa that may constitute non-obligate precursors of malignant melanoma, particularly when they show lentiginous and dysplastic features. The aim of this study was to investigate the repertoire of somatic genetic alterations in melanocytic naevi. DNA extracted from 12 melanocytic naevi and DNA from matching normal tissue were separately microdissected and subjected to targeted massively parallel sequencing of ≥300 cancer genes. A median of 5.5 (range 1-12) non-synonymous somatic mutations were detected, with 10 cases harbouring mutually exclusive BRAF V600E (6/12) or NRAS (4/12) clonal hotspot mutations. One of the two cases lacking BRAF and NRAS mutations was a dysplastic naevus harbouring an HRAS Q61L hotspot mutation. Analysis of the laser-capture microdissected components of a naevus synchronously diagnosed with in-situ and invasive malignant melanoma revealed a truncal, clonal BRAF V600E mutation, and the acquisition of a CDKN2A homozygous deletion in the invasive component, in conjunction with additional clonal mutations affecting NF2, FAT4 and KDR in both in-situ and invasive malignant components. Melanocytic naevi harbour recurrent BRAF V600E or NRAS hotspot mutations with low mutational burdens. Our findings also show that progression from naevi to malignant melanoma may be driven by the acquisition of additional genetic alterations, including CDKN2A homozygous deletions.

Abstract P3-06-08: Mucinous and neuroendocrine breast cancers: A spectrum of genetically-related lesions distinct from common forms of estrogen receptor-positive breast cancers

Abstract Introduction: Mucinous (MBCs) and neuroendocrine breast carcinomas (NBCs) are special histologic types of estrogen receptor (ER)-positive breast cancers. Together, MBCs and NBCs account for approximately 5% of invasive breast cancers. NBCs are defined by the expression of neuroendocrine markers (i. e., chromogranin and/ or synaptophysin), whereas MBCs consist of nests of cancer cells floating in pools of extracellular mucin and can be either hypocellular/type A (MBC-A) or hypercellular/type B (MBC-B). MBC-B may display neuroendocrine differentiation. We compared the repertoire of somatic genetic alterations in MBCs-A and MBCs-B, and in NBCs, and evaluated whether they differ from ER-positive/ HER2-negative invasive ductal carcinomas of no special type (IDC-NSTs). Materials and methods: Reanalysis of the targeted capture or whole exome sequencing data of 22 MBCs (12 MBCs-A and 10 MBCs-B), and of 15 NBCs was performed. The BAM files were retrieved and somatic mutations were detected with MuTect, indels with Strelka, Varscan2, Scalpel and Lancet, and copy number alterations using FACETS. The mutational repertoire of MBCs-A and MCBs-B was compared to that of NBCs, and of ER-positive/ HER2-negative IDC-NSTs from The Cancer Genome Atlas (TCGA) breast cancer study (n=310). Results: The most frequently mutated genes in MBCs-A were SF3B1 and FRG1B (17%, each); in MBCs-B were GATA3 and KMT2C (30%, each), and in NBCs were FOXA1 and TBX3 (20%, each) and KMT2C (20%). No significant differences were observed in single gene comparisons between MBCs-A, MBCs-B and NBCs (Fisher's exact tests, p&amp;gt;0.05). When compared with ER-positive/HER2-negative IDC-NSTs from TCGA, NBCs harbored a higher frequency of mutations targeting FOXA1 (20% vs 2.9%, Fisher's exact test, p&amp;lt;0.05) and TBX3 (20% vs 2.9%, Fisher's exact test, p&amp;lt;0.05), and a lower frequency of TP53 mutations (0% vs 23.9%; Fisher's exact test, p&amp;lt;0.05). We observed a lower frequency of PIK3CA mutations in both MBCs-A (8.3% vs 40%, Fisher's exact test, p&amp;lt;0.05) and MBCs-B (0% vs 40%; Fisher's exact test, p&amp;lt;0.01) compared to ER-positive/HER2-negative IDC-NSTs. Although not statistically significant, PIK3CA mutations were numerically less frequent in NBCs than in ER+/HER2- IDC-NSTs (13.3% vs 40%; Fisher's exact test, p&amp;gt;0.05). Concurrent 1q gains and 16q losses, the hallmark copy number alterations of ER-positive/HER2-negative breast cancer were found in 47% of the ER-positive/HER2-negative IDC-NSTs from TCGA, but only present in 20% of NBCs and in none of the MBCs-A or MBCs-B. Conclusion: Type A MBCs, type B MBCs and NBCs harbor similar patterns of genetic alterations, including a low frequency of PIK3CA mutations and of concurrent 1q gains/ 16q deletions. Taken together, our data suggest that both MBCs and the vast majority of NBCs constitute a spectrum of histologically and genomically related subtypes, distinct from ER-positive/HER2-negative IDC-NSTs. Citation Format: Pareja F, Da Cruz Paula A, Selenica P, Lee JY, Marchio C, Brown D, Geyer FC, Brogi E, Vincent-Salomon A, Weigelt B, Reis-Filho JS. Mucinous and neuroendocrine breast cancers: A spectrum of genetically-related lesions distinct from common forms of estrogen receptor-positive breast cancers [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-06-08.

Assessment of HMGA2 and PLAG1 rearrangements in breast adenomyoepitheliomas

Breast adenomyoepitheliomas (AMEs) are rare epithelial-myoepithelial neoplasms that may occasionally produce myxochondroid matrix, akin to pleomorphic adenomas (PAs). Regardless of their anatomic location, PAs often harbor rearrangements involving HMGA2 or PLAG1. We have recently shown that the repertoire of somatic genetic alterations of AMEs varies according to their estrogen receptor (ER) status; whilst the majority of ER-positive AMEs display mutually exclusive PIK3CA or AKT1 hotspot mutations, up to 60% of ER-negative AMEs harbor concurrent HRAS Q61 hotspot mutations and mutations affecting either PIK3CA or PIK3R1. Here, we hypothesized that a subset of AMEs lacking these somatic genetic alterations could be underpinned by oncogenic fusion genes, in particular those involving HMGA2 or PLAG1. Therefore, we subjected 13 AMEs to RNA-sequencing for fusion discovery (n = 5) and/or fluorescence in situ hybridization (FISH) analysis for HMGA2 and PLAG1 rearrangements (n = 13). RNA-sequencing revealed an HMGA2-WIF1 fusion gene in an ER-positive AME lacking HRAS, PIK3CA and AKT1 somatic mutations. This fusion gene, which has been previously described in salivary gland PAs, results in a chimeric transcript composed of exons 1–5 of HMGA2 and exons 3–10 of WIF1. No additional in-frame fusion genes or HMGA2 or PLAG1 rearrangements were identified in the remaining AMEs analyzed. Our results demonstrate that a subset of AMEs lacking mutations affecting HRAS and PI3K pathway-related genes may harbor HMGA2-WIF1 fusion genes, suggesting that a subset of breast AMEs may be genetically related to PAs or that a subset of AMEs may originate in the context of a PA.

Somatic genetic alterations in synchronous and metachronous low-grade serous tumours and high-grade carcinomas of the adnexa.

Low-grade serous carcinomas (LGSCs) and their precursors serous borderline tumours (SBTs) characteristically harbour mutations in BRAF, KRAS or NRAS but rarely in TP53, whereas high-grade serous carcinomas (HGSCs) are characterised by frequent TP53 mutations but rare BRAF, KRAS or NRAS mutations. In a small subset of cases, LGSCs and/or SBTs develop into high-grade tumours, including HGSCs and poorly differentiated carcinomas (PDCs). Here, we sought to define the repertoire of somatic genetic alterations in low-grade serous tumours and synchronous or metachronous high-grade adnexal carcinomas. DNA extracted from five SBTs/LGSCs and synchronous or metachronous HGSCs/PDCs and matched normal tissue was subjected to massively parallel sequencing targeting all exons and selected non-coding regions of 341 cancer-related genes. The low-grade and high-grade tumours from a given case were related, and shared mutations and copy number alterations. Progression from low-grade to high-grade lesions was observed, and involved the acquisition of additional mutations and/or copy number alterations, or shifts from subclonal to clonal mutations. Only two (an HGSC and a PDC) of the five high-grade tumours investigated harboured TP53 mutations, whereas NRAS and KRAS hotspot mutations were seen in two HGSCs and one HGSC, respectively. Our results suggest that progression from SBT to HGSC may take place in a subset of cases, and that at least some of the rare HGSCs lacking TP53 mutations may be derived from a low-grade serous precursor.

Mutation Profiling of Key Cancer Genes in Primary Breast Cancers and Their Distant Metastases.

Although the repertoire of somatic genetic alterations of primary breast cancers has been extensively catalogued, the genetic differences between primary and metastatic tumors have been less studied. In this study, we compared somatic mutations and gene copy number alterations of primary breast cancers and their matched metastases from patients with estrogen receptor (ER)-negative disease. DNA samples obtained from formalin-fixed paraffin-embedded ER-negative/HER2-positive (n = 9) and ER-, progesterone receptor (PR-), HER2-negative (n = 8) primary breast cancers and from paired brain or skin metastases and normal tissue were subjected to a hybridization capture-based massively parallel sequencing assay, targeting 341 key cancer genes. A large subset of nonsynonymous somatic mutations (45%) and gene copy number alterations (55%) was shared between the primary tumors and paired metastases. However, mutations restricted to either a given primary tumor or its metastasis, the acquisition of loss of heterozygosity of the wild-type allele, and clonal shifts of genes affected by somatic mutations, such as TP53 and RB1, were observed in the progression from primary tumors to metastases. No metastasis location-specific alterations were identified, but synchronous metastases showed higher concordance with the paired primary tumor than metachronous metastases. Novel potentially targetable alterations were found in the metastases relative to their matched primary tumors. These data indicate that repertoires of somatic genetic alterations in ER-negative metastatic breast cancers may differ from those of their primary tumors, even by the presence of driver and targetable somatic genetic alterations.Significance: Somatic genetic alterations in ER-negative breast cancer metastases may be distinct from those of their primary tumors, suggesting that for treatment-decision making, genetic analyses of DNA obtained from the metastatic lesion rather than from the primary tumor should be considered. Cancer Res; 78(12); 3112-21. ©2018 AACR.

Abstract PD1-15: The landscape of somatic genetic alterations in breast cancers from ATM germline mutation carriers

Abstract Introduction:Pathogenic and/or founder germline variants in the ataxia-telangiectasia mutated (ATM) gene confer an increased breast cancer (BC) risk. The protein kinase ATM plays a central role inDNA double-strand break-repair and in the activation of downstream targets such as p53 and BRCA1. We sought to define the repertoire of somatic genetic alterations of BCs from patients with pathogenic germline ATM mutations and whether somatic loss of heterozygosity (LOH) of ATM would be present in these cancers. Methods: 21 BCs from ATM germline mutation carriers were microdissected. Tumor and normal DNA samples were subjected to whole-exome sequencing (WES, n=12) or massively parallel sequencing targeting all coding regions and selected intronic and regulatory regions of 410 key cancer genes (n=9). Somatic mutations, copy number alterations, cancer cell fractions, large-scale state transitions (LSTs) and mutational signatures were defined using state-of-the-art bioinformatics algorithms. ABSOLUTE and FACETS were employed to assess LOH of the wild-type allele of ATM. Results: Of the patients included in this study, 71%, 24% and 5% of cases harbored ATM missense (all but one p.V2424G), frame-shift and nonsense germline mutations, respectively. All tumors were ER-positive and four (19%) were HER2-positive. The median age of the patients was 46 years (32–79 years). Our analyses revealed biallelic inactivation of ATM through LOH of the wild-type allele in 16 of 21 cases (76%), and second somatic ATM mutations were not found. The median number of non-synonymous somatic mutations was 38 (range 15-113) and 2 (range 0-8)in tumors subjected to WES and targeted sequencing, respectively. The repertoire of somatic genetic alterations of ATM-associated BCs was found to be heterogeneous, including clonal PIK3CA mutations (24%), GATA3 mutations (19%), FANCI amplifications (19%) and CCND1 amplifications (14%). Importantly, however, no somatic mutations affecting TP53 were found. Analysis of the WES data revealed that 5 (42%) ATM-associated BCs displayed high LST scores, all of which harbored bi-allelic ATM inactivation. In contrast to BRCA1- and BRCA2-associated BCs, which frequently display the mutational signature 3 associated with defective homologous recombination DNA repair, the ATM-associated BCs studied displayed the ageing mutational signature (i.e. signature 1). Comparison of the mutational profiles of the ATM--associated BCs subjected to WES (n=12) with those of BRCA1- (n=11) and BRCA2-associated (n=10) BCs from The Cancer Genome Atlas revealed that TP53 was more frequently mutated in BCs from BRCA1 germline mutation carriers (0% vs 72%, P&amp;lt;0.001), while no differences with BRCA2-associated BCs were found. Conclusion: ATM-associated BCs frequently display bi-allelic ATM inactivation through LOH of the wild-type allele and a subset of these cases displayed high levels of LSTs. These findings suggest that at least in a subset of ATM-associated BCs, biallelic inactivation of ATM rather than a dominant negative effect of the germline mutation may be the mechanism of inactivation of this tumor suppressor gene. The repertoire of somatic genetic alterations of ATM-associated BCs is heterogeneous, with a noticeable lack of TP53 somatic mutations. Citation Format: Weigelt B, Bi R, Kumar R, James PA, Thorne H, Couch FJ, Eccles DM, Blows F, Geyer FC, Li A, Selenica P, Lim RS, Blecua P, Shen R, Wen H, Robson ME, Reis-Filho JS, Chenevix-Trench G. The landscape of somatic genetic alterations in breast cancers from ATM germline mutation carriers [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD1-15.

The genetic landscape of endometrial clear cell carcinomas.

Clear cell carcinoma of the endometrium is a rare type of endometrial cancer that is generally associated with an aggressive clinical behaviour. Here, we sought to define the repertoire of somatic genetic alterations in endometrial clear cell carcinomas (ECCs), and whether ECCs could be classified into the molecular subtypes described for endometrial endometrioid and serous carcinomas. We performed a rigorous histopathological review, immunohistochemical analysis and massively parallel sequencing targeting 300 cancer-related genes of 32 pure ECCs. Eleven (34%), seven (22%) and six (19%) ECCs showed abnormal expression patterns for p53, ARID1A, and at least one DNA mismatch repair (MMR) protein, respectively. Targeted sequencing data were obtained from 30 of the 32 ECCs included in this study, and these revealed that two ECCs (7%) were ultramutated and harboured mutations affecting the exonuclease domain of POLE. In POLE wild-type ECCs, TP53 (46%), PIK3CA (36%), PPP2R1A (36%), FBXW7 (25%), ARID1A (21%), PIK3R1 (18%) and SPOP (18%) were the genes most commonly affected by mutations; 18% and 11% harboured CCNE1 and ERBB2 amplifications, respectively, and 11% showed DAXX homozygous deletions. ECCs less frequently harboured mutations affecting CTNNB1 and PTEN but more frequently harboured PPP2R1A and TP53 mutations than non-POLE endometrioid carcinomas from The Cancer Genome Atlas (TCGA). Compared to endometrial serous carcinomas (TCGA), ECCs less frequently harboured TP53 mutations. When a surrogate model for the molecular-based TCGA classification was used, all molecular subtypes previously identified in endometrial endometrioid and serous carcinomas were present in the ECCs studied, including POLE, MMR-deficient, copy-number high (serous-like)/p53 abnormal, and copy-number low (endometrioid)/p53 wild-type, which were significantly associated with disease-free survival in univariate analysis. These findings demonstrate that ECCs constitute a histologically and genetically heterogeneous group of tumours with varying outcomes. Furthermore, our data suggest that the classification of ECCs as being generally 'high-grade' or 'type II' tumours may not be warranted. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Genetic Heterogeneity in Therapy-Naïve Synchronous Primary Breast Cancers and Their Metastases.

Purpose: Paired primary breast cancers and metachronous metastases after adjuvant treatment are reported to differ in their clonal composition and genetic alterations, but it is unclear whether these differences stem from the selective pressures of the metastatic process, the systemic therapies, or both. We sought to define the repertoire of genetic alterations in breast cancer patients with de novo metastatic disease who had not received local or systemic therapy.Experimental Design: Up to two anatomically distinct core biopsies of primary breast cancers and synchronous distant metastases from nine patients who presented with metastatic disease were subjected to high-depth whole-exome sequencing. Mutations, copy number alterations and their cancer cell fractions, and mutation signatures were defined using state-of-the-art bioinformatics methods. All mutations identified were validated with orthogonal methods.Results: Genomic differences were observed between primary and metastatic deposits, with a median of 60% (range 6%-95%) of shared somatic mutations. Although mutations in known driver genes including TP53, PIK3CA, and GATA3 were preferentially clonal in both sites, primary breast cancers and their synchronous metastases displayed spatial intratumor heterogeneity. Likely pathogenic mutations affecting epithelial-to-mesenchymal transition-related genes, including SMAD4, TCF7L2, and TCF4 (ITF2), were found to be restricted to or enriched in the metastatic lesions. Mutational signatures of trunk mutations differed from those of mutations enriched in the primary tumor or the metastasis in six cases.Conclusions: Synchronous primary breast cancers and metastases differ in their repertoire of somatic genetic alterations even in the absence of systemic therapy. Mutational signature shifts might contribute to spatial intratumor genetic heterogeneity. Clin Cancer Res; 23(15); 4402-15. ©2017 AACR.

The Landscape of Somatic Genetic Alterations in Metaplastic Breast Carcinomas.

Purpose: Metaplastic breast carcinoma (MBC) is a rare and aggressive histologic type of breast cancer, predominantly of triple-negative phenotype, and characterized by the presence of malignant cells showing squamous and/or mesenchymal differentiation. We sought to define the repertoire of somatic genetic alterations and the mutational signatures of MBCs.Experimental Design: Whole-exome sequencing was performed in 35 MBCs, with 16, 10, and 9 classified as harboring chondroid, spindle, and squamous metaplasia as the predominant metaplastic component. The genomic landscape of MBCs was compared with that of triple-negative invasive ductal carcinomas of no special type (IDC-NST) from The Cancer Genome Atlas. Wnt and PI3K/AKT/mTOR pathway activity was assessed using a qPCR assay.Results: MBCs harbored complex genomes with frequent TP53 (69%) mutations. In contrast to triple-negative IDC-NSTs, MBCs more frequently harbored mutations in PIK3CA (29%), PIK3R1 (11%), ARID1A (11%), FAT1 (11%), and PTEN (11%). PIK3CA mutations were not found in MBCs with chondroid metaplasia. Compared with triple-negative IDC-NSTs, MBCs significantly more frequently harbored mutations in PI3K/AKT/mTOR pathway-related (57% vs. 22%) and canonical Wnt pathway-related (51% vs. 28%) genes. MBCs with somatic mutations in PI3K/AKT/mTOR or Wnt pathway-related genes displayed increased activity of the respective pathway.Conclusions: MBCs are genetically complex and heterogeneous, and are driven by a repertoire of somatic mutations distinct from that of triple-negative IDC-NSTs. Our study highlights the genetic basis and the importance of PI3K/AKT/mTOR and Wnt pathway dysregulation in MBCs and provides a rationale for the metaplastic phenotype and the reported responses to PI3K/AKT/mTOR inhibitors in these tumors. Clin Cancer Res; 23(14); 3859-70. ©2017 AACR.

Abstract P2-03-01: Mutational landscape of breast cancers from PALB2 germline mutation carriers

Abstract Background: The PALB2 gene encodes the partner and localizer of the BRCA2 protein, which participates in homologous recombination during DNA repair via an interaction with BRCA1 and BRCA2. Germline mutations in PALB2 are associated with an increased risk of breast cancer, with a cumulative risk of 35% by age 70 in female PALB2 mutant carriers. The aims of this project were to characterize the genomic landscape of PALB2 breast cancers and define the differences in the repertoire of somatic genetic alterations and mutational signatures between PALB2, BRCA1 and BRCA2 breast cancers. Methods: Representative samples from fourteen breast cancers from patients with known PALB2 germline mutations (seven frame-shift (2 H1170fs, 3 K346fs, 1 T841fs and 1 L531fs), five truncating (3 W1038* and 2 Q775*) and two missense (W1140G and L35P)) were microdissected to ensure a tumor cell content of &amp;gt;70%. DNA samples from microdissected tumors and their matched normal counterparts were subjected to whole exome sequencing on an Illumina HiSeq2000 to a median depth of 118x (range 33-193x). Somatic single nucleotide variations were detected using MuTect, and small insertions and deletions were identified using Strelka and Varscan2. Using ABSOLUTE and FACETS, we investigated the presence of loss of heterozygosity (LOH) of the PALB2 wild-type allele in these tumors. In addition, the mutational signatures and large scale state transitions (LSTs) were defined. The repertoire of somatic mutations identified in PALB2 breast cancers was compared to that of breast cancers from BRCA1 (n=11) and BRCA2 (n=10) germline mutation carriers from The Cancer Genome Atlas study. Results: PALB2 breast cancers were found to harbor a median of 80 somatic mutations (range 22-286) and one somatic mutation (range 0-13) affecting known cancer genes. Somatic loss of the PALB2 wild-type allele was found in five cases, and in three additional cases, a second PALB2 somatic mutation likely constituted the second 'hit' (two with truncating mutations, Q479* and Q61*, and one with a Q921fs frameshift mutation). Six PALB2 breast cancers displayed the BRCA mutations signature; of these, five had PALB2 bi-allelic inactivation (three LOH of the wild-type allele and two a second PALB2 somatic mutation). 71% of the samples were found to have LSTs, including all cases with a BRCA mutational signature. A significant association between PALB2 bi-allelic inactivation and concurrent BRCA signature and high LST was observed (p=0.015). Breast cancers from PALB2 mutation carriers had fewer somatic TP53 mutations than BRCA1 breast cancers (3/14, 21% vs 9/11, 82%, p=0.004), but no difference in the repertoire of somatic mutations compared to that of BRCA2 breast cancers. Conclusions: PALB2 breast cancers were found to harbor pathogenic mutations in driver genes, including TP53, PIK3CA, NF1 and NCOR1, however lacked highly recurrent somatic mutations. Unlike BRCA1/2 breast cancers, the majority of breast cancers from PALB2 germline mutation carriers lacked LOH of the PALB2 wild-type allele. Importantly, however, an association between PALB2 bi-allelic inactivation and the BRCA mutational signature and LSTs was observed, providing additional evidence for a homologous recombination-deficient phenotype at least in a subset of PALB2 cancers. Citation Format: Burke KA, Berman S, Geyer FC, Piscuoglio S, Ng CK, Wen YH, Mannermaa A, Peterlongo P, Tondini C, Janatova M, Soo Hwang T, Ng P-S, Looi LM, Chenevix-Trench G, Southey MC, Weigelt B, Foulkes W, Tischkowitz M, Reis-Filho JS, PALB2 Interest Group. Mutational landscape of breast cancers from PALB2 germline mutation carriers [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-03-01.

Genetic events in the progression of adenoid cystic carcinoma of the breast to high-grade triple-negative breast cancer

AbstractAdenoid cystic carcinoma of the breast is a rare histological type of triple-negative breast cancer with an indolent clinical behavior, often driven by the MYB-NFIB fusion gene. Here we sought to define the repertoire of somatic genetic alterations in two adenoid cystic carcinomas associated with high-grade triple-negative breast cancer. The different components of each case were subjected to copy number profiling and massively parallel sequencing targeting all exons and selected regulatory and intronic regions of 488 genes. Reverse transcription PCR and fluorescence in situ hybridization were employed to investigate the presence of the MYB-NFIB translocation. The MYB-NFIB fusion gene was detected in both adenoid cystic carcinomas and their associated high-grade triple-negative breast cancer components. Although the distinct components of both cases displayed similar patterns of gene copy number alterations, massively parallel sequencing analysis revealed intratumor genetic heterogeneity. In case 1, progression from the trabecular adenoid cystic carcinoma to the high-grade triple-negative breast cancer was found to involve clonal shifts with enrichment of mutations affecting EP300, NOTCH1, ERBB2 and FGFR1 in the high-grade triple-negative breast cancer. In case 2, a clonal KMT2C mutation was present in the cribriform adenoid cystic carcinoma, solid adenoid cystic carcinoma and high-grade triple-negative breast cancer components, whereas a mutation affecting MYB was present only in the solid and high-grade triple-negative breast cancer areas and additional three mutations targeting STAG2, KDM6A and CDK12 were restricted to the high-grade triple-negative breast cancer. In conclusion, adenoid cystic carcinomas of the breast with high-grade transformation are underpinned by the MYB-NFIB fusion gene and, akin to other forms of cancer, may be constituted by a mosaic of cancer cell clones at diagnosis. The progression from adenoid cystic carcinoma to high-grade triple-negative breast cancer of no special type may involve the selection of neoplastic clones and/or the acquisition of additional genetic alterations.

The Genomic Landscape of Male Breast Cancers

Male breast cancer is rare, and its genomic landscape has yet to be fully characterized. Lacking studies in men, treatment of males with breast cancer is extrapolated from results in females with breast cancer. We sought to define whether male breast cancers harbor somatic genetic alterations in genes frequently altered in female breast cancers. All male breast cancers were estrogen receptor-positive, and all but two were HER2-negative. Fifty-nine male breast cancers were subtyped by immunohistochemistry, and tumor-normal pairs were microdissected and subjected to massively parallel sequencing targeting all exons of 241 genes frequently mutated in female breast cancers or DNA-repair related. The repertoires of somatic mutations and copy number alterations of male breast cancers were compared with that of subtype-matched female breast cancers. Twenty-nine percent and 71% of male breast cancers were immunohistochemically classified as luminal A-like or luminal B-like, respectively. Male breast cancers displayed a heterogeneous repertoire of somatic genetic alterations that to some extent recapitulated that of estrogen receptor (ER)-positive/HER2-negative female breast cancers, including recurrent mutations affecting PIK3CA (20%) and GATA3 (15%). ER-positive/HER2-negative male breast cancers, however, less frequently harbored 16q losses, and PIK3CA and TP53 mutations than ER-positive/HER2-negative female breast cancers. In addition, male breast cancers were found to be significantly enriched for mutations affecting DNA repair-related genes. Male breast cancers less frequently harbor somatic genetic alterations typical of ER-positive/HER2-negative female breast cancers, such as PIK3CA and TP53 mutations and losses of 16q, suggesting that at least a subset of male breast cancers are driven by a distinct repertoire of somatic changes. Given the genomic differences, caution may be needed in the application of biologic and therapeutic findings from studies of female breast cancers to male breast cancers. Clin Cancer Res; 22(16); 4045-56. ©2016 AACR.