Hobnail variant of papillary thyroid carcinoma (HPTC) represents a recently described, aggressive and rare group of thyroid tumors with poorly understood pathogenesis. Molecular data about this group ...of cancers are few, and a more detailed molecular characterization of these tumors is needed. The main objective of the study is to define a comprehensive molecular typing of HPTC. Eighteen patients affected by HPTC, including eighteen primary tumors and four lymph node metastases, were screened for NRAS, KRAS, HRAS, BRAF, TP53, PIK3CA, hTERT, PTEN, CDKN2A, EGFR, AKT1, CTNNB1 and NOTCH1 gene mutations. Sequencing is conducted on the MiSEQ system, and molecular data are compared with clinical-pathologic data and follow-up. The patients include 14 women and 4 men. Ages range from 23 to 87 years. All 18 primary tumors of HPTC showed ≥30% hobnail features. BRAF and TP53 mutations are by far the most common genetic alterations in primary HPTC (72.2% and 55.6%, respectively), followed by hTERT (44.4%), PIK3CA (27.8%), CTNNB1 (16.7%), EGFR (11.1%), AKT1 (5.5%) and NOTCH1 (5.5%). The mutational pattern in primary tumors and metastasis was usually maintained. Univariate Cox regression analyses with bootstrap procedure indicated a significantly increased mortality risk in patients harboring BRAF mutation and BRAF mutation associated with TP53 and/or PIK3CA mutations. The detection of these multiple mutations appears to allow the identification of a subset of more aggressive tumors within the group and to bear information that should be useful for prognostic stratification of these patients including the planning of adjuvant therapy.
BackgroundHER2 is a therapeutic target for metastatic colorectal cancer (mCRC), as demonstrated in the pivotal HERACLES-A (HER2 Amplification for Colo-rectaL cancer Enhanced Stratification) trial ...with trastuzumab and lapatinib. The aim of HERACLES-B trial is to assess the efficacy of the combination of pertuzumab and trastuzumab-emtansine (T-DM1) in this setting.MethodsHERACLES-B was a single-arm, phase II trial, in patients with histologically confirmed RAS/BRAF wild-type and HER2+ mCRC refractory to standard treatments. HER2 positivity was assessed by immunohistochemistry and in situ hybridisation according to HERACLES criteria. Patients were treated with pertuzumab (840 mg intravenous load followed by 420 mg intravenous every 3 weeks) and T-DM1 (3.6 mg/kg every 3 weeks) until disease progression or toxicity. Primary and secondary end points were objective response rate (ORR) and progression-free survival (PFS). With a Fleming/Hern design (H0=ORR 10%; α=0.05; power=0.85), 7/30 responses were required to demonstrate an ORR ≥30% (H1).ResultsThirty-one patients, 48% with ≥4 lines of previous therapies, were treated and evaluable. ORR was 9.7% (95% CI: 0 to 28) and stable disease (SD) 67.7% (95% CI: 50 to 85). OR/SD ≥4 months was associated with higher HER2 immunohistochemistry score (3+ vs 2+) (p = 0.03). Median PFS was 4.1 months (95% CI: 3.6 to 5.9). Drug-related grade (G) 3 adverse events were observed in two patients (thrombocytopaenia); G≤2 AE in 84% of cycles (n = 296), mainly nausea and fatigue.ConclusionsHERACLES-B trial did not reach its primary end point of ORR; however, based on high disease control, PFS similar to other anti-HER2 regimens, and low toxicity, pertuzumab in combination with T-DM1 can be considered for HER2+mCRC as a potential therapeutic resource.Trial registration number2012-002128-33 and NCT03225937.
Cancers of unknown primary (CUPs), featuring metastatic dissemination in the absence of a primary tumor, are a biological enigma and a fatal disease. We propose that CUPs are a distinct, yet ...unrecognized, pathological entity originating from stem-like cells endowed with peculiar and shared properties. These cells can be isolated in vitro (agnospheres) and propagated in vivo by serial transplantation, displaying high tumorigenicity. After subcutaneous engraftment, agnospheres recapitulate the CUP phenotype, by spontaneously and quickly disseminating, and forming widespread established metastases. Regardless of different genetic backgrounds, agnospheres invariably display cell-autonomous proliferation and self-renewal, mostly relying on unrestrained activation of the MAP kinase/MYC axis, which confers sensitivity to MEK inhibitors in vitro and in vivo. Such sensitivity is associated with a transcriptomic signature predicting that more than 70% of CUP patients could be eligible to MEK inhibition. These data shed light on CUP biology and unveil an opportunity for therapeutic intervention.
Although reprogramming of cellular metabolism is a hallmark of cancer, little is known about how metabolic reprogramming contributes to early stages of transformation. Here, we show that the histone ...deacetylase SIRT6 regulates tumor initiation during intestinal cancer by controlling glucose metabolism. Loss of SIRT6 results in an increase in the number of intestinal stem cells (ISCs), which translates into enhanced tumor initiating potential in APC
mice. By tracking down the connection between glucose metabolism and tumor initiation, we find a metabolic compartmentalization within the intestinal epithelium and adenomas, where a rare population of cells exhibit features of Warburg-like metabolism characterized by high pyruvate dehydrogenase kinase (PDK) activity. Our results show that these cells are quiescent cells expressing +4 ISCs and enteroendocrine markers. Active glycolysis in these cells suppresses ROS accumulation and enhances their stem cell and tumorigenic potential. Our studies reveal that aerobic glycolysis represents a heterogeneous feature of cancer, and indicate that this metabolic adaptation can occur in non-dividing cells, suggesting a role for the Warburg effect beyond biomass production in tumors.
Summary Despite the marked improvement in the understanding of molecular mechanisms and classification of apocrine carcinoma, little is known about its specific molecular genetic alterations and ...potentially targetable biomarkers. In this study, we explored immunohistochemical and molecular genetic characteristics of 37 invasive apocrine carcinomas using immunohistochemistry (IHC), fluorescent in situ hybridization (FISH), multiplex ligation-dependent probe amplification (MLPA), and next-generation sequencing (NGS) assays. IHC revealed frequent E-cadherin expression (89%), moderate (16%) proliferation activity Ki-67, phosphohistone H3, infrequent (~10%) expression of basal cell markers CK5/6, CK14, p63, caveolin-1, loss of PTEN (83%), and overexpression of HER2 (32%), EGFR (41%), cyclin D1 (50%), and MUC-1 (88%). MLPA assay revealed gene copy gains of MYC , CCND1 , ZNF703 , CDH1 , and TRAF4 in 50% or greater of the apocrine carcinomas, whereas gene copy losses frequently affected BRCA2 (75%), ADAM9 (54%), and BRCA1 (46%). HER2 gain, detected by MLPA in 38% of the cases, was in excellent concordance with HER2 results obtained by IHC/FISH ( κ = 0.915, P < .001). TOP2A gain was observed in one case, while five cases (21%) exhibited TOP2A loss. Unsupervised hierarchical cluster analysis revealed two distinct clusters: HER2- positive and HER2- negative ( P = .03 and .04, respectively). NGS assay revealed mutations of the TP53 (2 of 7, 29%), BRAF/KRAS (2 of 7, 29%), and PI3KCA/PTEN genes (7 of 7, 100%). We conclude that morphologically defined apocrine carcinomas exhibit complex molecular genetic alterations that are consistent with the “luminal-complex” phenotype. Some of the identified molecular targets are promising biomarkers; however, functional studies are needed to prove these observations.
The classification of breast neuroendocrine neoplasms (Br-NENs) was modified many times over the years and is still a matter of discussion. In the present study, we aimed to evaluate the diagnostic ...reproducibility and impact on patient outcomes of the most recent WHO 2019 edition of breast tumor classification, namely, for neuroendocrine tumors (NETs) and neuroendocrine carcinomas (NECs). This multicentric observational study included 287 breast neoplasms with NE differentiation. The cases were blindly classified by three independent groups of dedicated breast and/or endocrine pathologists following the 2019 guidelines. Diagnostic concordance and clinical impact were assessed. We observed only a moderate overall diagnostic agreement across the three centers (Cohen’s kappa 0.4532) in distinguishing NET from solid papillary carcinomas (SPCs) and no special type carcinomas (NST) with NE differentiation. Br-NENs were diagnosed in 122/287 (42.5%) cases, subclassified as 11 NET G1 (3.8%), 84 NET G2 (29.3%), and 27 NEC (9.4%), the latter group consisting of 26 large-cell and 1 small-cell NECs. The remaining 165/287 (57.5%) cases were labeled as non-NEN, including SPC, mucinous, NST, and mixed NE carcinomas. While NET and non-NEN cases had a comparable outcome, the diagnosis of NECs showed negative impact on disease-free interval compared to NETs and non-NENs (
p
= 0.0109). In conclusion, the current diagnostic classification of Br-NENs needs further adjustments regarding morphological and immunohistochemical criteria to increase the diagnostic reproducibility among pathologists. Our data suggest that, apart from high-grade small- and large-cell NECs, Br-NENs behave like non-NEN breast carcinomas and should be managed similarly.
Somatic copy number alterations (SCNAs) are pervasive in advanced human cancers, but their prevalence and spatial distribution in early-stage, localized tumors and their surrounding normal tissues ...are poorly characterized. Here, we perform multi-region, single-cell DNA sequencing to characterize the SCNA landscape across tumor-rich and normal tissue in two male patients with localized prostate cancer. We identify two distinct karyotypes: 'pseudo-diploid' cells harboring few SCNAs and highly aneuploid cells. Pseudo-diploid cells form numerous small-sized subclones ranging from highly spatially localized to broadly spread subclones. In contrast, aneuploid cells do not form subclones and are detected throughout the prostate, including normal tissue regions. Highly localized pseudo-diploid subclones are confined within tumor-rich regions and carry deletions in multiple tumor-suppressor genes. Our study reveals that SCNAs are widespread in normal and tumor regions across the prostate in localized prostate cancer patients and suggests that a subset of pseudo-diploid cells drive tumorigenesis in the aging prostate.
Current multiplexing strategies for massively parallel sequencing of genomic DNA mainly rely on library indexing in the final steps of library preparation. This procedure is costly and ...time-consuming, because a library must be generated separately for each sample. Furthermore, library preparation is challenging in the case of fixed samples, such as DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissues. Here we describe CUTseq, a method that uses restriction enzymes and in vitro transcription to barcode and amplify genomic DNA prior to library construction. We thoroughly assess the sensitivity and reproducibility of CUTseq in both cell lines and FFPE samples, and demonstrate an application of CUTseq for multi-region DNA copy number profiling within single FFPE tumor sections, to assess intratumor genetic heterogeneity at high spatial resolution. In conclusion, CUTseq is a versatile and cost-effective method for library preparation for reduced representation genome sequencing, which can find numerous applications in research and diagnostics.
Background The "HER2-low" nomenclature identifies breast carcinomas (BCs) displaying a HER2 score of 1+/2+ in immunohistochemistry and lacking ERBB2 amplification. Whether HER2-low BCs (HLBCs) ...constitute a distinct entity is debated. Methods We performed DNA and RNA high-throughput analysis on 99 HLBC samples (n = 34 cases with HER2 score 1+/HLBC-1, n = 15 cases with HER2 score 2+ and ERBB2 not amplified/HLBC-2N, and n = 50 cases with score 2+ and ERBB2 copy number in the equivocal range/HLBC-2E). We compared the mutation rates with data from 1317 samples in the Memorial Sloan-Kettering Cancer Center (MSKCC) BC cohort and gene expression data with those from an internal cohort of HER2-negative and HER2-positive BCs. Results The most represented mutations affected PIK3CA (31/99, 31%), GATA3 (18/99, 18%), TP53 (17/99, 17%), and ERBB2 (8/99, 8%, private to HLBC-2E). Tumor mutational burden was significantly higher in HLBC-1 compared to HLBC-2E/N (P = 0.04). Comparison of mutation spectra revealed that HLBCs were different from both HER2-negative and HER2-positive BCs, with HLBC-1 resembling more HER2-negative tumors and HLBC-2 mutationally related to HER2-addicted tumors. Potentially actionable alterations (annotated by using OncoKB/ESCAT classes) affected 52 patients. Intra-group gene expression revealed overlapping features between HLBC-1 and control HER2-negative BCs, whereas the HLBC-2E tumors showed the highest diversity overall. The RNA-based class discovery analysis unveiled four subsets of tumors with (i) lymphocyte activation, (ii) unique enrichment in HER2-related features, (iii) stromal remodeling alterations, and (iv) actionability of PIK3CA mutations (LAURA classification). Conclusions HLBCs harbor distinct genomic features when compared with HER2-positive and HER2-negative BCs; however, differences across IHC classes were also unveiled thus dissecting the full picture of heterogeneity across HER2-low disease. The HLBC-2E category harbors most distinctive features, whereas HLBC-1 seems superimposable to HER2-negative disease. Further studies are needed to ascertain whether the four genomic-driver classes of the LAURA classification hold prognostic and/or predictive implications. Keywords: Breast cancer, HER2, HER2-low, Heterogeneity, Classification, Mutation, Actionable alteration, Gene expression, Transcriptome, Stratification
The role of forkhead-box A1 (FOXA1) and Androgen receptor (AR) in breast cancer (BC) has been extensively studied. However, the prognostic role of their co-expression in Estrogen receptor positive ...(ER+) BC has not been investigated so far. The aim of the present study was thus to assess the co-expression (protein and mRNA) of FOXA1 and AR in BC patients, in order to evaluate their prognostic impact according to ER status.
Immunohistochemical expression of AR and FOXA1 was evaluated on 479 consecutive BC, with complete clinical-pathological and follow up data. Fresh-frozen tissues from 65 cases were available. The expression of AR and FOXA1 with ER was validated using mRNA analyses. Survival and Cox proportional hazard analyses were used to evaluate the relationship between FOXA1, AR and prognosis.
Expression of ER, AR and FOXA1 was observed in 78, 60 and 85% of cases respectively. Most AR+ cases (97%) were also FOXA1+. The level of FOXA1 mRNA positively correlated with level of both AR mRNA (r = 0.8975; P < 0.001) and ER mRNA (r = 0.7326; P < 0.001). In ER+ BC, FOXA1 was associated with a good prognosis independently of AR expression in the three subgroups analyzed (FOXA1+/AR+; FOXA1+/AR-; FOXA1-/AR-). Multivariate analyses confirmed that FOXA1 may provide more information than AR in Disease-Free Interval (DFI) of ER+ BC patients.
Our results suggest that in BC the expression of FOXA1 is directly related to the expression of AR. Despite that, FOXA1 is found as superior predicting marker of recurrences compared to AR in ER+ BC patients.
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