The emergence of drug resistance limits the efficacy of targeted therapies in human tumors. The prevalent view is that resistance is a fait accompli: when treatment is initiated, cancers already ...contain drug-resistant mutant cells. Bacteria exposed to antibiotics transiently increase their mutation rates (adaptive mutability), thus improving the likelihood of survival. We investigated whether human colorectal cancer (CRC) cells likewise exploit adaptive mutability to evade therapeutic pressure. We found that epidermal growth factor receptor (EGFR)/BRAF inhibition down-regulates mismatch repair (MMR) and homologous recombination DNA-repair genes and concomitantly up-regulates error-prone polymerases in drug-tolerant (persister) cells. MMR proteins were also down-regulated in patient-derived xenografts and tumor specimens during therapy. EGFR/BRAF inhibition induced DNA damage, increased mutability, and triggered microsatellite instability. Thus, like unicellular organisms, tumor cells evade therapeutic pressures by enhancing mutability.
Mutations in
, the gene encoding epidermal growth factor receptor (EGFR) family member HER2, are common in and drive the growth of "HER2-negative" (not
amplified) tumors but are rare in ..."HER2-positive" (
amplified) breast cancer. We analyzed DNA-sequencing data from HER2-positive patients and used cell lines and a patient-derived xenograft model to test the consequence of HER2 mutations on the efficacy of anti-HER2 agents such as trastuzumab, lapatinib, and neratinib, an irreversible pan-EGFR inhibitor. HER2 mutations were present in ~7% of HER2-positive tumors, all of which were metastatic but not all were previously treated. Compared to HER2 amplification alone, in both patients and cultured cell lines, the co-occurrence of HER2 mutation and amplification was associated with poor response to trastuzumab and lapatinib, the standard-of-care anti-HER2 agents. In mice, xenografts established from a patient whose HER2-positive tumor acquired a D769Y mutation in HER2 after progression on trastuzumab-based therapy were resistant to trastuzumab or lapatinib but were sensitive to neratinib. Clinical data revealed that six heavily pretreated patients with tumors bearing coincident HER2 amplification and mutation subsequently exhibited a statistically significant response to neratinib monotherapy. Thus, these findings indicate that coincident HER2 mutation reduces the efficacy of therapies commonly used to treat HER2-positive breast cancer, particularly in metastatic and previously HER2 inhibitor-treated patients, as well as potentially in patients scheduled for first-line treatment. Therefore, we propose that clinical studies testing the efficacy of neratinib are warranted selectively in breast cancer patients whose tumors carry both amplification and mutation of
/HER2.
Objective
We evaluated whether the apparent diffusion coefficient (ADC) provided by diffusion-weighted imaging (DWI) varies according to biological features in breast cancer.
Methods
DWI was ...performed in 190 patients undergoing dynamic contrast-enhanced magnetic resonance imaging (MRI) for local staging. For each of the 192 index cancers we studied the correlation between ADC and classical histopathological and immunohistochemical breast tumour features (size, histological type, grade, oestrogen receptor ER and Ki-67 expression, HER2 status). ADC was compared with immunohistochemical surrogates of the intrinsic subtypes (Luminal A; Luminal B; HER2-enriched; triple-negative). Correlations were analysed using the Mann–Whitney
U
and Kruskal–Wallis
H
tests.
Results
A weak, statistically significant correlation was observed between ADC values and the percentage of ER-positive cells (-0.168,
P
= 0.020). Median ADC values were significantly higher in ER-negative than in ER-positive tumours (1.110 vs 1.050 × 10
-3
mm
2
/s,
P
= 0.015). HER2-enriched tumours had the highest median ADC value (1.190 × 10
-3
mm
2
/s, range 0.950–2.090). Multiple comparisons showed that this value was significantly higher than that of Luminal A (1.025 × 10
-3
mm
2
/s 0.700–1.340,
P
= 0.004) and Luminal B/HER2-negative (1.060 × 10
-3
mm
2
/s 0.470–2.420,
P
= 0.008) tumours. A trend towards statistical significance (
P
= 0.018) was seen with Luminal B/HER2-positive tumours.
Conclusions
ADC values vary significantly according to biological tumour features, suggesting that cancer heterogeneity influences imaging parameters.
Key Points
•
DWI may identify biological heterogeneity of breast neoplasms.
•
ADC values vary significantly according to biological features of breast cancer.
•
Compared with other types, HER2-enriched tumours show highest median ADC value.
•
Knowledge of biological heterogeneity of breast neoplasm may improve imaging interpretation.
Advanced biliary tract carcinomas (BTCs) have poor prognosis and limited therapeutic options. Therefore, it is crucial to combine standard therapies with molecular targeting. In this study EGFR, ...HER2, and their molecular transducers were analysed in terms of mutations, amplifications and over-expression in a BTC case series. Furthermore, we tested the efficacy of drugs targeting these molecules, as single agents or in combination with gemcitabine, the standard therapeutic agent against BTC.
Immunohistochemistry, FISH and mutational analysis were performed on 49 BTC samples of intrahepatic (ICCs), extrahepatic (ECCs), and gallbladder (GBCs) origin. The effect on cell proliferation of different EGFR/HER2 pathway inhibitors as single agents or in combination with gemcitabine was investigated on BTC cell lines. Western blot analyses were performed to investigate molecular mechanisms of targeted drugs.
EGFR is expressed in 100% of ICCs, 52.6% of ECCs, and in 38.5% of GBCs. P-MAPK and p-Akt are highly expressed in ICCs (>58% of samples), and to a lower extent in ECCs and GBCs (<46%), indicating EGFR pathway activation. HER2 is overexpressed in 10% of GBCs (with genomic amplification), and 26.3% of ECCs (half of which has genomic amplification). EGFR or its signal transducers are mutated in 26.5% of cases: 4 samples bear mutations of PI3K (8.2%), 3 cases (6.1%) in K-RAS, 4 (8.2%) in B-RAF, and 2 cases (4.1%) in PTEN, but no loss of PTEN expression is detected. EGI-1 cell line is highly sensitive to gemcitabine, TFK1 and TGBC1-TKB cell lines are responsive and HuH28 cell line is resistant. In EGI-1 cells, combination with gefitinib further increases the antiproliferative effect of gemcitabine. In TFK1 and TGBC1-TKB cells, the efficacy of gemcitabine is increased with addiction of sorafenib and everolimus. In TGBC1-TKB cells, lapatinib also has a synergic effect with gemcitabine. HuH28 becomes responsive if treated in combination with erlotinib. Moreover, HuH28 cells are sensitive to lapatinib as a single agent. Molecular mechanisms were confirmed by western blot analysis.
These data demonstrate that EGFR and HER2 pathways are suitable therapeutic targets for BTCs. The combination of gemcitabine with drugs targeting these pathways gives encouraging results and further clinical studies could be warranted.
Background
Trastuzumab is the only approved targeted therapy in patients with
HER2
-amplified metastatic gastric cancer (GC). Regrettably, in clinical practice, only a fraction of them achieves ...long-term benefit from trastuzumab-based upfront strategy. To advance precision oncology, we investigated the therapeutic efficacy of different HER2-targeted strategies, in
HER2
“hyper”-amplified (≥ 8 copies) tumors.
Methods
We undertook a prospective evaluation of HER2 targeting with monoclonal antibodies, tyrosine kinase inhibitors and antibody–drug conjugates, in a selected subgroup of
HER2
“hyper”-amplified gastric patient-derived xenografts (PDXs), through the design of ad hoc preclinical trials.
Results
Despite the high level of
HER2
amplification, trastuzumab elicited a partial response only in 2 out of 8 PDX models. The dual-HER2 blockade with trastuzumab plus either pertuzumab or lapatinib led to complete and durable responses in 5 (62.5%) out of 8 models, including one tumor bearing a concomitant
HER2
mutation. In a resistant PDX harboring
KRAS
amplification, the novel antibody–drug conjugate trastuzumab deruxtecan (but not trastuzumab emtansine) overcame
KRAS
-mediated resistance. We also identified a HGF-mediated non-cell-autonomous mechanism of secondary resistance to anti-HER2 drugs, responsive to MET co-targeting.
Conclusion
These preclinical randomized trials clearly indicate that in HER2-driven gastric tumors, a boosted HER2 therapeutic blockade is required for optimal efficacy, leading to complete and durable responses in most of the cases. Our results suggest that a selected subpopulation of
HER2
-“hyper”-amplified GC patients could strongly benefit from this strategy. Despite the negative results of clinical trials, the dual blockade should be reconsidered for patients with clearly HER2-addicted cancers.
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
Unresectable metastatic bone sarcoma and soft-tissue sarcomas (STS) are incurable due to the inability to eradicate chemoresistant cancer stem-like cells (sCSC) that are likely responsible for ...relapses and drug resistance. In this study, we investigated the preclinical activity of patient-derived cytokine-induced killer (CIK) cells against autologous bone sarcoma and STS, including against putative sCSCs. Tumor killing was evaluated both in vitro and within an immunodeficient mouse model of autologous sarcoma. To identify putative sCSCs, autologous bone sarcoma and STS cells were engineered with a CSC detector vector encoding eGFP under the control of the human promoter for OCT4, a stem cell gene activated in putative sCSCs. Using CIK cells expanded from 21 patients, we found that CIK cells efficiently killed allogeneic and autologous sarcoma cells in vitro. Intravenous infusion of CIK cells delayed autologous tumor growth in immunodeficient mice. Further in vivo analyses established that CIK cells could infiltrate tumors and that tumor growth inhibition occurred without an enrichment of sCSCs relative to control-treated animals. These results provide preclinical proof-of-concept for an effective strategy to attack autologous sarcomas, including putative sCSCs, supporting the clinical development of CIK cells as a novel class of immunotherapy for use in settings of untreatable metastatic disease.
Demethylation of the long interspersed nuclear element (LINE‐1; L1) antisense promoter can result in transcription of neighboring sequences as for the L1‐MET transcript produced by the L1 placed in ...the second intron of MET. To define the role of L1‐MET, we investigated the sequence and the transcription of L1‐MET in vitro models and heterogeneous breast cancers, previously reported to show other L1‐derived transcripts. L1‐MET expressing cell lines were initially identified in silico and investigated for L1‐MET promoter methylation, cDNA sequence and cell fraction mRNA. The transcriptional level of L1‐MET and MET were then evaluated in breast specimens, including 9 cancer cell lines, 41 carcinomas of different subtypes, and 11 normal tissues. In addition to a L1‐MET transcript ending at MET exon 21, six novel L1‐MET splice variants were identified. Normal breast tissues were negative for the L1‐MET expression, whereas the triple‐negative breast cancer (TNBC) and the high‐grade carcinomas were enriched with the L1‐MET mRNA (p = 0.005 and p = 0.018, respectively). In cancer cells and tissues the L1–MET expression was associated with its promoter hypomethylation (ρ = −0.8 and −0.9, respectively). No correlation was found between L1‐MET and MET mRNA although L1‐MET expressing tumors with higher L1‐MET/MET ratio were negative for the MET protein expression (p = 0.006). Besides providing the first identification and detailed description of L1‐MET in breast cancer, we clearly demonstrate that higher levels of this transcript specifically recognize a subset of more aggressive carcinomas, mainly TNBC. We suggest the possible evaluation of L1‐MET in the challenging diagnosis of early TNBCs.
What's new?
The transposable element L1 can generate some troublesome transcripts, occasionally leading to cancer. Here, the authors set out to thoroughly characterize the L1‐MET transcript and investigate its role in breast cancer. They identified numerous cell types expressing L1‐MET, including a variety of cancer subtypes. They found that L1‐MET transcripts are present in breast cancers but not in untransformed breast cells, with highest levels occurring in the more aggressive tumors, particularly triple‐negative cancers.
Formalin fixation and paraffin embedding (FFPE) represent the standard method to preserve tissue specimens for diagnostic pathology, however formalin fixation induces severe fragmentation of nucleic ...acids. We investigated whether formalin fixation at 4°C could preserve DNA integrity in FFPE specimens. Paired samples from 38 specimens were formalin fixed at room temperature (
) and at 4°C (
), respectively. Two independent cohorts were prospectively collected, cohort A (collected 6 years prior to the study,
= 21), cohort B (collected at time of the study,
= 17). DNA was extracted and its integrity evaluated with a qPCR-based assay that produces a normalized integrity index, the QC score (ratio between the quantity of a long and a short amplicon of the same gene). We observed higher QC scores in
compared to
samples (mean values: 0.69 vs. 0.36,
< 0.0001) and
breast cancer specimens showed the most detrimental effect overall. Comparable QC scores were obtained between
tissues of both cohorts; conversely, DNA integrity of
was significantly lower in cohort A compared to cohort B (
< 0.0001). Of note, QC scores of
(but not of
) samples were significantly reduced following 6 months of storage (
= 0.0001). Monitored formalin fixation at 4°C outperforms standard fixation in ensuring high-quality DNA, which is key to feasibility of downstream high-throughput molecular analyses. An important effect was observed over storage time, thus suggesting a likely better preservation of archival samples when this cold fixation protocol is used.
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