High-risk human papillomaviruses (HPVs) have been proposed to be associated with a subset of head and neck cancers (HNSCCs). However, clear biological evidence linking HPVmediated oncogenesis to the development of HNSCC is hardly available. An important biological mechanism underlying HPV-mediated carcinogenesis is the inactivation of p53 by the HPV E6 oncoprotein. In the present study we investigated this biological relationship between HPV and HNSCC. In total 84 HNSCC tumors were analyzed for the presence of high-risk HPV nucleic acids by DNA polymerase chain reaction-enzyme immunoassay (PCR-EIA) and E6 reverse transcriptase (RT)-PCR as well as for the presence of mutations in the p53 gene. We found 20/84 HPV16 DNA-positive cases with one or more DNA assays, 10 of which were consistently positive with all assays. Only 9/20 cases showed E6 mRNA expression, indicative for viral activity. Only these nine E6 mRNA-positive cases all lacked a p53 mutation, whereas both the other HPV DNA-positive and HPV-DNA negative tumors showed p53 mutations in 36% and 63% of the cases, respectively. Moreover, only in lymph node metastases of HPV E6 mRNA-positive tumors both viral DNA and E6 mRNA were present. Our study provides strong biological evidence for a plausible etiological role of high-risk HPV in a subgroup of HNSCC. Analysis of E6 mRNA expression by RT-PCR or alternatively, semiquantitative analyses of the viral load, seem more reliable assays to assess HPV involvement in HNSCC than the very sensitive DNA PCR analyses used routinely. © 2001 Wiley-Liss, Inc. Key words: papillomavirus; head and neck cancer; squamous cell carcinoma; p53 mutations; E6 oncoproteinMucosotropic high-risk human papillomaviruses (HPVs), known to cause cervical and other anogenital cancers, have been proposed to play a role in the etiology of head and neck squamous cell carcinomas (HNSCCs). 1 The presence of high-risk HPV DNA in a subgroup of HNSCCs has supported this hypothesis. [2][3][4][5] Molecular studies have provided important data on the role and oncogenic mechanism of high-risk HPV in carcinogenesis. 6 -8 By expression of the viral oncoproteins E6 and E7, the virus dysregulates crucial cellular mechanisms such as the cell cycle and the apoptotic pathway. The E6 oncoprotein specifically inactivates wild-type p53, and the E7 oncoprotein inactivates Rb. In this way the high-risk HPV E6-mediated degradation of the p53 protein should be considered an alternative pathway for "classical" mutation to knock-out the p53 regulated pathways; it provides the biological basis to expect that tumors originating from HPV infection will show wild-type p53. Indeed, this general biological mechanism is supported by the finding that p53 mutations hardly occur in cervical carcinomas. 9,10 However, in most studies on head and neck cancer, HPV DNA presence and p53 mutations were overlapping, 2,11,12 an observation that gave rise to a long debate as to whether HPV is causally related to the development of a subset of these tumors.On the other hand, the inconsi...

In total, 10-30% of patients with head and neck squamous cell carcinoma (HNSCC) develop local recurrences despite seemingly adequate tumour resection. This may result from minimal residual cancer (MRC): small numbers of tumour cells left behind in the surgical margins, undetectable by routine histopathology. In recent studies, p53 mutations have been considered as selective and sensitive DNA markers of cancer cells. There are two potential problems in using mutated-p53 DNA as a marker. Firstly, p53 mutations occur early in progression and might therefore detect unresected precursor lesions besides tumour cells. Secondly, DNA is a very stable biomolecule that might lead to false-positive results. These two potential problems have been evaluated in this study. Fifty patients with a radical tumour resection were included, of whom 30 showed a p53 mutation in the primary tumour. Histopathologically tumour-free surgical margins were quantitatively analysed for mutated p53 by molecular diagnosis (plaque assay) and subsequent (immuno)histopathology. p53 mutated DNA was detected in the surgical margins of 19/30 patients. Immunohistochemistry confirmed the presence of small tumour foci in 2/19 mutated p53-positive cases. In 7/19 cases, the tumour-specific p53 mutation was found in unresected dysplastic mucosal precursor lesions. Moreover, in a number of cases small p53-immunostained patches were detected, but the mutations found were never tumour-related. By screening contralateral exfoliated cells and plaque assays on RNA it was shown that detection of mutated-p53 DNA is prone to false-positive results. In conclusion, using p53 mutations as a marker, both MRC and unresected mutated p53-positive mucosal precursor lesions are detected within surgical margins. Molecular assessment of surgical margins using p53 mutations enables the selection of HNSCC patients at high risk for tumour recurrence, but tumour RNA seems at present to be a more specific biomolecule for analysis than tumour DNA.

Despite high remission rates after therapy, 60% to 70% of patients with acute myeloid leukemia (AML) do not survive 5 years after their initial diagnosis. The main cause of treatment failures may be insufficient eradication of a subpopulation of leukemic stem-like cells (LSC), which are thought to be responsible for relapse by giving rise to more differentiated leukemic progenitors (LP). To address the need for therapeutic targets in LSCs, we compared microRNA (miRNA) expression patterns in highly enriched healthy CD34

Standard induction chemotherapy, consisting of an anthracycline and cytarabine, has been the first-line therapy for many years to treat acute myeloid leukemia (AML). Although this treatment induces complete remissions in the majority of patients, many face a relapse (adaptive resistance) or have refractory disease (primary resistance). Moreover, older patients are often unfit for cytotoxic-based treatment. AML relapse is due to the survival of therapy-resistant leukemia cells (minimal residual disease, MRD). Leukemia cells with stem cell features, named leukemic stem cells (LSCs), residing within MRD are thought to be at the origin of relapse initiation. It is increasingly recognized that leukemia “persisters” are caused by intra-leukemic heterogeneity and non-genetic factors leading to plasticity in therapy response. The BCL2 inhibitor venetoclax, combined with hypomethylating agents or low dose cytarabine, represents an important new therapy especially for older AML patients. However, often there is also a small population of AML cells refractory to venetoclax treatment. As AML MRD reflects the sum of therapy resistance mechanisms, the different faces of treatment “persisters” and LSCs might be exploited to reach an optimal therapy response and prevent the initiation of relapse. Here, we describe the different epigenetic, transcriptional, and metabolic states of therapy sensitive and resistant AML (stem) cell populations and LSCs, how these cell states are influenced by the microenvironment and affect treatment outcome of AML. Moreover, we discuss potential strategies to target dynamic treatment resistance and LSCs.

Key Points• EVI-1-positive AML cases are sensitive to ATRA.Enhanced expression of ecotropic viral integration site 1 (EVI-1) occurs in ∼10% of acute myeloid leukemia (AML) patients and is associated with a very poor disease outcome.Patients with EVI-1-positive AML have poor initial responses to chemotherapy and high relapse rates, indicating an urgent need for alternative treatment strategies improving clinical outcome for these patients. Because treatment of acute promyelocytic patients with all-trans retinoic acid (ATRA) has improved the survival of these patients substantially, we investigated whether ATRA might also be effective for the subgroup of AML patients with EVI-1 overexpression. Here, we show that a substantial part of the EVI-1-positive AML cases respond to ATRA by induction of differentiation and decreased clonogenic capacity of myeloid blasts. Most importantly, we demonstrate that in vivo treatment of primary EVI-1-positive AML with ATRA leads to a significant reduction in leukemic engraftment. Altogether, our results show that a considerable part of the EVI-1-positive primary AML cases are sensitive to ATRA, suggesting that combining ATRA with the currently used conventional chemotherapy might be a promising treatment strategy decreasing relapse rates and enhancing complete remissions in this poor prognostic subgroup of AML patients. (Blood. 2016;127(4):458-463) Introduction Acute myeloid leukemia (AML) is a heterogeneous disease that can be classified based on morphology, immunophenotypic features, and, more importantly, cytogenetic aberrations, molecular abnormalities, gene expression, and methylation signatures.1-3 Aberrant expression of the transcriptional regulator ecotropic viral integration site 1 (EVI-1) occurs in ;10% of human adult AML patients and is associated with particularly aggressive disease and a very poor outcome. Up to 95% of EVI-1-positive patients have an overall survival of ,1 year, 4-6 and novel treatment strategies to improve the survival of AML patients with aberrant expression of EVI-1 are urgently needed. For more than 40 years, most AML patients, including the subgroup with EVI-1 overexpression, have been treated with a combination chemotherapy consisting of cytarabine-arabinoside and an antracycline like daunorubicin. 7,8 Importantly, EVI-1-positive AML cases have an extremely poor initial response to the currently used combination chemotherapy; 39% of EVI-1-positive patients do not achieve complete remission after induction therapy, which is in sharp contrast to the 18% of patients in the other AML subgroups. 5To date, none of other tested alternative regimens have proved to be more effective in AML treatment than the combination of cytarabine and an anthracycline. The exception to this is the treatment of acute promyelocytic (APL) patients with all-trans retinoic acid (ATRA), which increased their survival chances significantly and has turned APL from a poor prognostic leukemia to one that can be cured. 9,10 Because treatment of APL patients with ATRA is very s...