Comparative analysis of diagnostic ultrasound and histopathology for detecting cervical lymph node metastases in head and neck cancer.

PURPOSE: We evaluated the current performance of diagnostic ultrasound (US) for detecting cervical lymph node (LN) metastases based on objective measures and subjective findings in comparison to the gold standard, histopathological evaluation. PATIENTS AND METHODS: From 2007 to 2016, we prospectively included patients with head and neck cancer who were scheduled for surgical therapy including neck dissection. LNs were examined by multimodal US by a level III head and neck sonologist and individually assigned to a map containing six AAO-HNS neck LN levels preoperatively. During the operation, LNs were dissected and then assessed by routine histopathology, with 86% of them examined individually and the remaining LNs (14%) per AAO-HNS neck LN level. The optimal cutoff points (OCPs) of four defined LN diameters and 2D and 3D roundness indices per AAO-HNS neck LN level were determined. RESULTS: In total, 235 patients were included, and 4539 LNs were analyzed by US, 7237 by histopathology and 2684 by both methods. Of these, 259 (9.65%) were classified as suspicious for metastasis by US, whereas 299 (11.14%) were found to be positive by histopathology. Subjective US sensitivity and specificity were 0.79 and 0.99, respectively. The OCPs of the individual LN diameters and the 2D and 3D roundness index were determined individually for all AAO-HNS neck LN levels. Across all levels, the OCP for the 2D index was 1.79 and the 3D index was 14.97. The predictive performance of all distances, indices, and subjective findings improved with increasing metastasis size. Anticipation of pN stage was best achieved with subjective US findings and the smallest diameter (Cohen's κ = 0.713 and 0.438, respectively). CONCLUSION: Our LN mapping and meticulous 1:1 node-by-node comparison reveals the usefulness of US for detecting metastatic involvement of neck LNs in head and neck carcinomas as compared to histopathology. The predictive ability for small tumor deposits less than 8 mm in size remains weak and urgently needs improvement.

MacroH2A impedes metastatic growth by enforcing a discrete dormancy program in disseminated cancer cells.

MacroH2A variants have been linked to inhibition of metastasis through incompletely understood mechanisms. Here, we reveal that solitary dormant disseminated cancer cells (DCCs) display increased levels of macroH2A variants in head and neck squamous cell carcinoma PDX in vivo models and patient samples compared to proliferating primary or metastatic lesions. We demonstrate that dormancy-inducing transforming growth factor-ß2 and p38α/ß pathways up-regulate macroH2A expression and that macroH2A variant overexpression is sufficient to induce DCC dormancy and suppress metastasis in vivo. Notably, inducible expression of the macroH2A2 variant in vivo suppresses metastasis via a reversible growth arrest of DCCs. This state does not require the dormancy-regulating transcription factors DEC2 and NR2F1; instead, transcriptomic analysis reveals that macroH2A2 overexpression inhibits cell cycle and oncogenic signaling programs, while up-regulating dormancy and senescence-associated inflammatory cytokines. We conclude that the macroH2A2-enforced dormant phenotype results from tapping into transcriptional programs of both quiescence and senescence to limit metastatic outgrowth.

Genetic analysis of single disseminated tumor cells in the lymph nodes and bone marrow of patients with head and neck squamous cell carcinoma.

Considering the limited information on the biology and molecular characteristics of disseminated tumor cells (DTCs) in head and neck squamous cell carcinoma (HNSCC), we examined the genomic alterations in DTCs from HNSCCs and their potential clinical relevance. To analyze both the lymphatic and hematogenous routes of tumor cell dissemination, we investigated samples from lymph nodes (LNs) and bone marrow (BM) of 49 patients using immunofluorescence double staining for epithelial cells expressing cytokeratin 18 (KRT18) and/or epithelial cell adhesion molecules (EpCAM, CD326). The identified marker-positive cells were isolated by micromanipulation followed by single-cell whole-genome amplification and metaphase-based comparative genomic hybridization (mCGH) to determine genome-wide copy number alterations. The findings were correlated with clinical parameters and follow-up data. We detected chromosomal aberrations in KRT18- and EpCAM-positive cells from both compartments; BM-derived cells showed a significantly higher percentage of aberrant genome (PAG) per cell than cells detected in LNs. No significant association was found between DTC data and clinical follow-up. Genomic profiling of BM-DTCs revealed genomic alterations typical for HNSCC, suggesting hematogenous dissemination of subclones around the time of surgery. In contrast, DTC data in LNs revealed that several marker-positive cells were not of malignant origin, indicating the presence of epithelial glandular inclusions in parts of the processed neck LN samples. Therefore, DTC detection of LNs in the neck based only on epithelial markers is not advisable and requires detection of chromosomal instability (CIN), gene mutations, or additional markers, which have yet to be identified. Nevertheless, our investigation paves the way for larger studies to focus on HNSCC BM-DTCs with high-resolution methods to gain deeper insights into the biology of hematogenous metastasis in this cancer.

An NR2F1-specific agonist suppresses metastasis by inducing cancer cell dormancy.

We describe the discovery of an agonist of the nuclear receptor NR2F1 that specifically activates dormancy programs in malignant cells. The agonist led to a self-regulated increase in NR2F1 mRNA and protein and downstream transcription of a novel dormancy program. This program led to growth arrest of an HNSCC PDX line, human cell lines, and patient-derived organoids in 3D cultures and in vivo. This effect was lost when NR2F1 was knocked out by CRISPR-Cas9. RNA sequencing revealed that agonist treatment induces transcriptional changes associated with inhibition of cell cycle progression and mTOR signaling, metastasis suppression, and induction of a neural crest lineage program. In mice, agonist treatment resulted in inhibition of lung HNSCC metastasis, even after cessation of the treatment, where disseminated tumor cells displayed an NR2F1hi/p27hi/Ki-67lo/p-S6lo phenotype and remained in a dormant single-cell state. Our work provides proof of principle supporting the use of NR2F1 agonists to induce dormancy as a therapeutic strategy to prevent metastasis.

Is the prediction of one or two ipsilateral positive lymph nodes by computerized tomography and ultrasound reliable enough to restrict therapeutic neck dissection in oral squamous cell carcinoma (OSCC) patients?

INTRODUCTION: Proper management of the clinically involved neck in OSCC patients continues to be a matter of debate. Our aim was to analyze the accuracy of computerized tomography (CT) and ultrasound (US) in anticipating the exact location of lymph node (LN) metastases of OSCC patients across the AAO-HNS (American Academy of Otolaryngology-Head and Neck Surgery) levels ipsi- and contralaterally. Furthermore, we wanted to assess the suitability of therapeutic selective neck dissection (SND) in patients with one or two ipsilateral positive nodes upon clinical staging (cN1/cN2a and cN2b(2/x) patients). METHODS: We prospectively analyzed the LN status of patients with primary OSCC using CT and US from 2007 to 2013. LNs were individually assigned to a map containing the AAO-HNS levels; patients bearing a single or just two ipsilateral positive nodes (designated cN1/cN2a or cN2b(2/x) patients either by CT (CT group) or US alone (US group) or in a group combining findings of CT and US (CTUS group)) received an ipsi-ND (I-V) and a contra-ND (I-IV). 78% of the LNs were sent individually for routine histopathological examination; the remaining were dissected and analyzed per neck level. RESULTS: Upon the analysis of 1.670 LNs of 57 patients, the exact location of pathology proven LN metastases in cN1 patients was more precisely predicted by US compared to CT with confirmed findings only in levels IA, IB und IIA. Clearly decreasing the number of missed lesions, the findings in the CTUS group nearly kept the spatial reliability of the US group. The same analysis for patients with exactly two supposed ipsilateral lesions (cN2b(2/x)) yielded confirmed metastases from levels I to V for both methods individually and in combination and, therefore, render SND insufficient for these cases. CONCLUSION: Our findings stress the importance of conducting both, CT and US, in patients with primary OSCC. Only the combination of their findings warrants the application of therapeutic SND in patients with a single ipsilateral LN metastasis (cN1/cN2a patients) but not in patients with more than one lesion upon clinical staging (≥ cN2b).

CT-scan is a valuable tool to detect mandibular involvement in oral cancer patients.

In patients with oral squamous cell carcinomas (OSSC) it is desirable to avoid unnecessary bone resection without neglecting the overall surgical treatment goal of tumor-free margins. Whereas computed tomography (CT) is most commonly used to detect mandibular invasion, there are conflicting reports regarding the accuracy of CT. Therefore, the aim of this study was to reinvestigate the accuracy of CT in predicting mandibular involvement by OSSC. One hundred and seven patients with OSSC who received a mandibulectomy were included. Before treatment all patients underwent a contrast-enhanced multi-detector CT. Axial 3 or 1.25 mm thick images were reconstructed for evaluation in overlapping technique and displayed in a bone (1400/400 HU) and a soft tissue window (350/50 HU). CT scans were examined by three investigators and compared with the histological findings. The radiological examination showed a high interrater reliability (Cronbachs alpha 0.982). Comparing the radiological findings with the histological results the CT showed 8 false-positive results and 8 false-negative patients. The quality criteria for detecting bone involvement of OSSC by CT were calculated as follows: sensitivity 82.6%; specificity 86.9%; positive predictive value 82.6%; negative predictive value 86.9%. However, in all false-positive patients a sagittal bone defect of 15.1mm could be found presumably caused by pressure of the tumor, but no histologically detectable bone infiltration. Modern CT (1-2 mm sections) is a valuable tool for surgical treatment planning. If bone invasion is detected, a mandibulectomy seems always reasonable. In radiologically negative cases histological assessment is necessary to detect mandibular involvement.

Nonvascularized iliac bone grafts for mandibular reconstruction--requirements and limitations.

BACKGROUND: Treatment of intraoral malignant tumors often leads to continuity defects of the mandible. Whereas the use of free vascularised flaps has shortcomings regarding donor site morbidity and a worse-fitting bone geometry, the nonvascularized iliac crest graft could be an alternative option. The purpose of this study was to describe the treatment outcome with nonvascularized iliac crest grafts over a 10-year period and to determine possible limitations of their use. PATIENTS AND METHODS: Eighty-four patients with bicortical nonvascularized iliac crest grafts for mandibular reconstruction were examined at least one year after reconstruction. Patients' records and the radiological and/or surgical data were analyzed. RESULTS: Sixty-three patients (75%) showed complete healing, in 20 patients the treatment was not successful and in one patient the treatment result was unclear. Interestingly, comparing the successfully and the unsuccessfully treated patients, only the irradiation dose played a crucial role. Neither defect length nor defect localisation, nor time interval between resection and reconstruction were statistically significant parameters in graft success. Comparing only patients with malignancies, the non-irradiated patients had a higher success rate (77.3%). CONCLUSION: The nonvaslcularized iliac crest graft seems to be a reasonably reliable treatment option for reconstruction of mandibular defects up to about 5-6 cm in size. Radiotherapy is a strong confounder reducing the success rate. Necessary constraints are sufficient soft tissue conditions. However, primary reconstruction by free flaps (e.g. fibula flap) has a higher success rate in literature and should be preferred whenever possible.

Is there a role for the Fas-/Fas-Ligand pathway in chemoresistance of human squamous cell carcinomas of the head and neck (SCCHN)?

The aim of the present investigation was to determine the expression of the Fas-receptor/ligand system in established cell lines of squamous cell carcinomas of the head and neck (SCCHN), and to study it's functional impact on chemotherapy-induced apoptosis in these SCCHN cell lines. We observed constitutive expression of Fas and FasL in 13 SCCHN cell lines by RT-PCR, Southern-blotting and immunocytochemistry, respectively. Administration of the agonistic Fas-antibody CH-11 led to a significant reduction of viable cells in the colorimetric MTT-assay in 5 out of 13 (38%) cell lines tested and preincubation with Interferon-gamma (IFN-gamma) rendered 3 (23%) primarily resistant cell lines sensitive. Cisplatin (cDDP) and bleomycin (BLM) caused dose-dependent cytotoxicity in all cell lines as determined by the 50% inhibitory concentration (IC(50)) and induction of apoptosis. Furthermore, both antineoplastic agents led to an enhanced surface expression of Fas and FasL in all cell lines, and this effect was independent of the respective p53-status. This upregulation of Fas/FasL surface expression increased preexisting Fas-sensitivity only, but failed to make primarily resistant cell lines undergo Fas-mediated growth reduction or apoptosis. Vice versa, blockade of Fas-receptor-ligand-interactions by monoclonal antibodies directed against FasL was able to attenuate the cytotoxic effect of cDDP and BLM in 2 out of 5 (40%) cell lines tested only. In conclusion, in contrast to many other solid tumors, the Fas/FasL-system does not seem to play an exclusive role in anticancer drug mediated apoptosis in SCCHN.