Christoph Sproll

Diagnostic leukapheresis enables reliable detection of circulating tumor cells of nonmetastatic cancer patients

Significance The infrequent detection of circulating tumor cells (CTCs) has hindered their clinical implication and their potential use in the sense of a “liquid biopsy” for cancer diagnosis and therapy. Hypothesizing that the limited blood volume commonly used for CTC analysis (1–10 mL) accounts for variable detection rates, we used leukapheresis to screen large blood volumes for CTCs. This enabled a more reliable detection of CTCs at high frequency even in nonmetastatic cancer patients. Thus, diagnostic leukapheresis may facilitate the routine clinical use of CTCs as biomarkers for personalized medicine. Combined with technologies for single-cell molecular genetics or cell biology, it may significantly improve prediction of therapy response and monitoring, especially in early systemic cancer. Circulating tumor cells (CTCs) are promising biomarkers for diagnosis and therapy in systemic cancer. However, their infrequent and unreliable detection, especially in nonmetastatic cancer, currently impedes the clinical use of CTCs. Because leukapheresis (LA) targets peripheral blood mononuclear cells, which have a similar density to CTCs, and usually involves processing the whole circulating blood, we tested whether LA could substantially increase CTC detection in operable cancer patients. Therefore, we screened LA products generated from up to 25 L of blood per patient in two independent studies, and found that CTCs can be detected in more than 90% of nonmetastatic breast cancer patients. Interestingly, complete white blood cell sampling enabled determining an upper level for total CTC numbers of about 100,000 cells (median, 7,500 CTCs) per patient and identified a correlation of CTC numbers with anatomic disease spread. We further show that diagnostic leukapheresis can be easily combined with the US Food and Drug Administration-approved CellSearch system for standardized enumeration of CTCs. Direct comparison with 7.5 mL of blood revealed a significantly higher CTC frequency in matched LA samples. Finally, genomic single-cell profiling disclosed highly aberrant CTCs as therapy-escaping variants in breast cancer. In conclusion, LA is a clinically safe method that enabled a reliable detection of CTCs at high frequency even in nonmetastatic cancer patients, and might facilitate the routine clinical use of CTCs as in the sense of a liquid biopsy. Combined with technologies for single-cell molecular genetics or cell biology, it may significantly improve prediction of therapy response and monitoring of early systemic cancer.

Genomic High-Resolution Profiling of Single CKpos/CD45neg Flow-Sorting Purified Circulating Tumor Cells from Patients with Metastatic Breast Cancer

BACKGROUND Circulating tumor cells (CTCs) are promising surrogate markers for systemic disease, and their molecular characterization might be relevant to guide more individualized cancer therapies. To enable fast and efficient purification of individual CTCs, we developed a work flow from CellSearch(TM) cartridges enabling high-resolution genomic profiling on the single-cell level. METHODS Single CTCs were sorted from 40 CellSearch samples from patients with metastatic breast cancer using a MoFlo XDP cell sorter. Genomes of sorted single cells were amplified using an adapter-linker PCR. Amplification products were analyzed by array-based comparative genomic hybridization, a gene-specific quantitative PCR (qPCR) assay for cyclin D1 (CCND1) locus amplification, and genomic sequencing to screen for mutations in exons 1, 9, and 20 of the phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) gene and exons 5, 7, and 8 of the tumor protein p53 (TP53) gene. RESULTS One common flow-sorting protocol was appropriate for 90% of the analyzed CellSearch cartridges, and the detected CTC numbers correlated positively with those originally detected with the CellSearch system (R(2) = 0.9257). Whole genome amplification was successful in 72.9% of the sorted single CTCs. Over 95% of the cells displayed chromosomal aberrations typical for metastatic breast cancers, and amplifications at the CCND1 locus were validated by qPCR. Aberrant CTCs from 2 patients harbored mutations in exon 20 of the PIK3CA gene. CONCLUSIONS This work flow enabled effective CTC isolation and provided insights into genomic alterations of CTCs in metastatic breast cancer. This approach might facilitate further molecular characterization of rare CTCs to increase understanding of their biology and as a basis for their molecular screening in the clinical setting.

Role of Brush Biopsy and DNA Cytometry for Prevention, Diagnosis, Therapy, and Followup Care of Oral Cancer

Late diagnosis resulting in late treatment and locoregional failure after surgery are the main causes of death in patients with oral squamous cell carcinomas (SCCs). Actually, exfoliative cytology is increasingly used for early detection of oral cancer and has been the subject of intense research over the last five years. Significant advances have been made both in relation to screening and evaluation of precursor lesions. As this noninvasive procedure is well tolerated by patients, more lesions may be screened and thus more oral cancers may be found in early, curable stages. Moreover, the additional use of DNA image cytometry is a reasonable tool for the assessment of the resection margins of SCC. DNA image cytometry could help to find the appropriate treatment option for the patients. Finally, diagnostic DNA image cytometry is an accurate method and has internationally been standardized. In conclusion, DNA image cytometry has increasing impact on the prevention, diagnostic, and therapeutical considerations in head and neck SCC.

RAD51C – A new human cancer susceptibility gene for sporadic squamous cell carcinoma of the head and neck (HNSCC)

INTRODUCTION Head and neck squamous cell carcinomas (HNSSCs) are one of the leading causes of cancer-associated death worldwide. Although certain behavioral risk factors are well recognized as tumor promoting, there is very little known about the presence of predisposing germline mutations in HNSCC patients. METHODS In this study, we analyzed 121 individuals with HNSCCs collected at our institution for germline alterations in the newly identified cancer susceptibility gene RAD51C. RESULTS Sequencing of all exons and the adjacent introns revealed five distinct heterozygous sequence deviations in RAD51C in seven patients (5.8%). A female patient without any other risk factors carried a germline mutation that disrupted the canonical splice acceptor site of exon 5 (c.706-2A>G). CONCLUSIONS As there are only a few publications in the literature identifying germline mutations in head and neck cancer patients, our results provide the first indication that paralogs of RAD51, recently described as mutated in breast and ovarian cancer patients, might also be candidates for genetic risk factors in sporadic squamous cell carcinomas of the head and neck.

Bilateral multifocal Warthin's tumors in upper neck lymph nodes. report of a case and brief review of the literature

Cystadenolymphomas (Warthins tumors) are the second most frequent lesions of the parotid gland. Due to their benign clinical behavior, the low rates of recurrence and malignant transformation they were classified as tumor-like lesions. In addition, a polyclonal growth of the epithelial components of the tumor could be detected. Warthins tumors occur bilateral in 7-10%, whereas a multifocal appearance is extremely rare. Even if the pathogenesis is still unclear a heterotopia of salivary tissue during embryogenesis is the most likely explanation for the origin of these tumors in the upper neck and periparotideal region. Here we present a rare case of bilateral, multifocal, extraglandular Warthins tumors in lymph nodes of the upper neck and give a brief review of the literature. If a primary malignancy can be excluded by a careful staging procedure prior to the operation an isolated excision of the lesions of the neck is the adequate treatment.

Pathogenesis of medication-related osteonecrosis of the jaw: a comparative study of in vivo and in vitro trials

Objective This study was performed to determine whether the results of prevailing in vivo and in vitro studies offer a reliable model for investigation of medication-related osteonecrosis of the jaw (MRONJ). Methods Embase, Medline, and the Cochrane Library were searched for articles published from September 2003 to June 2017 involving experimental approaches to the pathogenesis of MRONJ. In vivo and in vitro trials were analyzed with respect to the scientific question, study design, methodology, and results. Results Of 139 studies, 87, 46, and 6 conducted in vivo, in vitro, and both in vivo and in vitro experiments, respectively. Rats, mice, dogs, minipigs, sheep, and rabbits were the preferred animal models used. Osteoblasts, osteoclasts, fibroblasts, keratinocytes, macrophages, and human umbilical vein endothelial cells were the preferred cell types. Zoledronate, alendronate, ibandronate, and risedronate were the most frequent bisphosphonates used. MRONJ was most reliably induced in minipigs because of the close relationship with human bone physiology. In vitro studies showed that reduced viability, growth, and migration of cells in the bone and soft tissues were causative for MRONJ. Other than exposed jawbone after tooth extraction, no reliable cofactors were found. Conclusion The minipig is the most suitable animal model for MRONJ.

Trajectory-guided biopsy of orbital tumor – Technology, principal considerations and clinical implementation

Intraorbital space-occupying lesions always pose a challenge, both in terms of definite surgical removal as well as preoperative sampling for histopathological examination. Despite the use of modern high-resolution imaging techniques, the dignity of orbital lesions can often not be determined with sufficient certainty preoperatively. As the amount and complexity of treatment possibilities continue to increase, detailed diagnostics in advance of treatment choice are essential. Histological classification of orbital lesions can still be considered the gold standard for reliable diagnoses, leading to appropriate treatment. Over recent years minimally invasive surgical approaches have gained more importance in the treatment and diagnosis of cranio-maxillo-facial tumor and trauma. The aim of our study was to adapt and establish a precise procedure for orbital biopsies. 23 patients suffering from space-occupying lesions of unknown dignity were included. Trajectory-guided procedures were pre-planned for all cases. In most cases minimally invasive procedures were suitable for taking biopsies of the orbit. For only two patients a conventional, non-minimally invasive, lateral orbitotomy had to be performed. Further evaluation of the presented procedure demonstrates clearly that trajectory-guided biopsies of the orbit can be performed correctly and effectively, regardless of the suspected lesions size.

Extraskeletal chondroma walling the temporomandibular joint: Report of a rare case and review of the literature

Highlights • Extraskeletal or soft-tissue chondroma is a rare cartilaginous benign neoplasm.• Affects usually hands and feet, preauricular region is extremely rare.• Diagnosis is challenging since even histological features may be misleading to other pathologies.• Report describes a rare case of extraskeletal chondroma surrounding the temporomandibular joint in a 55-year-old woman.

Extensive Craniocervical Abscess after Transoral Ganglionic Local Opioid Analgesia at the Superior Cervical Ganglion for Atypical Trigeminal Neuralgia: Report of a Severely Complicated Case

Ganglionic local opioid analgesia (GLOA) describes the application of low-dose opioids close to sympathetic as, for example, to the superior cervical ganglion. GLOA can be effective in different pain syndromes affecting the head and face region and has been considered to be a safe technique with few complications reported so far. We present the case of a patient who received a single, transoral GLOA for a refractory trigeminal neuralgia. The patient subsequently developed an extensive epidural abscess at the craniocervical junction, requiring ultimately transoral odontoid resection and dorsal stabilisation. This severe complication challenges the role of transoral infiltration therapies in analgetic medicine.

A female traveler returning from Peru with an erythematous infraorbital swelling: A diagnostic challenge

After a vacation trip to tropical Peru a 54-year-old Caucasian woman presented at our clinic with sensations of pressure, irritation and pain located at a skin area with close proximity to her right infraorbital foramen. Clinical inspection of the said region showed a papular erythematous swelling with a diameter of approximately 2 cm. A little nodule in the center of the lesion appeared to be the opening of a fistula. In the case history we elicited that these symptoms already began in Peru and reached a maximum after she had returned home. The patient could not recall any traumas or infections in the facial region. At first, a general practitioner was consulted and prescribed a 7-day treatment with antibiotics (amoxicillin + clavulanic acid). These therapeutic measures did not have any positive effects on the lesion. In cooperation with the department for tropical medicine our clinic further investigated the case. We conducted an ultrasound examination displaying a space-occupying mass underneath the skin (see picture below). In analogy with the patients travel history an infestation with fly larvae was deemed most likely. In a surgical procedure under general anesthesia a superficial incision was made to carefully prepare the underlying tissue. Here a live insect larva was found which was extracted from the wound (Fig. 1). In a subsequent morphometric analysis the genus of the larva was categorized as dermatobia hominis. This species is indigenous to tropical and subtropical regions with a hot and humid local climate causing a disease termed myasis. Myasis is characterized by destructive processes of skin and other tissues infested by fly larvae just as described in our patient. We conclude that for a fast diagnosis of myasis early clinical examinations must be supported by sonographic imaging in patients with signs of persistent skin inflammation especially with a history of tropical travel destinations.