Immunohistochemical expression of CD44 in oral squamous cell carcinoma in relation to histomorphological parameters and clinicopathological factors.

AIMS: Oral squamous cell carcinoma (OSCC) is characterised by its variable clinical course. In addition to the routinely used TNM and Union for International Cancer Control systems, patient-specific prognostic/predictive biomarkers are needed. Promising biomarkers include the determination of the cancer stem cell compartment, which can be identified by CD44 expression (among other things). The aim of this study was to evaluate the impact of CD44 in OSCC in terms of correlation with histomorphology, especially targeting features of EMT, and its influence on patient prognosis. METHODS AND RESULTS: A well-characterised cohort of 108 therapy-naive OSCCs with complete long-term follow-up and matched lymph node metastases were evaluated for CD44 expression by immunohistochemistry. CD44 expression was correlated with histomorphological characteristics (including tumour differentiation and tumour budding), clinicopathological parameters, and follow-up data. Overexpression of CD44 was detected in 37% of OSCCs within the tumour centre, in 39% of OSCCs at the invasive margin, and in 16% of lymph node metastases. CD44 overexpression at the invasive margin was significantly correlated with poor histopathological differentiation, and specifically with high tumour budding activity and single-cell invasion as signs of epithelial-mesenchymal transition (EMT). CD44 overexpression within the tumour core region and in lymph node metastases was identified as an independent prognostic factor for poor overall, disease-specific and disease-free survival in subsets of patients with advanced OSCC. CONCLUSION: Our study demonstrates the association of CD44 with tumour aggressiveness and EMT, as well as the independent prognostic impact of CD44 in a subset of OSCCs, which underlines the role of tumour cell stemness as a key factor in malignant behaviour in this disease.

Tumour budding activity and cell nest size determine patient outcome in oral squamous cell carcinoma: proposal for an adjusted grading system.

AIMS: Oral squamous cell carcinoma (OSCC) is a common malignancy with a variable clinical course. One of the established survival predictors in carcinomas in general is tumour grade; in OSCC, however, grading according to the World Health Organization (WHO) has no independent prognostic impact. Recently, a novel grading scheme associated with high impact on patient outcome has been proposed for squamous cell carcinoma of the lung. METHODS AND RESULTS: To probe whether this scheme could be applied to the upper aerodigestive tract, we retrospectively evaluated 157 chemo- and radiotherapy-naive OSCCs with complete clinical follow-up data and standardized treatment for tumour budding activity (BA), cell nest size (CNS), extent of keratinization, stromal content, nuclear size and mitotic count. Histomorphological characteristics were correlated with clinicopathological data and patient outcome. As in squamous cell carcinoma of the lung, high BA and small CNS were correlated significantly with shortened overall, disease-specific and disease-free survival. A three-tiered grading system based on a sum score of these two prognostic markers proved to be a strong age-, stage- and sex-independent prognosticator for survival with a hazard ratio for overall survival of 2.1 for intermediately differentiated (G2) tumours and 3.4 for poorly differentiated (G3) tumours compared to well-differentiated (G1) tumours (P < 0.001). CONCLUSIONS: We recapitulated and validated almost exactly the strong prognostic impact of a grading algorithm proposed recently for squamous cell carcinoma of the lung in OSCC. Our data may pave the way for a prognostically highly relevant future squamous cell carcinoma grading system broadly applicable in the aerodigestive tract.

Eight free flaps in 24 hours: a training concept for postgraduate teaching of how to raise microvascular free flaps.

Microvascular free flaps are complex but important tools in oral and maxillofacial surgery (OMFS), and though techniques to raise flaps are challenging surgeons often have little structured training. In this study we have evaluated a structured, three-day, hands-on, practical training course on raising flaps. Five human, Thiel-embalmed cadavers were used for training in how to raise the following flaps: radial forearm, anterolateral thigh, lateral arm, fibular, latissimus dorsi, scapular, iliac crest, and rectus abdominis. The total duration of the course was 24 hours over three days. All participants were asked to evaluate the design and conduct of the course, their own learning curve, and general questions about their knowledge of how to raise flaps and microsurgery. There was a significant increase in participants' assessments of how they raised all free flaps, except the lateral arm flap (4.74 (0.68) compared with 2.42 (0.81); p=0.052) before and after the course. The radial forearm flap was thought to be the most relevant in clinical practice (n=40; 75%), followed by the anterolateral thigh (n=5; 9%) and fibular (n=4; 8%) flaps. Comparisons between residents and consultants showed unsurprising differences in experience with microsurgery and self-assessment in raising particular free flaps before the course. We have shown that a structured, hands-on course using a well-established simulation model can significantly improve postgraduate surgeons' skills in raising free flaps.

Evaluation of human amniotic membrane as a wound dressing for split-thickness skin-graft donor sites.

Human amniotic membrane (HAM) has been used as a biomaterial in various surgical procedures and exceeds some qualities of common materials. We evaluated HAM as wound dressing for split-thickness skin-graft (STSG) donor sites in a swine model (Part A) and a clinical trial (Part B). Part A: STSG donor sites in 4 piglets were treated with HAM or a clinically used conventional polyurethane (PU) foil (n = 8 each). Biopsies were taken on days 5, 7, 10, 20, 40, and 60 and investigated immunohistochemically for alpha-smooth muscle actin (αSMA: wound contraction marker), von Willebrand factor (vWF: angiogenesis), Ki-67 (cell proliferation), and laminin (basement membrane integrity). Part B: STSG donor sites in 45 adult patients (16 female/29 male) were treated with HAM covered by PU foam, solely by PU foam, or PU foil/paraffin gauze (n = 15 each). Part A revealed no difference in the rate of wound closure between groups. HAM showed improved esthetic results and inhibitory effects on cicatrization. Angioneogenesis was reduced, and basement membrane formation was accelerated in HAM group. Part B: no difference in re-epithelialization/infection rate was found. HAM caused less ichor exudation and less pruritus. HAM has no relevant advantage over conventional dressings but might be a cost-effective alternative.

Can tissue spectrophotometry and laser Doppler flowmetry help to identify patients at risk for wound healing disorders after neck dissection?

OBJECTIVE: Microcirculation and oxygen supply in cervical skin were measured with an optical, noninvasive method in patients with or without radiotherapy before neck dissection. The course of wound healing was monitored after the surgical procedure to identify predictive factors for postoperative wound healing disorders. STUDY DESIGN: Tissue spectrophotometry and laser Doppler flowmetry were used to determine capillary oxygen saturation, hemoglobin concentration, blood flow, and blood velocity at 2-mm and 8-mm depths in the cervical skin of 91 patients before neck dissection in a maxillofacial unit of a university hospital in Munich, Germany. Parameters were evaluated for differences between patients with irradiation (24) and without (67) and patients with wound healing disorders (25) and without (66) (univariate or multivariate statistical analyses). RESULTS: Velocity at 2 mm was lower in irradiated skin (P = .016). Flow at 2 mm was higher in patients with wound healing disorders (P = .018). CONCLUSIONS: High flow values could help to identify patients at risk for cervical wound healing disorders.

Repair of oronasal fistulae by interposition of multilayered amniotic membrane allograft.

BACKGROUND: Oronasal fistulas are a frequent complication after cleft palate surgery. Numerous repair methods have been described, but wound-healing problems occur often. The authors investigated, for the first time, the suitability of multilayered amniotic membrane allograft for fistula repair in a laboratory experiment (part A), a swine model (part B), and an initial patient series (part C). METHODS: In part A, one-, two-, and four-layer porcine and human amniotic membranes (n = 20 each) were fixed in a digital towing device and the force needed for rupture was determined. In part B, iatrogenic oronasal fistulas in 18 piglets were repaired with amniotic membrane allograft, autofetal amniotic membrane, or small intestinal submucosa (n = 6 each). Healing was evaluated by probing and visual inflammation control (no/moderate/strong) on postoperative days 3, 7, 10, and 76. Histological analysis was performed to visualize tissue architecture. In part C, four patients (two women and two men, ages 21 to 51 years) were treated with multilayered amniotic membrane allograft. RESULTS: In part A, forces needed for amniotic membrane rupture increased with additional layers (p < 0.001). Human amniotic membrane was stronger than porcine membrane (p < 0.001). In part B, fistula closure succeeded in all animals treated with amniotic membrane with less inflammation than in the small intestinal submucosa group. One fistula remained persistent in the small intestinal submucosa group. In part C, all fistulas healed completely without inflammation. CONCLUSIONS: Amniotic membrane is an easily available biomaterial and can be used successfully for oronasal fistula repair. The multilayer technique and protective plates should be utilized to prevent membrane ruptures. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Secondary correction of posttraumatic orbital wall adhesions by membranes laminated with amniotic membrane.

The objective of the study was to find out if human amniotic membrane could be used for corrective surgery after trauma to the orbital wall. Because of its proposed antiadhesive qualities, it seemed to be potentially suitable. We studied 8 men (mean age 37 (range 19-74) years) who had deficient ocular movement after fractures of the orbital floor. Five of them had already been operated on. Inclusion criteria were trauma dating back more than 4 months and a soft tissue stricture in the orbital floor diagnosed by magnetic resonance imaging. Patients were treated secondarily with lysis of adhesions and insertion of allogeneic human amniotic membrane laminated on to polyglactin 910/polydioxanone foil, which functioned as the carrier material. Patients were followed up for 3 months, by which time disorders of motility of the ocular bulb had disappeared completely in 5. Two patients had improved motility and a reduction in both their subjective and objective symptoms. One patient had no improvement. The considerable reduction in adhesions and scarring after insertion of the membrane confirms previous assumptions, according to which the epithelial side of the human amniotic membrane has an antiadhesive effect because of its smooth surface.

Autonomization of free flaps in the oral cavity: A prospective clinical study.

Controversy exists over how long a free flap is dependent on its pedicle and if neovascularization is different between flap types, recipient sites, and irradiated and nonirradiated patients. An understanding of the timing of this process should optimize the safety of secondary procedures involving the flap. In a prospective clinical study, hemoglobin oxygenation and capillary flow were measured in 50 flaps (25 forearm flaps, 15 osteocutaneous fibula flaps, and 10 anterolateral thigh flaps) 4 and 12 weeks postoperatively. The flaps were located at the floor of the mouth, cheek, or tongue (n = 39) or at the hard or soft palate (n = 11). Measurements were carried out using the O2C monitoring system under temporary digital occlusion of the pedicle. After 4 weeks, 17 free flaps were found to be autonomized indicated by the O2C measurements comparing both values before and after digital compression of the vascular pedicle. After 12 weeks, 41 patients had completion of free flap autonomization, as indicated by the HbO(2) and CF before and after pedicle compression. The location of free flap in the lower jaw (P < 0.0001 after 4 weeks, P = 0.013 after 12 weeks), fasciocutaneous radial forearm flaps after 4 weeks (P < 0.0001), and not irradiated recipient site after 4 weeks (P = 0.014) were found to be positive factors significantly influencing autonomization. In conclusion, free flap autonomization depends on several variables which should be considered before further surgery after free flap reconstruction as the transferred tissue can be still dependent on its pedicle.

Further experience with the free soleus perforator flaps using CT-angiography as a planning tool - a preliminary study.

INTRODUCTION: The soleus perforator flap is a soft tissue flap with minimal donor site morbidity however is not frequently utilised due to the unpredictability of the perforating vessel to serve as the vascular pedicle. We have trialed the use of CT-angiography as a planning tool to predict location, length, course, and calibre of the pedicle to make this a more reliable choice. METHODS: Twenty consecutive patients with intraoral squamous cell carcinomas were assessed with CT-angiography to examine the peroneal perforators before considering soleus flap raising. If a sizeable perforator could be visualised at the upper half of the lower leg, flap raising was carried out, and the result of the CT-angiography was compared with the intra-operative findings. RESULTS: CT-angiography allowed for visualisation of perforators measuring 1mm in diameter and could predict location, length and course of the vessel. Accordingly, eight of the 20 patients had to be excluded from flap raising due to missing, too fine or too far distally located perforators. Intra-operative findings corresponded well with the results of the CT-scans. CONCLUSION: CT-angiography is a useful tool for planning the soleus perforator flap and allows selection of the most suitable perforator making the use of this flap more reliable.

Peroneal perforator flap for intraoral reconstruction.

Thin and pliable flaps with long, high calibre pedicles are ideally suited to lining the inside of the mouth. The radial forearm free flap has been our flap of choice until now, but we are unhappy with its potential for complications at the donor site. As an alternative, 30 patients have been treated in our unit with peroneal perforator flaps. Magnetic resonance (MR) angiography is necessary preoperatively to identify major perforating vessels. Flaps were raised using a lateral approach after the position of the most suitable perforator had been marked on the skin. The skin flaps were outlined in the proximal half of the lower leg with a maximum width of 5 cm to allow for direct closure of the wound. Five patients (of the original 35) were excluded after the results of MR angiography were known. All perforators identified on MR angiography could be exposed in the proximal half of the lower leg and most had a septocutaneous course. Reconstructions were in the floor of the mouth (n=16), tongue (n=11), and buccal mucosa (n=3). All but one flap survived with satisfactory functional results. The donor site morbidity was low. With the aid of MR angiography the peroneal perforator flap is a safe option for intraoral reconstruction. For small and medium sized defects we think that this flap is a good alternative to others, particularly if direct closure at an inconspicuous donor site is desired.

Quantitative comparison of the expression of antimicrobial peptides in the oral mucosa and extraoral skin.

Antimicrobial peptides (AMP) defend epithelial surfaces against pathological micro-organisms. We know of no comparison of their expression between the oral mucosa and extraoral epithelium, but knowledge of differences in their quantities is of interest, possibly as a starting point for new treatments. Expression of AMP human beta-defensin (hBD)-1/-2/-3 and psoriasin in the oral mucosa and extraoral epithelium of the head and neck were measured by real-time polymerase chain reaction (RT-PCR) (n=14), immunohistochemistry (n=6), and western blot (n=8). RT-PCR showed that all the genes investigated were expressed significantly more in the oral mucosa than in the skin (hBD-1: p=0.002; hBD-2: p=0.006; hBD-3: p=0.035; psoriasin: p=0.02). Immunohistochemistry and western blot showed differential concentrations of proteins: hBD-2 (p=0.021) and hBD-3 (p=0.043) were pronounced in the oral mucosa, whereas psoriasin was raised in the extraoral skin (p=0.021). There was no difference in protein concentrations for hBD-1 (p=0.08). The observed differences in the expression of AMP may be important for new treatments such as topical application of AMP derivatives.

Decreased cognitive functioning in patients with advanced oral squamous cell carcinoma.

Cognitive impairment causes a delay in diagnosis and treatment of the various cancer entities, resulting in reduced surgical outcomes and patient survival. However, no investigations have been carried out as to whether an association exists between cognitive functioning and tumour size in patients with oral squamous cell carcinoma (OSCC). In this study, 46 patients with OSCC were evaluated by using a screening test for dementia, consisting of a combination of the mini-mental state examination and the clock test (81% sensitivity and 90% specificity). Test scores were correlated with tumour size according to the TNM staging system, which was categorized as being either limited (T1, T2; n=24) or advanced (T3, T4; n=22). No difference in age (P=0.172), sex (P=0.330), the percentage of drinkers (P=0.090) or the percentage of smokers (P=0.484) was evident between the groups. Patients with advanced tumour size scored significantly lower (median 5.5 of 9 possible points) when compared with those having tumours of a limited size (median 9 of 9 possible points; P=0.005). The median score of patients with T3/T4 tumours suggested the need for comprehensive neuropsychological evaluations for dementia. In conclusion, this study has demonstrated the correlation of reduced cognitive functioning in patients with advanced OSCC. As a consequence, instructions for the identification of early signs and of symptoms of oral cancer are strongly recommended for relatives and nursing staff of patients with cognitive impairment. Such patients might need immediate treatment for oral cancer but might not be able to understand the significance of their symptoms and therefore present late, often too late.

Raising the osteocutaneous fibular flap for oral reconstruction with reduced tissue alteration.

Raising the osteocutaneous fibular flap offers excellent possibilities for oral reconstructive surgery but is associated with specific donor-site risks. Moreover, with inadequate surgical technique, flap-specific complications can occur, such as loss of the skin paddle or inadequate pedicle length. A flap-raising technique has been used to decrease surgical damage but provide maximal pedicle length. Sixty-six osteocutaneous fibular flaps were raised by the lateral approach with the following modifications: 1) flap-raising was carried out without a tourniquet, 2) only the amount of bone needed was removed, leaving the rest of the fibula intact, 3) only a minimal muscle cuff was included, and 4) the complete pedicle was dissected along the posterior intermuscular septum without opening the interosseous membrane and without touching the deep flexor muscles proximal to the osteotomized fibular segment. The skin paddle was placed distally in the leg, perfused by only 1 perforator in most cases. Medical records were analyzed and patients were examined postoperatively for up to 32 months to evaluate the above-mentioned complications. Of the 66 reconstructions, 44 were performed in a previously operated or irradiated neck. Three flaps and 1 skin paddle were lost. The most common donor-site complications were temporary wound-healing disturbances of the skin graft (n = 17) and transient pain or sensory alterations (n = 12). No compartment syndrome, ankle instability, or need for walking aids was recorded. A hammertoe deformity developed in 1 patient. On average, pedicle length was 9 cm and flap-raising took 130 minutes. In conclusion, maximal pedicle length and minimal bone and muscle resections can be achieved with a small number of donor-site complications. The skin paddle is highly reliable based on only 1 perforator. Perforators can be precisely controlled when raising the flap in the perfused leg.

Use of microvascular flap technique in older adults with head and neck cancer: a persisting dilemma in reconstructive surgery?

OBJECTIVES: To compare perioperative problems and outcomes of reconstructive surgery with microvascular flaps of a group of older (≥ 70) and younger adults (20-69). DESIGN: Prospective clinical cohort study. SETTING: Maxillofacial surgical unit of a university teaching hospital in Munich, Germany. PARTICIPANTS: Two hundred fifteen people with head and neck carcinoma (older: n = 54, mean age 75.8, range 70-96; younger: n = 161, mean age 55.5, range 20-69) who underwent surgery between 2007 and 2009. MEASUREMENTS: Participant characteristics: age, sex, American Society of Anesthesiologists (ASA) status, tumor type, preoperative radiation or chemotherapy, medical comorbidities. Surgical variables: flap type, type of reconstruction (primary/secondary), length of operation (minutes). Postoperative variables: length of stay (minutes) on intensive care unit (ICU), reasons for ICU stay longer than 1,500 minutes (surgical or medical), length of hospitalization (days), and reasons for hospitalization longer than 20 days (surgical or /medical). Short-term outcome within 30 days: revisions, flap success, overall complication rate, mortality. RESULTS: Older adults had a higher ASA class (P < .001) and shorter duration of surgery (P = .02). Age as an independent factor prolonged stay on ICU (P = .008) and was associated with a higher complication rate (P = .003) but had no influence on length of hospitalization, flap success, need for revisions, or mortality. CONCLUSION: Although higher rates of peri- and postoperative difficulties must be expected when microvascular reconstructive surgery is considered for older adults, careful surgical technique, adequate postoperative surveillance, and immediate management of complications can facilitate outcomes comparable with those for younger adults.

Secondary maxillofacial reconstruction with the radial forearm free flap: a standard operating procedure for the venous microanastomoses.

BACKGROUND: The radial forearm free flap (RFFF) is commonly used in reconstructive surgery. With respect to the maxillofacial region, several venous anastomotic techniques (e.g., single or double anastomoses) have been described, but the debate as to which alternative is preferable is ongoing. A complicating factor is the unpredictable anatomical situation in patients undergoing secondary operation. No recommendations are available for the surgical strategy in such cases. We present a standard operating procedure (SOP) applicable for secondary reconstructions, postulating double anastomoses as the method of choice, and evaluate its efficacy. METHODS: The following parameters were retrospectively analyzed for 120 patients with secondary (41; 34.2%) or primary (79; 65.8%) reconstruction following the instructions of SOP and compared between the study groups: age; sex; history of radiotherapy; side of the donor arm; flap size; preparation and use of the cephalic vein and reasons for its non-inclusion; included venae comitantes; recipient veins; arterial anastomoses; revisions, flap survival, and mortality within thirty days after operation. RESULTS: The method of choice was applicable in 26 (63.4%) secondary and 52 (65.8%) primary reconstructions (no difference; P = 0.841), resulting in 100% flap viability in both groups. In the remaining cases, single venous anastomoses were performed, resulting in 73.3% flap viability in secondary and 100% in primary reconstructions. Flap survival in secondary reconstructions was significantly higher when double anastomoses were conducted (P = 0.012). CONCLUSIONS: The results suggest the necessity of double venous anastomoses in secondary maxillofacial reconstructions with RFFF.

Thiel embalming technique: a valuable method for microvascular exercise and teaching of flap raising.

Because of its high requirements on dexterity and microsurgical skills and the need of complete understanding of flap anatomy, microvascular free flap transfer belongs to the most demanding surgical procedures. Therefore, courses for flap raising and microvascular exercise are considered a prerequisite to prepare for clinical practise. To achieve teaching conditions as realistic as possible we used a novel cadaver embalming method enabling tissue dissection comparable to the living body. Thirty cadavers which were offered to us by the Institute of Anatomy for the purpose of running flap raising courses were embalmed in the technique described by Thiel. On each cadaver, nine free flaps were dissected according to a structured protocol by each course participant and afterwards used for microvascular exercise. The conservation of fine vascular structures and the suitability of the embalmed tissue for microvascular suturing were observed and photographically documented. The Thiel embalming technique provided flap raising procedures to be performed under realistic conditions similar to the living body. Vessels and nerves could be exposed and dissected up to a diameter of 1 mm and allowed for microvascular suturing even after weeks like fresh specimens. The Thiel embalming method is a unique technique and ideally suited to teach flap raising and microvascular suturing on human material.