Variations of salivary concentration of cytokines and chemokines in presence of oral squamous cell carcinoma. A case-crossover longitudinal prospective study.

Salivary biomarkers represent a promising diagnostic tool. Non-invasive and stress-free sampling results in objective parameters able to overcome the need of clinical expertise required for a correct interpretation of early oral squamous cell carcinoma (OSCC). The present study aims at evaluating potential variations in the salivary concentration of cytokines in presence of OSCC. Twenty patients who underwent surgical resection of OSCC were enrolled in order to assess the variation of the salivary concentration of cytokines and chemokines in a longitudinal prospective case-crossover setting. The salivary concentration of 27 salivary cytokines/chemokines was assessed before and after surgical resection of OSCC. In cases of relapsing/recurrent disease further assessments were performed. In presence of OSCC an increased concentration of IL-8(p = 0.004), IL-6(p = 0.005), VEGF(p = 0.014), MIP-1ß(p = 0.033), IP-10(p = 0.047), IL-1ß(p = 0.049) was observed; conversely the concentration of IFN-γ(p = 0.036) and IL-5(P = 0.048) decreased. In cases with relapsing/recurrent disease IL-5, IL-6 and MIP-1ß showed variations consistent with the previously reported results. Further studies are needed in order to confirm these results and to determine reference values to determine the presence or absence of disease.

Physics and Histologic Evaluation of Rotary, Ultrasonic, and Sonic Instruments.

Rotary instruments (RIs) are the most commonly used to perform osteotomies in many fields of medicine. Owing to a new interest in performing a minimally invasive surgery, over last fifteen years new devices have been used in oral surgery such as ultrasonic instruments (UIs) and, lately, sonic instruments (SIs). Nowadays, bone preservation and regeneration are paramount in many clinical situations and, consequently, it is crucial to rely upon instruments, which cause the least tissue damage during the surgery. Concerning SIs, there is still few information about workload to be applied and related temperature increases; furthermore, there are no comparative in-vivo studies, which analyze the thermal and mechanical effects on bone. Thus, SIs have been compared with UIs and RIs in terms of heat generation, operating time, accuracy, and tissue damage. Decalcification and sectioning procedure resulted in no significant differences between the applied instruments in terms of bone damage. RIs resulted more efficient than UIs (P < 0.001), but demonstrated low accuracy (NRS 4.9), whereas SIs (P = 0.005) required more time to perform the osteotomy. The maximum temperature increase occurred in the ultrasonic group. Even though SI were the slowest, they have proved to be the most accurate (NRS 8.4) in comparison with UI (NRS 7.6) and RI (NRS 4.9). Within the limit of this study, sonic instruments could be considered a safe alternative to ultrasonic instruments.

A novel genomic signature reclassifies an oral cancer subtype.

Verrucous carcinoma of the oral cavity (OVC) is considered a subtype of classical oral squamous cell carcinoma (OSCC). Diagnosis is problematic, and additional biomarkers are needed to better stratify patients. To investigate their molecular signature, we performed low-coverage copy number (CN) sequencing on 57 OVC and exome and RNA sequencing on a subset of these and compared the data to the same OSCC parameters. CN results showed that OVC lacked any of the classical OSCC patterns such as gain of 3q and loss of 3p and demonstrated considerably fewer genomic rearrangements compared to the OSCC cohort. OVC and OSCC samples could be clearly differentiated. Exome sequencing showed that OVC samples lacked mutations in genes commonly associated with OSCC (TP53, NOTCH1, NOTCH2, CDKN2A and FAT1). RNA sequencing identified genes that were differentially expressed between the groups. In silico functional analysis showed that the mutated and differentially expressed genes in OVC samples were involved in cell adhesion and keratinocyte proliferation, while those in the OSCC cohort were enriched for cell death and apoptosis pathways. This is the largest and most detailed genomic and transcriptomic analysis yet performed on this tumour type, which, as an example of non-metastatic cancer, may shed light on the nature of metastases. These three independent investigations consistently show substantial differences between the cohorts. Taken together, they lead to the conclusion that OVC is not a subtype of OSCC, but should be classified as a distinct entity.

Peculiar alexithymic traits in burning mouth syndrome: case-control study.

OBJECTIVES: The present case-control study aims to assess the occurrence of alexithymic traits in burning mouth syndrome (BMS) subjects and to correlate alexithymic traits to anxious and depressive traits in BMS subjects. MATERIALS AND METHODS: Prospectively enrolled BMS and control subjects were administered the 20-item Toronto Alexithymia Scale (TAS-20). Anxiety and depressive traits were assessed using the Hamilton Anxiety Rating Scale and the Montgomery and Asberg Depression Rating Scale. Occurrence of alexithymic traits was compared between BMS and control subjects. Correlation tests were used to measure the importance of alexithymic traits related to demographic characteristics, pain intensity (VAS score), and to the other psychometric scores. RESULTS: Fifty-eight BMS subjects (46 females and 12 males) had a mean TAS-20 score significantly higher when compared to controls (p < 0.001; r = 0.72), corresponding to an occurrence rate of alexithymic traits of 79.3 versus 6.9%. Alexithymic traits in BMS subjects were just related to depressive traits (p = 0.02; ρ = 0.31). CONCLUSIONS: The high occurrence of alexithymia in BMS is an adjunctive issue in favor of its multifactorial pathogenesis, with a not negligible role for somatization. CLINICAL RELEVANCE: Clinicians should be aware of the high occurrence of alexithymic traits among BMS subjects as such traits may affect the doctor-patient relationship.

Microbiopsy a novel sampling technique to early detect dysplastic/malignant alterations in oral mucosal lesions: practicability by general dentists.

BACKGROUND: When used in oral medicine clinics, microbiopsy is able to obtain tissue fragments suitable for a highly sensitive first-level diagnosis of dysplastic/malignant alterations in oral mucosal lesions. If feasible by general dentists, this sampling technique could reduce the diagnostic delay for oral malignant and premalignant lesions. This study assesses the adequacy of microbiopsy samples when taken by general dentists. METHODS: Fifty dentists, without specific training on oral medicine, volunteered for enrolment. They were given brief training and asked to prospectively sample any mucosal lesion observed during their routine practice. The sample adequacy features were assessed. RESULTS: The dentists sampled 152 lesions; there were 92.1% of adequate samples (140/152), and the BMZ was visible in 78.6% of these (110/140). Neither the clinical aspect nor lesion site affected either the adequacy or the presence of BMZ. CONCLUSIONS: The high adequacy rate observed and the advantages histological specimens have over cytological ones go to support the feasibility of microbiopsy taken by general dentists for the characterization of oral mucosal lesions and in selecting those requiring further assessment in specialized oral medicine centres.

Differential expression of HER2, STAT3, SOX2, IFI16 and cell cycle markers during HPV-related head and neck carcinogenesis.

The aim of this study was to analyze protein and gene expression of HER2 in 224 head and neck precancerous and malignant lesions by immunohistochemistry and FISH analysis. In parallel, expression of pStat3, Sox2, IFI16 and p16, Ki67 was evaluated. Immunohistochemical analysis was assessed on formalin-fixed paraffin-embedded (FFPE) tissue specimens. A combined method for HPV detection consisting of p16 immunostaining and two PCR probes was applied. HER2 gene status was evaluated by FISH analysis. HPV DNA was detected in 24% of cases with predominant HPV16 genotype. HPV-positive lesions had higher HER2, pStat3 and within carcinoma group, and higher IFI16 expression compared to the HPV-negative group (Fig. 1A-B-C). A strong positive correlation between Sox2 and proliferative activity was observed, whereas IFI16 expression displayed a negative relationship with Sox2 and Ki67 activity. The most striking result was higher pStat3 expression in HPV-positive lesions and its strong positive correlation with IFI16 expression. The presence of HPV may induce upregulation of HER2/neu, pStat3 and IFI16. High levels and a strong positive correlation between pStat3 and IFI16 suggest their synergistic pro-apoptotic effects in HPV-positive lesions.

Impact of academic and continuing education on oral cancer knowledge, attitude and practice among dentists in north-western Italy.

The present study aims to assess the knowledge, attitude and practice of dentists practicing in the Turin Province (north-western Italy) regarding oral cancer prevention and early detection, to weigh the impact of academic and continuing education and to compare actual and perceived knowledge/practice. A survey was prospectively carried out using an anonymous 23-item questionnaire. Bivariate analyses, multivariate logistic regression analyses and Spearman's correlation analyses examined the overall effect of demographic/background characteristics of responders, with particular emphasis on academic and continuing education. The responder group was formed by 450 dentists representative of the Turin Province Council of Dentists. Both academic and continuing education have a significant impact on knowledge, with a significant association between the time elapsed from continuing education and the degree of knowledge. Knowledge acquired during graduation is seen to significantly weaken in the absence of continuing education. The present study highlights the need of both thorough academic curricula and compulsory current continuing education.

Choristoma involving the floor of the mouth and the anterior tongue: a case of teratoid cyst with gastric and respiratory epithelia.

Oral dysontogenic cysts result from defective embryonic development. Among them teratoid cysts are the most unusual presentation and may be lined by gastric, intestinal, respiratory, squamous, ciliated epithelium or even pancreatic structures. Teratoid cysts containing respiratory and gastrointestinal epithelium have typically been called choristomas. This article describes a 15-year-old boy presenting a choristoma involving both the floor of the mouth and the anterior tongue and characterized by the presence of squamous epithelium with skin adnexa, gastric and respiratory epithelium.

Predictors of malignant transformation in oral leukoplakia and proliferative verrucous leukoplakia: An observational prospective study including the DNA ploidy status.

BACKGROUND: This prospective observational study investigated the determinants of malignant transformation (MT) in localized oral leukoplakia (OL) and proliferative verrucous leukoplakia (PVL). METHODS: Demographic, clinical, histological, and DNA ploidy status data were collected at enrolment. Survival analysis was performed (MT being the event of interest). RESULTS: One-hundred and thirty-three patients with OL and 20 patients with PVL entered the study over 6 years (mean follow-up 7.8 years). The presence of OED, DNA ploidy, clinical presentation, and lesion site were associated with MT in patients with OL in a univariate analysis. In a multivariate model, OED was the strongest predictor of MT in patients with OL. Adding DNA ploidy increased the model's predictive power. None of the assessed predictors was associated with MT in patients with PVL. CONCLUSIONS: DNA ploidy might identify a subset OL with low risk or minimal risk of MT, but it does not seem to be a reliable predictor in patients with PVL.

Field effect in oral precancer as assessed by DNA flow cytometry and array-CGH.

OBJECTIVE: 'Field cancerization' is an accepted model for oral carcinogenesis. So far, genetically altered fields have been just reported in the presence of carcinomas. This study assessed the distant mirror fields (MFs) of oral precancer by DNA high-resolution flow cytometry (hr DNA-FCM) and array-Comparative Genomic Hybridization (a-CGH). METHODS: Five leukoplakias without dysplasia (OLs), seven dysplastic leukoplakias (DOLs), and 12 corresponding visually normal and non-dysplastic MFs were analyzed. DNA aneuploidy (DNA Index, DI ≠ 1) was detected by hr DNA-FCM on DAPI stained nuclei suspensions. The epithelial DNA aneuploid subclones were FCM-sorted to obtain genomic DNA for a-CGH. RESULTS: Mirror fields, OLs, and DOLs showed increasing prevalence of DNA aneuploidy of, respectively, 8%, 20%, and 57%. The average number of chromosome aberrations (Ch-Abs) was 2.8 in MFs, 3 in OLs, and 10.6 in DOLs. MFs relative to OLs and DOLs had average numbers of Ch-Abs, respectively, of 1.8 and 3.6. Ch-Abs were also observed in DNA diploid sublines, and often the same aberrations were observed in both MFs and corresponding OLs/DOLs. CONCLUSION: DNA aneuploidy and Ch-Abs in MFs, the last ones being mainly gains, indicate an early onset of field effect in oral carcinogenesis.

Chromosomal instability, aneuploidy and routine high-resolution DNA content analysis in oral cancer risk evaluation.

Carcinogen exposure of the oral cavity is thought to create an extensive 'field cancerization'. According to this model, a very early precursor of oral cancer is a patch of normal-appearing mucosa in which stem cells share genetic/genomic aberrations. These precancerous fields then become clinically visible as white and red lesions (leuko- and erythro-plakias), which represent the vast majority of the oral potentially malignant disorders. This review focuses on aneuploidy (where it is from) and on biomarkers associated with DNA aneuploidy in oral mucosa and oral potentially malignant disorders, as detected by DNA image and flow cytometry. Data from the literature strongly support the association of DNA ploidy with dysplasia. However, work is still needed to prove the clinical value of DNA ploidy in large-scale prospective studies. Using high-resolution DNA flow cytometry with fresh/frozen material and the degree of DNA aneuploidy (DNA Index) might improve the prediction of risk of oral cancer development.

Clinical and histological features of gingival lesions: a 17-year retrospective analysis in a northern Italian population.

OBJECTIVES: Only few studies on gingival lesions considered large enough populations and contemporary literature does not provide a valid report regarding the epidemiology of gingival lesions within the Italian population. The histopathological and clinical appearance of 538 gingival lesions from northern Italians are described and discussed here. STUDY DESIGN: The case records of patients referred for the diagnosis and management of gingival lesions, from October 1993 to October 2009, were reviewed. Data regarding the histological type of lesion were also obtained from the biopsy register for each case, and blindly re-examined. RESULTS: We reported a greater frequency of benign lesions (reactive and/or inflammatory) in non-plaque/non-calculus induced gingival disorders. We confirmed an unambiguous prevalence of oral squamous cell carcinoma above all other malignant neoplasia, and a prevalence of neoplastic malignant lesions in the maxilla, with a slight increase in females and a drift of the incidence peak from the seventh to the eighth decade. There was a prevalence of precancerous gingival lesions in the maxilla, with a higher incidence in females and with a drift from the sixth to the seventh decade. We also reported a prevalence of oral lichen planus and lichenoid lesions as major manifestations of desquamative gingivitis. CONCLUSIONS: The high frequency of gingival involvement of such different diseases emphasizes the importance of histological characterization and differential diagnosis for periodontists, but more prospective studies are needed to better describe the true incidence of the non-plaque related gingival diseases.

Distinctive chromosomal instability patterns in oral verrucous and squamous cell carcinomas detected by high-resolution DNA flow cytometry.

BACKGROUND: Oral verrucous carcinomas (OVCs) are characterized by better prognosis than oral squamous cell carcinomas (OSCCs). Because chromosomal instability (CIN) in solid tumors is indicative of prognosis, this study investigated whether OVCs and OSCCs were characterized by differences in CIN biomarkers. METHODS: Fresh or frozen multiple tissue samples were submitted to high-resolution DNA flow cytometry (hr DNA-FCM). RESULTS: DNA aneuploid sublines were detected in 6 of 9 OVCs (66.7%) and in 20 of 25 OSCCs (80.0%). Multiple DNA aneuploid sublines were observed, respectively, in 2 of 6 (33.3%) DNA aneuploid OVCs and in 14 of 20 (70%) DNA aneuploid OSCCs (P = .163). OVCs were mainly characterized by DNA Index (DI) values in the near-diploid region (DI≠1 and DI < 1.4), whereas aneuploid OSCCs carried most frequently multiple aneuploid sublines with high DI values (DI ≥ 1.4). DNA near-diploid and high aneuploid sublines were, respectively, 87.5% and 12.5% for the OVCs versus 30% and 70% for the OSCCs (P = .004). CONCLUSIONS: Present data suggest that OVCs are characterized by a lower degree of CIN and tumor heterogeneity than OSCCs, such that they appear as "frozen" in an early stage of DNA near-diploid aneuploidy, as previously observed for oral preneoplastic lesions. These DI characteristics, which can easily be obtained by hr DNA-FCM, appear to reflect the well-known differences in aggressiveness and prognosis of OVCs and OSCCs.

Chromosomal aberrations and aneuploidy in oral potentially malignant lesions: distinctive features for tongue.

BACKGROUND: The mucosae of the oral cavity are different at the histological level but appear all equally exposed to common genotoxic agents. As a result of this exposure, changes in the mucosal epithelia may develop giving rise to Oral Potentially Malignant Lesions (OPMLs), which with time may in turn progress to Oral Squamous Cell Carcinomas (OSCCs). Therefore, much effort should be devoted to identify features able to predict the likeliness of progression associated with an OPML. Such features may be helpful in assisting the clinician to establish both appropriate therapies and follow-up schedules. Here, we report a pilot study that compared the occurrence of DNA aneuploidy and chromosomal copy number aberrations (CNAs) in the OPMLs from different oral anatomical subsites. METHODS: Samples from histologically diagnosed OPMLs were processed for high resolution DNA flow cytometry (hr DNA-FCM) in order to determine the relative DNA content expressed by the DNA index (DI). Additionally, array-Comparative Genomic Hybridization (a-CGH) analysis was performed on DNA obtained from diploid nuclei suspensions directly. When aneuploid nuclei were detected, these were physically separated from diploid nuclei on the base of their DI values by means of a DNA-FCM-Sorter in order to improve the a-CGH analysis. RESULTS: Tongue OPMLs were more frequently associated with DNA aneuploidy and CNAs than OPMLs arising from all the other mucosal subsites. CONCLUSIONS: We suggest that the follow-up and the management of the patients with tongue OPMLs should receive a distinctive special attention. Clearly, this hypothesis should be validated in a prospective clinical study.

Evidence for a possible anatomical subsite-mediated effect of tobacco in oral potentially malignant disorders and carcinoma.

PURPOSE: To test the hypothesis that cigarette smokers develop oral potentially malignant disorders or carcinomas in preferential anatomical subsites. METHODS: The association of smoking habit with the presence of oral lesions in specific anatomical subsites was assessed in 123 patients using the odds ratio analysis. RESULTS: When compared to all the other subsites, the relative frequency of smokers with lesions was higher in the buccal mucosa and in the floor of the mouth (FOM) (P=0.002 and P=0.005), while it was lower in the tongue (P<0.0005). Smokers were about 7 years younger than non-smokers (P=0.008). CONCLUSIONS: The association of smoking and age suggests that smoking may contribute to generate a field of injury that leads to lesions in shorter periods than other causes. The stronger relationship of smoking with lesions in the buccal mucosa and FOM than in the tongue suggests that tissue characteristics mediate the effects of tobacco.

Immunohistochemical expression of basement membrane proteins of verrucous carcinoma of the oral mucosa.

Squamous cell carcinoma (SCC) of the oral cavity is an extremely invasive tumour of stratified squamous epithelium that spreads throughout degradation of the basement membrane (BM) and extra-cellular matrix. Oral verrucous carcinoma (VC) is a rare low-grade variant of oral SCC that penetrates into the subepithelial connective tissue. It also has a different clinical behaviour from classical oral SCC. We investigated the immunohistochemical expression of laminin, laminin-5, collagen IV and fibronectin in VC, severe epithelial dysplasia (SED) and SCC in order to analyse if the pattern of these molecules expression contributes to the differences in the biological behaviour of these diseases. The staining pattern of laminin was less intensive in SCC compared with SED and VC, and collagen IV expression was increased in VC compared with SED. Discontinuities of laminin, collagen IV and fibronectin were more evident in SED than in VC. This study indicates that VC has a biological behaviour different from SED or SCC, observable by immunohistochemistry in the BM zone.

Outcome of oral dysplasia: a retrospective hospital-based study of 207 patients with a long follow-up.

The aim of this retrospective hospital-based study was to review and evaluate the long-term outcome of patients with oral epithelial dysplasia (OED), with or without surgical intervention, to identify factors affecting clinical course and malignant evolution. Patients with a follow-up of at least 12 months were included. Data collected were statistically analyzed. The mean age was 63.58 years for women (n = 100) and 64.17 years for men (n = 107). One hundred and thirty-five of the patients had lesions with histopathological features of mild OED, 50 had moderate OED and 22 had severe OED. Gender and risk factors seemed not to be related with the development of OED. One hundred and thirty-three patients underwent active treatment. During the period considered, 39.4% of the 207 lesions disappeared; 19.66% remained stable and 33.7% of the total cases showed a new dysplastic event after treatment. Fifteen (7.24%) out of 207 developed a squamous cell carcinoma during follow-up. Our data showed that speckled lesions are more often associated with high histological grade. The risk of malignant development does not seem to be predictable. Surrounded by the limitations of the retrospective designs, we have showed that there is no eminent benefit of surgical intervention of OED in preventing recurrences and malignant development.

Proliferative verrucous leukoplakia: a potentially malignant disorder involving periodontal sites.

BACKGROUND: Proliferative verrucous leukoplakia (PVL) is a rare oral mucosa disorder, frequently involving periodontal sites, with a high rate of progression to oral squamous cell carcinoma (SCC) and verrucous carcinoma (VC). This article describes the clinical features and follow-up of a group of patients with PVL, with attention to the involvement of the mucosa covering the alveolar crest and its malignant transformation. METHODS: Patients were retrospectively evaluated for demographic data, risk habits (smoking and drinking), locations of PVL lesions, incidence, and locations of malignant transformation. Patients with malignant transformation were compared to a control group (patients affected by oral carcinoma without PVL). RESULTS: Forty-seven patients were enrolled; PVL lesions were most frequently observed on the alveolar crest (41/47 [87.2%]), with gingival involvement in 22 of 47 (46.8%) cases. Nineteen patients (40.4%) developed 41 malignant lesions; the alveolar crest was the most affected site (12/41 [29.3%]). Compared to controls, patients with PVL were more likely to develop VC (odds ratio [OR] = 6.61; 95% confidence interval [CI]: 1.23 to 65.52) than SCC (OR = 0.15; 95% CI: 0.02 to 0.82), and they showed a higher incidence of cancer on masticatory mucosa (OR = 6.49; 95% CI: 1.78 to 29.12), particularly gingiva (P = 0.007) and the hard palate (P = 0.017). CONCLUSIONS: The importance of PVL awareness for periodontists is underscored by the frequency of gingival involvement and the high prevalence of malignant transformation on masticatory mucosa, which usually can be suspected because of the onset of warning signs, such as rapid growth of verrucosity, area of erosion or ulceration, acquisition of red areas, induration, and positive response to toluidine blue staining.

Interobserver and intraobserver variability affecting the assessment of loss of autofluorescence of oral mucosal lesions.

OBJECTIVES: The assessment of loss of tissue autofluorescence (LAF) has been proposed as an adjunct to comprehensive oral examination to enhance the detection of mucosal lesions harbouring dysplasia or carcinoma. The assessment of LAF is not based on completely objectified parameters therefore intraobserver and interobserver variability cannot be neglected alongside the issue of correct interpretation of LAF. The present study evaluated intraobserver and interobserver variability in the clinical assessment of LAF as performed by oral medicine practitioners (OMPs) or general dental practitioners (GDPs). MATERIALS AND METHODS: Couples of clinical pictures, acquired under white incandescent dental operatory light and during the assessment of LAF performed by VELscope were retrieved. Four OMPs and eight GDPs were asked to assess the pictures and to score the LAF. Kappa statistics allowed the assessment of intra- and inter-observer related variability. RESULTS: Pictures of 109 lesions representative of all oral mucosal sites and clinical appearances were selected. OMPs had a better intraobserver agreement than GDPs (substantial versus moderate). The moderate (k = 0.506) interobserver agreement observed among both OMPs and GDPs in a 2-score model (positive versus negative), lowered down to poor values only among GDPs when a 3-score or 4-score model (including uncertain judgements) was applied. CONCLUSIONS: A good agreement (k>0.8) was never observed and the present results are similar to previously reported data about conventional oral examination. Irrespective of the diagnostic accuracy, the assessment of AF seems not to be able to improve observer-related variability in the clinical assessment of oral mucosal lesions.

Oral potentially malignant lesions: first-level micro-histological diagnosis from tissue fragments sampled in liquid-based diagnostic cytology.

BACKGROUND: Scalpel biopsy may under-diagnose oral dysplasia/carcinoma in potentially malignant lesions (PMLs) because samples represent only one or a few sites. It is possible that small tissue specimens obtained from over the whole area of PMLs, by scraping with a dermatological curette, could be treated histologically and used as 'micro' biopsies. This study values the accuracy of micro-biopsies in the detection of dysplasia/carcinoma in oral PMLs. METHODS: A prospective study was carried out on 164 patients with PMLs, with both scalpel and micro-biopsies, for the presence of dysplasia/carcinoma. The most severe diagnosis (obtained by either method) was used as the reference standard. The presence/absence of the basement membrane zone (BMZ) in the micro-biopsy specimens correlated with the site, the clinical features of the PMLs and the operator. RESULTS: Micro-biopsy gave six of 164 (3.66%) inadequate specimens. Of 158 of 164 adequate samples, dysplasia/carcinoma was diagnosed in 85 of 158 cases; micro-biopsy diagnosis was in agreement with scalpel biopsy in 144 of 158 (91.14%) cases and showed a better sensitivity than did scalpel biopsy (97.65% vs. 85.88%), corresponding to two of 158 false-negative cases by micro-biopsy vs. 12 of 158 by scalpel biopsy. The BMZ was observed in 110 of 158 (69.62%) of all micro-biopsies and had no relationship with the sampling site, the clinical features of the PMLs or the operator. CONCLUSIONS: The negative predictive value (97.33%) suggests that micro-biopsy may well be an effective first-level diagnostic procedure for PMLs (especially in follow-ups and multiple lesions); moreover, in carcinoma (17% of cases) a definitive diagnosis could be made without further investigation.