Comparative study of the PD-L1 status between surgically resected specimens and matched biopsies of NSCLC patients reveal major discordances: a potential issue for anti-PD-L1 therapeutic strategies.

BACKGROUND: High expression of programmed death ligand-1 (PD-L1) on tumor cells (TC) and/or on tumor-infiltrating immune cells (IC) is associated with a high response rate in patients with advanced nonsmall-cell lung cancer (NSCLC) treated with PD-L1 inhibitors. The use of a PD-L1 immunohistochemical (IHC) test in determining the responsiveness to immunotherapy has raised the question of the reliability and reproducibility of its evaluation in lung biopsies compared with corresponding resected surgical specimens. PATIENTS AND METHODS: PD-L1 expression in TC and IC was assessed in 160 patients with operable NSCLC on both whole surgical tissue sections and matched lung biopsies, by using a highly sensitive SP142 IHC assay. The specimens were scored as TC 0-3 and IC 0-3 based on increasing PD-L1 expression. RESULTS: PD-L1 expression was frequently discordant between surgical resected and matched biopsy specimens (the overall discordance rate = 48%; 95% confidence interval 4.64-13.24) and κ value was equal to 0.218 (poor agreement). In all cases, the biopsy specimens underestimated the PD-L1 status observed on the whole tissue sample. PD-L1-positive IC tumors were more common than PD-L1-positive TC tumors on resected specimens. The discrepancies were mainly related to the lack of a PD-L1-positive IC component in matched biopsies. CONCLUSIONS: Our results indicate relatively poor association of the PD-L1 expression in TC and IC between lung biopsies and corresponding resected tumors. Although these results need to be further validated in larger cohorts, they indicate that the daily routine evaluation of the PD-L1 expression in diagnostic biopsies can be misleading in defining the sensitivity to treatment with PD-L1 targeted therapy.

Reflectance confocal microscopy for the diagnosis of vulvar naevi: six cases.

BACKGROUND AND OBJECTIVES: The differential diagnosis between vulvar naevi and melanoma is challenging. In vivo reflectance-mode confocal microscopy (RCM) is an emerging technique that allows non-invasive high-resolution imaging of the skin and mucosa. It has recently been used for the study of vulvar melanosis and melanoma, but it has not been so far employed for the diagnosis of genital naevi. The objective of this study is to evaluate RCM features of vulvar naevi and to compare them with dermoscopical and histopathological aspects. METHODS: Clinical, dermoscopical, in vivo RCM and histological features of six vulvar naevi were evaluated. RESULTS: The clinical and/or dermoscopical aspects were suspicious in all six cases. RCM showed a blue naevus, an atypical genital naevus, a junctional naevus and three compound naevi that were later confirmed by histological examination. In one compound naevus, RCM showed focal cytological atypia and architectural irregularity without clear features of malignancy, confirmed by histological examination. CONCLUSIONS: Reflectance-mode confocal microscopy can play a role in non-invasive diagnosis of vulvar naevi, but further broader studies are required to validate our observations.

Why and how immunohistochemistry should now be used to screen for the BRAFV600E status in metastatic melanoma? The experience of a single institution (LCEP, Nice, France).

BACKGROUND: Knowledge of the BRAFV600E status is mandatory in metastatic melanoma patients (MMP). Molecular biology is currently the gold standard method for status assessment. OBJECTIVES: We assessed and compared the specificity, sensibility, cost-effectiveness and turnaround time (TAT) of immunohistochemistry (IHC) and molecular biology for detection of the BRAFV600E mutation in 188 MMP. METHODS: IHC, with the VE1 antibody, and pyrosequencing analysis were performed with formalin fixed paraffin embedded tumour samples. RESULTS: The BRAFV600E mutation was detected by pyrosequencing in 91/188 (48%) patients. IHC was strongly positive (3+) in all of these 91 cases. IHC was strongly positive in 9/188 (5%) cases in which the molecular testing failed due to non-amplifiable DNA. Weak or moderate staining was noted in 10/188 (5%) cases in which the molecular biology identified BRAF wild-type tumours. The ratio of the global cost for IHC/molecular biology testing was 1 : 2.2. The average TAT was 48 h vs. 96 h, for IHC vs. molecular biology testing, respectively. CONCLUSIONS: This study showed that VE1 IHC should be a substitute for molecular biology in the initial assessment of the BRAFV600E status in MPP. This methodology needs to be set up in pathology laboratories in accordance with quality control/quality assurance accreditation procedures. Under these strict conditions the question is to know if BRAFV600E-IHC can serve not only as a prescreening tool, but also as a stand-alone test (at least in cases displaying an unequivocally staining pattern) as well as an alternative predictive test for samples for which the molecular biology failed.

Discrepancies between FISH and immunohistochemistry for assessment of the ALK status are associated with ALK 'borderline'-positive rearrangements or a high copy number: a potential major issue for anti-ALK therapeutic strategies.

BACKGROUND: Patients with advanced lung adenocarcinomas expressing ALK rearrangements are highly responsive to crizotinib, a dual ALK/c-MET inhibitor. Immunohistochemistry (IHC) is an easy clinically and routinely applicable cost-effective assay for ALK, c-MET and ROS1 protein expression for potential treatment with crizotinib. The purpose of this study was to evaluate the percentage and the pattern of ALK-rearranged cells, the variation in the native ALK copy number, as well as ALK, c-MET and ROS1 protein expression, and their significance on outcome of crizotinib-treated lung adenocarcinoma patients. PATIENTS AND METHODS: Consecutive lung adenocarcinoma specimens (n = 176) 'double-negative' (wild-type EGFR and KRAS) were tested for ALK rearrangements/copy number alterations and for ALK, c-MET and ROS1 protein expression using automated standardized protocols. Preliminary data on the outcome of crizotinib-treated patients were recorded. RESULTS: FISH analysis identified 26/176 (15%) cases with ALK rearrangements. Seven cases had discordant results between the ALK FISH and IHC. Five cases with discordant FISH-positive/IHC-negative revealed FISH 'borderline' positivity (15%-20%). Three cases overexpressed c-MET and responded to crizotinib, and two cases with ALK-'borderline' rearranged cells only, not associated with c-MET expression, progressed under crizotinib. Two cases with discordant FISH-negative/IHC-positive revealed ALK gene amplification without associated c-MET or ROS1 protein expression. CONCLUSIONS: The discrepancies observed between the IHC and FISH data revealed unexpected biological events, rather than technical issues, which potentially can have a strong impact on the therapeutic strategy with crizotinib.

[Atypical histiocytoma in a child]. / Histiocytofibrome atypique chez une enfant.

BACKGROUND: Histiocytoma (HC) is a very common benign tumour generally seen in the lower limbs of adults, particularly women. There are, however, atypical forms of HC that behave like locally aggressive tumours, occasionally with relapse or even metastasis. Herein we report a case of locally aggressive HC in a child, which, on account of its clinical extension, required seven surgical procedures to achieve complete excision. PATIENTS AND METHODS: A 13-year-old child consulted for a hard purplish papule measuring 8 mm in diameter located in the right lumbar region. Punch biopsy revealed a poorly delineated dermal-hypodermic tumour comprising randomly distributed moderately pleomorphic fusiform cells, arranged in bands or with storiform architecture, certain of which were multi-nucleated. The mitotic index was high (11 mitoses in 10 fields at high magnification). There was no expression by the tumour of melanocytic markers (PS100, Melan-A), histiocytic markers (CD68) or CD34. FISH analysis showed the absence of COL1A1-PDGFB fusion gene. Based on these immunohistochemical and molecular findings, a diagnosis was made of atypical HC with high cellular density. Since the lower margins of the section showed tumoural foci, surgical excision was performed with 5-mm margins. Because the lateral and vertical limits were reached in all cases, a series of five further procedures (the last of was preceded by multiple peripheral biopsies) was necessary to achieve complete excision. These multiple excision procedures resulted in total excision of 25 cm across the longest side. No clinical relapse was seen after 25 months. DISCUSSION: Cellular or atypical forms of HC carry a high likelihood of post-surgical relapse. They are characterised by marked pleomorphism and high cellular density. In our patient, the extent of the lesion had been greatly underestimated initially, resulting in the need for several surgical procedures in order to achieve complete excision. It is thus important to highlight the predictive factors for this type of tumour in order to enable sufficiently extensive excision, or excision guided by previous biopsies, to be contemplated from the outset. These predictive factors are: young patient age, unusual location (trunk, face, neck), high cellularity, marked mitotic activity and deep extension.

[Malignant pleural mesothelioma after radiation treatment for Hodgkin lymphoma]. / Mésothéliome pleural malin après irradiation pour maladie de Hodgkin.

Malignant mesothelioma is a relatively uncommon malignancy. Although the pathogenesis is primarily related to asbestos, the role of ionizing radiation is more controversial. We report the case of a 41-year-old male who developed pleural mesothelioma. He had both, a prior short asbestos exposure and a thoracic radiotherapy for Hodgkin's disease 26years before. The evidence for radiotherapy as cause for mesothelioma is expanding and the diagnosis of mesothelioma in patients who had previous irradiation should be kept in mind.

ALK-gene rearrangement: a comparative analysis on circulating tumour cells and tumour tissue from patients with lung adenocarcinoma.

BACKGROUND: A subgroup of anaplastic lymphoma kinase (ALK)-rearranged lung tumours can respond to ALK inhibitors. Until now, the ALK status in circulating tumour cells (CTCs) isolated from patients with lung cancer has not been characterised. We assessed the ALK status in CTCs detected in patients with lung cancer and correlated the results to the ALK status defined in the corresponding tumour tissue. PATIENTS AND METHODS: A total of 87 patients with lung adenocarcinoma showing CTCs isolated using the isolation by size of epithelial tumour cell method were screened for their ALK status both in tumour samples and in CTCs. ALK break-apart fluorescence in situ hybridisation (FISH) and immunoreactivity analyses using an anti-ALK antibody (5A4 clone) were carried out on CTCs and compared with the results obtained in the corresponding tissue specimens. RESULTS: A total of five patients showed ALK-gene rearrangement and strong ALK protein expression in CTCs and in the corresponding tumour samples. Both ALK-FISH and ALK immunoreactivity analyses show negative results in CTCs and corresponding tumour samples for 82 patients. Conclusions We demonstrated that the ALK status can be determined in CTCs isolated from patients with lung cancer by immunocytochemistry and FISH analyses. These results favour non-invasive, ALK-gene status pre-screening on a routine basis on CTCs isolated from patients with lung cancer and open new avenues for real-time monitoring for adapted targeted therapy.

Morphological analysis of circulating tumour cells in patients undergoing surgery for non-small cell lung carcinoma using the isolation by size of epithelial tumour cell (ISET) method.

BACKGROUND AND OBJECTIVE: Recurrence rates after surgery for non-small cell lung cancer (NSCLC) range from 25 to 50% and 5-year survival is only 60-70%. Because no biomarkers are predictive of recurrence or the onset of metastasis, pathological TNM (pTNM) staging is currently the best prognostic factor. Consequently, the preoperative detection of circulating tumour cells (CTCs) might be useful in tailoring therapy. The aim of this study was to characterize morphologically any circulating non-haematological cells (CNHCs) in patients undergoing surgery for NSCLC using the isolation by size of epithelial tumour cell (ISET) method. METHODS: Of 299 blood samples tested, 250 were from patients with resectable NSCLC and 59 from healthy controls. The presence of CNHCs was assessed blindly and independently by 10 cytopathologists on May-Grünwald-Giemsa stained filters and the cells classified into three groups: (i) malignant cells, (ii) uncertain malignant cells, and (iii) benign cells. We assessed interobserver agreement using Kappa (κ) analysis as the measure of agreement. RESULTS: A total of 123 out of 250 (49%) patients showed CNHCs corresponding to malignant, uncertain malignant and benign cells, in 102/250 (41%), 15/250 (6%) and 6/250 (2%) cases, respectively. No CNHCs were detected in the blood of healthy subjects. Interobserver diagnostic variability was absent for CNHCs, low for malignant cells and limited for uncertain malignant and benign cells. CONCLUSION: Identification of CTCs in resectable NSCLC patients, using ISET technology and according to cytopathological criteria of malignancy, appears to be a new and promising field of cytopathology with potential relevance to lung oncology.

[Six cases of pulmonary MALT lymphoma: a heterogeneous therapeutic management]. / Six cas de lymphomes pulmonaires de type MALT : une prise en charge thérapeutique hétérogène.

Pulmonary mucosa-associated lymphoid tissue lymphomas (PMALT) account for around 1% of lymphomas. Clinical and radiological presentations, and the treatment of six PMALT were collected from 1993 to 2008. All patients received chemotherapy before disease progression. Two patients had a lobectomy and one received thoracic radiotherapy. In 2008, all the patients were alive and three were in remission. A "watch and wait" strategy is widely accepted for stable, asymptomatic patients and patients with low tumour mass. Surgery may be proposed for symptomatic patients who have localised PMALT. When a chemotherapy treatment is to be suggested, chlorambucil-based chemotherapy is preferred. There may be room for rituximab alone or in combination, but this remains to be precisely defined. Several larger studies are currently ongoing to assess the role of monoclonal antibodies and chemotherapy in MALT lymphomas. Subgroup analysis should help us to define the optimal treatment for PMALT.

Effect of surgical modality and hypofractionated split-course radiotherapy on local control and survival from sinonasal mucosal melanoma.

AIMS: To assess the efficacy of surgery and high-dose split-course radiotherapy in sinonasal head and neck mucosal melanoma (SHNMM). MATERIAL AND METHODS: Between 1991 and 2006, 23 patients (median age 73 years, male:female ratio 0.4) with non-metastatic SHNMM underwent surgery and postoperative radiotherapy, two had exclusive radiotherapy. Radiotherapy consisted of three series of 18Gy (3×6Gy every other day for 1 week) with 3 week planned treatment breaks. Chi-squared tests, Kaplan-Meyer method and Log-rank test were used to assess prognostic factors for survival and local control. RESULTS: There were 20 nasal cavity tumours; 12 of these involved more than one sinonasal site. One patient (4%) had lymphadenopathies at diagnosis. Six SHNMMs (24%) were amelanotic. The median follow-up was 39 months. Fourteen patients had en bloc surgery, 16 underwent radiation (14 postoperative, two exclusive). Eleven patients had local relapse, three had regional relapse and three had bone or liver metastases. Five year local control was 49±12%. Five year overall and SHNMM-specific survival was 38±12% and 62±12%, respectively. Five patients were alive without disease after 5 years and three after 10 years. En bloc excision (tumour removed in one piece) was prognostic for survival. CONCLUSIONS: En bloc surgery was a prognostic factor on outcomes for local control and survival in this series. Data from the literature have shown that postoperative radiation therapy improves local control. Most series were carried out with conventional fractionation. The effect of planned breaks (split-course radiotherapy) may be deleterious, as suggested in this series. Therefore, split-course radiotherapy cannot be recommended for SHNMM.

Basaloid squamous cell carcinoma of the head and neck: role of HPV and implication in treatment and prognosis.

Basaloid squamous cell carcinoma (BSCC) is a rare variant of squamous cell carcinoma (SCC) of the head and neck. Wain's criteria (peripheral palisading, association with SCC, high nuclear-cytoplasmic ratio, high mitotic rate, solid growth), anti-34BE12 and CK 5/6 staining, and absence of neuroendocrine markers are mandatory for the diagnosis of BSCC. Its increasing incidence parallels that of human papilloma virus (HPV)-positive tumours for the oropharyngeal subsite. On the other hand, BSCC is frequently considered a high-grade carcinoma of poorer prognosis than its SCC counterparts, mostly due to a higher rate of distant metastases. However, BSCC has similar or better locoregional control rates and a relatively better radiosensitivity than SCC. BSCC seems to have a dual behaviour depending, at least partly, on its recently described association with HPV. The basaloid subtype of SCC, owing to its particular behaviour, should be systematically investigated along with HPV and smoking status, as those factors may be determinant in the response to treatment.

[Basaloid squamous cell carcinomas of the head and neck]. / Etat des lieux sur les carcinomes basaloïdes ORL.

Basaloid squamous cell carcinomas (SCC) are a rare variant of SCC of the head and neck. Their histological characteristics have been described by Wain in 1986 and are reported in the 2005 WHO classification. A poorer prognosis of BSCC has been reported. Two recent case-control studies have shown a higher rate of distant metastases (15-40%, mean over 30%). Conversely, BSCC have similar or better locoregional control rates, a relatively good radiosensitivity and locoregional control. The role of chemotherapy in the neoadjuvant, concomitant or adjuvant setting needs to be redefined due to high metastatic failure rates; chest CT or PET CT are recommended at baseline and every 6-month during follow-up. Some subgroups of BSCC (oropharynx in particular) are more likely to be associated with oncogenic human papilloma virus HPV16. The determination of BSCC head and neck subgroups by HPV status is critical for the prognosis. The basaloid sub-type of squamous cell carcinomas owing to its particular behavior, should be taken into account while deciding the optimal therapeutic strategy.

[Anti-MAG paraproteinemic demyelinating polyneuropathy: a clinical, biological, electrophysiological and anatomopathological descriptive study of a 13-patients' cohort]. / Les polyneuropathies avec IgM monoclonale anti-MAG : étude descriptive clinique, biologique, électrophysiologique et anatomopathologique d'une cohorte de 13 patients.

INTRODUCTION: Polyneuropathies associated with IgM paraproteinemia and anti-myelin associated glycoprotein (MAG) antibodies (MAG-PN) have to be differentiated from chronic inflammatory demyelinating polyneuropathies. METHODS: In a retrospective study, we have analyzed clinical, electrophysiological, biological and pathological data from MAG-PN patients. RESULTS: Seven male and six female patients were followed in the department for a mean 2 years (0.5-6.5 years). Mean age at diagnosis was 61 years (44.5-85.5 years). Patients had symmetrical bilateral paresthesia (11/13) and hypoesthesia (11/13) prominent in the lower limbs. Nine patients developed gait ataxia and four patients had moderate distal weakness in the lower limbs. Mean Overall Neuropathy Limitation Scale was 2.3 (0-5). Nerve conduction study showed demyelinating features though delayed distal motor latency on median (206 % of normal value) and ulnar nerves (150% of normal value). Seven out of thirteen patients had at least two nerves with terminal latency index below 0.25. IgM paraproteinemia was of undetermined significance in ten cases and three patients had non-Hodgkin lymphoma. IgM deposits and widening of the peripheral myelin were observed in 5/7 sural nerve biopsies. Anti-MAG antibodies were detected in the sera of all patients using enzyme-linked immunosorbent assay and in 8/12 patients using western blot analysis. CONCLUSIONS: MAG-PN have distinctive clinical, electrophysiological and pathological features. It is a chronic, slowly progressive, predominantly sensory and ataxic neuropathy. Disability is usually moderate. Electrophysiological study shows distal demyelinating process and is highly suggestive of MAG-PN in more than one half of our patients. Several techniques may detect anti-MAG antibodies, they have to be associated to improve sensitivity and specificity of the test.

LANA-1, Bcl-2, Mcl-1 and HIF-1alpha protein expression in HIV-associated Kaposi sarcoma.

Human herpesvirus 8 (HHV8) is necessary for Kaposi sarcoma (KS) to develop, but whether the tissue viral load is a marker of KS progression is still unclear. Little is known about the level of expression of apoptosis-regulating proteins and of hypoxia-inducible factors (HIFs) in KS tumour cells relative to HHV8 expression. We therefore investigated the expression of the latency-associated nuclear antigen (LANA-1) of HHV8, Bcl-2, Mcl-1, Bax, Bcl-xL, caspase 3 and HIF-1alphain KS tissue specimens at different stages of the disease. The expression of these proteins was evaluated immunohistochemically using tissue microarrays (TMAs) in tissue specimens from 245 HIV-positive patients at different stages of the disease. Both LANA-1 and HIF-1alpha were expressed in KS biopsies taken at different stages, but their level increased throughout tumour progression. Additionally, the levels of Bcl-2 and Mcl-1 were higher in visceral KS lesions compared to levels observed in cutaneous and mucosal KS. This study demonstrates that late tumour stages of KS in tissues from HIV-positive patients are associated with high levels of LANA-1, HIF-1alpha and of the anti-apoptotic proteins, Bcl-2 and Mcl-1. Finally, the expression of these proteins can be potentially used as a tissue biomarker in defining patients with a higher risk of disease progression.

[McArdle disease (gycogenosis type V): analysis of clinical, biological and genetic features of five French patients]. / Maladie de McArdle (glycogénose de type V) : étude clinique, biochimique et génétique d'une cohorte de cinq patients français.

INTRODUCTION: McArdle disease (glycogenosis type V) is an autosomal recessive metabolic myopathy. Defect in glycogen breakdown is due to mutations of the gene for myophosphorylase (PYGM). Among patients of the department, we searched for correlations between disease phenotype, biochemistry analysis of muscle samples and PYGM genotype. METHODS: We included five patients whose muscle biopsy showed deposits of glycogen and negative histochemical staining for myophosphorylase. RESULTS: All patients exhibited exercise intolerance and high serum CK levels (mean 4400). Two of them had an acute renal insufficiency caused by rhabdomyolysis. One patient developed moderate late-onset muscle weakness of the proximal part of upper limbs. Muscle glycogen concentration was high (three times the normal). Myophosphorylase activity was undetectable in four muscle samples out of five. Two patients were homozygous and two other heterozygous for the R50X mutation of PYGM. The other one had a novel missense mutation S814N. Patients homozygous for R50X mutation had higher CK levels (8080 versus 1457, p=0.046), but disease severity and muscle glycogen concentrations were equivalent. CONCLUSIONS: Our patients had typical clinical and laboratory features of McArdle disease. Diagnosis was suggested by exercise intolerance with high CK levels. The R50X mutation was the most common (60% of the mutated alleles). We found no relationship between clinical severity, PYGM genotype and biochemistry analysis of muscle samples.

Prognostic significance of cortactin levels in head and neck squamous cell carcinoma: comparison with epidermal growth factor receptor status.

Cortactin is an actin-binding Src substrate involved in cell motility and invasion. In this study, we sought to examine the prognostic importance of cortactin protein expression in head and neck squamous cell carcinoma (HNSCC). To do so, cortactin and EGF receptor (EGFR) expression was retrospectively evaluated by immunohistochemistry in a tissue microarray composed of 176 HNSCCs with a mean follow-up time of 5 years. Cortactin immunoreactivity was weak to absent in normal epithelial tissue. Overexpression of the protein in 77 out of 176 tumours (44%) was associated with more advanced tumour-node-metastasis stage and higher histologic grade. Cortactin overexpression was associated with significantly increased local recurrence rates (49 vs 28% for high and low expressing carcinomas, respectively), decreased disease-free survival (17 vs 61%), and decreased the 5-year overall survival of (21 vs 58%), independently of the EGFR status. In multivariate analysis, cortactin expression status remained an independent prognostic factor for local recurrence, disease-free survival, and overall survival. Importantly, we identified a subset of patients with cortactin-overexpressing tumours that displayed low EGFR levels and a survival rate that equalled that of patients with tumoral overexpression of both EGFR and cortactin. These findings identify cortactin as a relevant prognostic marker and may have implications for targeted therapies in patients with HNSCC.

[Angiomyolipoma of the parotid gland]. / Angiomyolipome de la glande parotide.

INTRODUCTION: Angiomyolipoma is a rare benign mesenchymal tumor. It often arises in the kidney and in association with tuberous sclerosis or lymphangioléiomyomatosis. Extrarenal locations have been described, especially in the liver. Only a few cases have been described in the head and neck region and these are usually not associated with tuberous sclerosis or lymphangioléiomyomatosis. MATERIAL AND METHODS: We report a case of angiomyolipoma located in the parotid gland. RESULTS: A 43-year-old man consulted for treatment with a slow-growing nodule located in his right parotid gland. Ultrasound examination and magnetic resonance imaging revealed a well-limited lesion. Fine needle aspiration cytology was not suspicious. Partial parotidectomy was performed and the tumor showed the characteristic appearance of angiomyolipoma, with an admixture of fat smooth cells and tortuous thick-walled blood vessels. Genetic analysis showed anomalies on chromosomes 7 and 18. CONCLUSION: This article discusses the presentation and management associated with this exceptional tumor.

[Report of four women with Duchenne or Becker muscular dystrophy]. / A propos de quatre cas féminins de dystrophie musculaire de Duchenne et Becker.

INTRODUCTION: Duchenne and Becker muscular dystrophy are X-linked and affect mainly males. The authors report four female cases. EXEGESIS: Four patients presented muscular deficiency predominant to lower limbs and chronic disease. Female distrophinopathy is understandable by three mechanisms: Turner's syndrome, translocation X-chromosome with an autosome and skewed X-chromosome inactivation. CONCLUSION: Diagnosis of female Duchenne and Becker muscular dystrophy is really difficult if there is not male case in family.