DLL3 expression in large cell neuroendocrine carcinoma (LCNEC) and association with molecular subtypes and neuroendocrine profile.

OBJECTIVES: For stage IV pulmonary large cell neuroendocrine carcinoma (LCNEC), the only therapeutic option is palliative chemotherapy. DLL3 is a new therapeutic target, which seems to be often expressed in SCLC and LCNEC. It has recently been reported that DLL3 mRNA expression is particularly upregulated in the LCNEC subgroup with STK11/KEAP1 and TP53 co-mutations, in contrast to lower expression levels in RB1 and TP53 co-mutated LCNEC. Our aim was to investigate DLL3 protein expression in stage IV LCNEC and correlate data with mutational profiles (i.e.STK11/KEAP1/RB1), immunostaining results (pRb, NE markers) and clinical characteristics. MATERIALS AND METHODS: Immunohistochemical analysis for DLL3 (SC16.65) and ASCL1 (SC72.201) was performed on 94 and 51 FFPE tissue sections, respectively, of pathologically reviewed stage IV LCNEC. DLL3 and ASCL1 were scored positive if ≥1% of the tumor cells showed cytoplasmic/membranous or dotlike (DLL3) or nuclear (ASCL1) immunostaining. Data were correlated with available sequencing (TP53, RB1, STK11, KEAP1), immunostaining (pRb, NE markers) and clinical data. RESULTS: DLL3 was expressed in 70/94 (74%) LCNEC, 56 (80%) of which showed cytoplasmic/membranous staining. Median H-score was 55 (interquartile range 0-160). DLL3 staining was not different in pRb immunohistochemistry negative and positive patients (DLL3+ in 53/70 (76%) vs. 14/21 (67%), p = 0.409) or RB1 mutated and wildtype patients (DLL3+ in 27/34 (79%) vs. 23/33 (70%), p = 0.361). Nevertheless, 6/6 (100%) STK11 mutated, 10/11 (91%) KEAP1 mutated and 9/9 (100%) TP53 wildtype tumors were DLL3+ . Furthermore, DLL3 expression was associated with expression of ASCL1 and at least 2 out of 3 neuroendocrine markers. CONCLUSION: The high percentage (74%) of DLL3 expression in stage IV LCNEC denotes the potential of DLL3 targeted therapy in this patient group.

Prevalence and prognostic value of PD-L1 expression in molecular subtypes of metastatic large cell neuroendocrine carcinoma (LCNEC).

BACKGROUND: Pulmonary large cell neuroendocrine carcinoma (LCNEC) is a rare tumor with high mutational burden. Two subtypes of LCNEC are recognized, the co-mutated TP53 and RB1 group and the TP53 and STK11/KEAP1 group. We investigated PD-L1 and CD8 expression in a well characterized stage IV LCNEC cohort and compared expression in the two subtypes. METHODS: Immunohistochemical (IHC) analysis for PD-L1 and CD8 was performed on pathological reviewed pretreatment tumor samples for 148 stage IV LCNEC. Data about targeted next generation sequencing (TNGS) (TP53, RB1, STK11, KEAP1) and IHC for RB1 were available for most tumors. IHC staining for PD-L1 (DAKO 28-8) was performed and scored positive if tumors showed ≥1% membranous staining. CD8 was scored for intra-tumor T-cells and stromal cells. RESULTS: PD-L1 IHC expression data could be generated in 98/148 confirmed LCNEC samples along with RB1 IHC (n = 97) of which 77 passed quality control for TNGS. PD-L1 expression was positive in 16/98 cases (16%); 5 (5%) with ≥50%. PD-L1 expression was equal in RB1 mutated and RB1 wildtype tumors. None of STK11 mutated tumors (n = 7) expressed PD-L1. PD-L1 expression was correlated with superior overall survival (OS), hazard ratio 0.55 ((95% Confidence Interval 0.31-0.96), p = 0.038). Intra-tumor CD8 was associated with PD-L1 expression (p = 0.021) and stromal and intra-tumor CD8 were correlated with improved OS (p = 0.037 and p = 0.026 respectively). CONCLUSIONS: PD-L1 expression was positive in 16% of stage IV LCNEC tumors. This was independent of molecular subtype but associated with CD8 expression. In LCNEC patients with PD-L1 and/or CD8 expression superior OS was observed.

Uniform and blinded cause of death verification of the NELSON lung cancer screening participants.

Primary outcome of the Dutch-Belgian lung cancer screening trial (NELSON) is lung cancer-specific mortality. Accurate assessment of the cause of death (CoD) is crucial. As death certificates regarding the CoD can be inaccurate, a clinical expert committee (CEC) was formed to assign the CoD. In this study, the medical files of deceased lung cancer patients were reviewed and the outcomes were compared with official death certificates. The first 266 completed medical files of Dutch deceased participants who were diagnosed with lung cancer during the study or of those with lung cancer on the death certificate were selected and blinded towards arms and patients identity. The end product of the review process consisted of six possible categories which defined the graduation of certainty that lung cancer was the primary CoD. The percentage agreement and the Cohen's kappa statistics between the two CEC-memberswere calculated. The sensitivity and specificity of the official death certificates were determined. The results indicated that, the overall concordance and the Cohen's kappa between the CEC-memberswere 86.1% and 0.57(0.45-0.69, p<0.001), respectively. This level increased with the numbers of cases evaluated. The sensitivity and the specificity of the official death certificate were 92.6% and 98.8%; 6.5% cases were reclassified to lung cancer specific death, which is lower than in the National Lung Screening trial(22.0%). Concluding, each death should be reviewed by at least two members. So far, in the NELSON trial, possible biases related to lung cancer death seem relatively small.

[Is histopathology still the gold standard?] / Histopathologisch onderzoek als gouden standaard?

In the diagnostic process, microscopical analysis is considered to be the gold standard in determining the presence and nature of a disease. In some cases, in particular in rare diseases and in precursor lesions in which pathological changes have not yet fully developed, histological assessment is subject to diagnostic uncertainty. This uncertainty may be further compounded by the increasing tendency to submit small biopsies which may only harbour part of a pathological process. Several strategies may be adopted to increase the precision of histological analysis. While it is reasonable to assume that these strategies will lead to improved performance, systematic research to confirm this assumption is lacking. It is important to be aware of the limitations of histopathological testing.

[A rare form of obstructive pulmonary disease]. / Een zeldzame vorm van obstructief longlijden.

BACKGROUND: Lymphangioleiomyomatosis (LAM) is characterised by progressive dyspnoea, spontaneous pneumothorax and cystic pulmonary destruction. The disease may show similarities with emphysema clinically, radiologically and on lung function tests. CASE DESCRIPTION: A 44-year-old woman was referred for lung transplantation because of a 6-year history of dyspnoea and severe obstructive pulmonary function disorder with decreased diffusion capacity. Both her relatively young age and the fact that she had never smoked made us doubt the diagnosis 'COPD'. The pulmonary cysts seen on high-resolution CT (HRCT) suggested LAM. This was confirmed when we revised a pulmonary biopsy that had previously been performed. CONCLUSION: CT investigation should be carried out in patients with severe obstructive pulmonary disease without a risk profile appropriate for COPD. Diffuse, homogenous cysts on CT scan can indicate LAM, particularly in women. Conflict of interest and financial support: none declared.

Neuroendocrine tumours--challenges in the diagnosis and classification of pulmonary neuroendocrine tumours.

Pulmonary neuroendocrine (NE) proliferations are a diverse group of disorders which share distinct cytological, architectural and biosynthetic features. Tumours composed of NE cells are dispersed among different tumour categories in the WHO classification of tumours and as such do not conform to a singular group with regards to treatment and prognosis. This is reflected by the highly variable behaviour of NE proliferations, ranging from asymptomatic, for instance in diffuse idiopathic pulmonary NE cell hyperplasia and tumourlets, to highly malignant cancers such as small cell lung cancer and large cell NE carcinoma. In this review NE proliferations are described as distinct entities ranging from low grade lesions to high grade cancers. The differential diagnoses are considered with each of the entries. Finally, mention is made of tumours which may show some NE features.

A new approach for rapid and reliable enumeration of circulating endothelial cells in patients.

BACKGROUND: Mature circulating endothelial cells (CECs) are surrogate markers of endothelial damage/dysfunction. A lack of standardized assays and consensus on CEC phenotype has resulted in a wide variation of reported CEC numbers (4-1300 per mL). OBJECTIVES: Given the need for a quick, reliable, robust and validated CEC assay at an affordable price, we present a novel approach to enumerate CECs using a multi-parameter flow cytometric (FCM) method without immunological pre-enrichment. METHODS: CECs were defined as CD34+, CD45neg, CD146+ and DNA+ events based on the immunophenotype of endothelial cells from vein-wall dissections. As CECs express high levels of CD34, we based our assay on absolute CD34 counts after analyzing all CD34 positive events in a total blood volume of 4 mL needed for a precise enumeration of CECs at a frequency of < 1 cell µL(-1). RESULTS: The endothelial origin of CECs was confirmed by morphology, immunohistochemistry and gene expression. The new FCM assay was tested in parallel with a validated assay (i.e. CellSearch). CEC levels ranged from 4 to 79 CEC mL(-1) in healthy individuals and were significantly higher in patients with advanced solid malignancies (P = 0.0008) and in patients with hematological malignancies (P < 0.0001). CONCLUSIONS: This flow cytometric method should be useful as a fast and economical assay to enumerate and characterize CECs.

Outcomes of first-line chemotherapy in patients with advanced or metastatic leiomyosarcoma of uterine and non-uterine origin.

Although leiomyosarcomas (LMSs) form the largest subgroup of soft tissue sarcomas (STSs), the efficacy of chemotherapy in this group is largely unclear, partly because older studies are contaminated with gastrointestinal stromal tumors (GISTs). In this retrospective study we investigated the outcome of first line chemotherapy in 65 patients with unresectable or metastatic LMS. The overall response rate (ORR) was 18%; and the median progression-free (PFS) and overall survival (OS) were 3.8 and 9.7 months respectively. No statistically significant differences in outcomes for uterine and non-uterine LMS were found. In non-uterine LMS, however, the PFS and OS seemed to be longer for females than for males, potentially negatively affecting outcomes in this group. If our observations are confirmed in other series, they would suggest that studies performed in STS patients should not only stratify for histological subtype but also for uterine versus non-uterine LMS and for gender.

Perivascular epithelioid cell tumor of the retroperitoneum in a young woman resulting in an abdominal chyloma.

Perivascular epithelioid cell tumor (PEComa) is an extremely rare neoplasm which appears to have predominancy for young, frequently Asian, women. The neoplasm is composed chiefly of HMB-45-positive epithelioid cells with clear to granular cytoplasm and usually showing a perivascular distribution. These tumors have been reported in various organs under a variety of designations. Malignant PEComas exist but are very rare. The difficulty in determining optimal therapy, owing to the sparse literature available, led us to present this case. We report a retroperitoneal PEComa discovered during emergency surgery for abdominal pain in a 28-year-old Asian woman. The postoperative period was complicated by chylous ascites that was initially controlled by a wait-and-see policy with total parenteral nutrition. However, the chyle production gradually increased to more than 4 l per day. The development of a bacterial peritonitis resulted in cessation of production of abdominal fluid permitting normal nutrition without chylous leakage. Effective treatment for this rare complication of PEComa is not yet known; therefore, we have chosen to engage in long-term clinical follow-up.

Selective neck dissection for N0 and N1 oral cavity and oropharyngeal cancer: are skip metastases a real danger?

OBJECTIVES: To contribute to insight in therapeutic safety of selective neck dissections for oral cavity and oropharyngeal cancer with a special focus on the risk of skip metastases. DESIGN: Retrospective data analysis. SETTING: Tertiary referral centre. PARTICIPANTS: A total of 291 patients operated for oral cavity or oropharyngeal squamous cell cancer between 1999 and 2004. MAIN OUTCOME MEASURES: Incidence of skip metastases in both pathologically N0 and N+ necks for oral cavity and oropharyngeal cancer. RESULTS: Of all neck dissections (n = 226) performed for oral cavity cancer, skip metastases to level III or level IV occurred in 14 cases (6%). Ten skip metastases occurred in level III only (10/226 = 4%). Thus, four necks had metastases in level IV, which would not have been removed in case of a Selective neck dissection level I-III (supraomohyoid neck dissection). In case of oropharyngeal cancer, skip metastases to level III or level IV occurred in six of 92 cases (7%). Five skip metastases occurred in level III only (5/92 = 5%). This means that of the necks containing skip metastases, only one neck (1%): had metastases in level IV, which would not have been removed in case of a Selective neck dissection level I-III (Supraomohyoid neck dissection). CONCLUSIONS: The question whether level IV should be included in the treatment of N0 and even N1 necks of patients with cancer of the oral cavity and oropharynx cannot be answered by all data available to us now. The fear of skip metastases including level IV does not seem to be justified.

Interobserver variation in the classification of thymic tumours--a multicentre study using the WHO classification system.

AIMS: To test the reproducibility of the current World Health Organization (WHO) classification of thymic epithelial tumours and to determine the level of interobserver variation within a group of pathologists, all with experience and expertise in thoracic pathology. METHODS AND RESULTS: Ninety-five thymic tumours were circulated to a group of 17 pathologists in the UK and The Netherlands over a 1-year period. Participants were asked to classify them according to WHO criteria. The diagnoses were subjected to statistical analysis and kappa values calculated. The overall level of agreement was moderate (kappa 0.45). When the categories were reduced in number by creating two groups, (A + AB + B1 + B2 and B3 + C), the level of agreement increased to 0.62. An alternative grouping (A + AB + B1 and B2 + B3 + C) increased it slightly further. The best agreement was in tumour types A and AB. Difficulties arose in distinguishing B1 tumours from B2 tumours and B2 tumours from B3 tumours. CONCLUSIONS: Although the WHO system describes a number of well-defined tumour types with clear diagnostic criteria, the overall level of agreement was moderate and improved if some groups were amalgamated.

Circulating endothelial cells in oncology: pitfalls and promises.

Adequate blood supply is a prerequisite in the pathogenesis of solid malignancies. As a result, depriving a tumour from its oxygen and nutrients, either by preventing the formation of new vessels, or by disrupting vessels already present in the tumour, appears to be an effective treatment modality in oncology. Given the mechanism by which these agents exert their anti-tumour activity together with the crucial role of tumour vasculature in the pathogenesis of tumours, there is a great need for markers properly reflecting its impact. Circulating endothelial cells (CEC), which are thought to derive from damaged vasculature, may be such a marker. Appropriate enumeration of these cells appears to be a technical challenge. Nevertheless, first studies using validated CEC assays have shown that CEC numbers in patients with advanced malignancies are elevated compared to healthy controls making CEC a potential tool for among other establishing prognosis and therapy-induced effects. In this review, we will address the possible clinical applications of CEC detection in oncology, as well as the pitfalls encountered in this process.

The incidence of parametrial tumor involvement in select patients with early cervix cancer is too low to justify parametrectomy.

OBJECTIVE: To determine the incidence of parametrial involvement in a select group of patients with early cervical cancer. METHODS: We retrospectively reviewed the records of patients with cervical cancer and a maximum tumor diameter of 2 cm, infiltration depth<10 mm and negative pelvic lymph nodes who underwent a radical hysterectomy in two university hospitals. In addition, the literature was reviewed. RESULTS: 103 patients were identified in our databases that met the abovementioned criteria. In two of these patients (1.94%), parametrial involvement was found. Both patients had LVSI. Literature review revealed 696 patients described in three studies that satisfied the selection criteria. Three (0.43%) of these patients had parametrial involvement. In patients with early stage cervical carcinoma, tumor size<2 cm, infiltration depth<10 mm, negative pelvic lymph nodes and absent LVSI the risk of parametrial involvement is 0.63%. CONCLUSION: Because of a very low risk on parametrial involvement, patients who fulfil strict selection criteria could be candidates for conization and pelvic lymphadenectomy instead of more extensive surgery. Morbidity and pregnancy complications may decrease while it is unlikely that survival will be compromised.

Molecular analysis of human endometrium: short-term tibolone signaling differs significantly from estrogen and estrogen + progestagen signaling.

Tibolone, a tissue-selective compound with a combination of estrogenic, progestagenic, and androgenic properties, is used as an alternative for estrogen or estrogen plus progesterone hormone therapy for the treatment of symptoms associated with menopause and osteoporosis. The current study compares the endometrial gene expression profiles after short-term (21 days) treatment with tibolone to the profiles after treatment with estradiol-only (E(2)) and E(2) + medroxyprogesterone acetate (E(2) + MPA) in healthy postmenopausal women undergoing hysterectomy for endometrial prolapse. The impact of E(2) treatment on endometrial gene expression (799 genes) was much higher than the effect of tibolone (173 genes) or E(2) + MPA treatment (174 genes). Furthermore, endometrial gene expression profiles after tibolone treatment show a weak similarity to the profiles after E(2) treatment (overlap 72 genes) and even less profile similarity to E(2) + MPA treatment (overlap 17 genes). Interestingly, 95 tibolone-specific genes were identified. Translation of profile similarity into biological processes and pathways showed that ER-mediated downstream processes, such as cell cycle and cell proliferation, are not affected by E2 + MPA, slightly by tibolone, but are significantly affected by E(2). In conclusion, tibolone treatment results in a tibolone-specific gene expression profile in the human endometrium, which shares only limited resemblance to E(2) and even less resemblance to E2 + MPA induced profiles.

Extra-axial chordoma.

A chordoma which occurs as a primary tumour outside the axial skeleton is known as an extra-axial chordoma, parachordoma or chordoma periphericum. It is extremely rare and therefore survival, recurrence and the rates of metastasis are not known. Whilst few recurrences have been described, the extra-axial chordoma has the potential for late recurrence at up to 12 years. Metastases are even less frequent. We report the case of a 56-year-old woman who developed an extra-axial chordoma of the right thoracic wall in close relationship with the tenth rib. The tumour was completely removed and the prognosis is excellent.

"Cutaneous-type" angiosarcoma arising in a mature cystic teratoma of the ovary.

Benign and malignant somatic tumours arising in mature cystic teratomas of the ovary are a rare but recognised phenomenon. Squamous cell carcinoma is the most common somatic malignancy arising in ovarian teratomas, although many other types of tumour have been described. An angiosarcoma with "cutaneous" type typical features arising in a dermoid cyst of the ovary is reported. Vascular tumours have only rarely been described as secondary somatic tumours in ovarian teratomas. The diagnostic features and complications of such tumours are described.

A critical appraisal of prognostic and predictive factors for common lung cancers.

The outlook for patients with lung cancer remains poor despite advances in the understanding of the pathology and biology of this disease. To optimize treatment protocols prognostic data are essential. The current era with molecular research on mRNA expression analysis and proteomics will lead to a plethora of new molecular markers, which are likely to be correlated, at least in part, with each other and with disease activity, progression and survival. However, although the number of prognostic factors analysed in published systematic reviews on lung cancer is large, the scope of these factors in individual studies is often narrow. In daily practice prognostic factors other than general TNM staging are not implemented. To assess the efficacy of new prognostic factors for the management of individual patients with non-small cell lung cancer, studies with clinically relevant modelling are required. In this review arguments are provided to use a model combining radiological and histopathological growth rate, histopathological diagnosis and molecular characteristics as markers for metastatic capacity, tumour volume doubling time and expected response to targeted therapy. This may reveal time-related predictive information useful for treatment guidance of the individual patient.