Fine needle aspiration cytology and flow cytometry immunophenotyping of non‐Hodgkin lymphoma: can we do better?

P. Zeppa, E. Vigliar, I. Cozzolino, G. Troncone, M. Picardi, A. De Renzo, F. Grimaldi, F. Pane, A. Vetrani and L. Palombini
Fine needle aspiration cytology and flow cytometry immunophenotyping of non‐Hodgkin lymphoma: can we do better? Objective: To evaluate the diagnostic efficiency of fine needle aspiration cytology/flow cytometry (FNAC/FC) in the diagnosis and classification of non‐Hodgkin lymphoma (NHL) in a series of 446 cases and to compare the results with those of previous experiences to evaluate whether there had been an improvement in FNAC/FC diagnostic accuracy. Methods: FNAC/FC was used to analyse 446 cases of benign reactive hyperplasia (BRH), NHL and NHL relapse (rNHL) in 362 lymph nodes and 84 extranodal lesions. When a diagnosis of NHL was reached, a classification was attempted combining FC data and cytological features. Sensitivity, specificity and positive and negative predictive values (PPV and NPV) of FNAC/FC in the diagnosis and classification of NHL were calculated and compared with those available in the literature. Results: FNAC/FC provided a diagnosis of NHL and rNHL in 245 cases and of BRH in 188 cases. In nine cases, the diagnosis was ‘suggestive of NHL’ (sNHL) and in four cases was inadequate. Histology and clinical follow‐up confirmed 102 cases of NHL and detected one false positive. In 18 cases of BRH diagnosed by FNAC/FC, histological examination revealed 14 BRH and four NHL (false negatives). All nine cases diagnosed as sNHL were confirmed by histology. Including sNHL cases as false negatives, statistical analysis showed 94.9% sensitivity, 99.4% specificity, 99.6% PPV and 93.4% NPV in the diagnosis of NHL. A specific subtype was diagnosed in 125 cases and confirmed in 67 of 70 cases that had histological biopsies. Statistical analysis did not demonstrate significant improvements between the present series and previous studies either in diagnosis or in classification of NHL. Conclusions: FNAC/FC is a fundamental tool in the diagnosis and classification of NHL but the exiguity of diagnostic material and other technical and clinical limitations will probably continue to limit further improvement of the technique.     

Can the combination of electrochemical regeneration of NAD+, selectivity of L‐α‐amino‐acid dehydrogenase,and reductive amination of α‐keto‐acid be applied to the inversion of configuration of a L‐α‐amino‐acid?

The inversion of configuration of L‐alanine can be carried out by combining its selective oxidation in the presence of NAD⁺ and L‐alanine dehydrogenase, electrochemical regeneration of the NAD⁺ at a carbon felt anode, and reductive amination of pyruvate, i.e., reduction of its imino derivative at a mercury cathode, the reaction mixture being buffered with concentrated ammonium/ammonia (1.28M / 1.28M). The dehydrogenase exhibits astonishing activity and stability under such extreme conditions of pH and ionic strength. The main drawback of the process is its slowness. At best, the complete inversion of a 10 mM solution of L‐alanine requires 140 h. A careful and detailed quantitative analysis of each of the key steps involved shows that the enzyme catalyzed oxidation is so thermodynamically uphill that it can be driven efficiently to completion only when both the coenzyme regeneration and the pyruvate reduction are very effective. The first condition is easily fulfilled. Under the best conditions, it is the rate of the chemical reaction producing the imine which controls the whole process kinetically. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 101–107, 1999.     

Genomic‐scale comparison of sequence‐ and structure‐based methods of function prediction: Does structure provide additional insight?

A function annotation method using the sequence-to-structure-to-function paradigm is applied to the identification of all disulfide oxidoreductases in the Saccharomyces cerevisiae genome. The method identifies 27 sequences as potential disulfide oxidoreductases. All previously known thioredoxins, glutaredoxins, and disulfide isomerases are correctly identified. Three of the 27 predictions are probable false-positives. Three novel predictions, which subsequently have been experimentally validated, are presented. Two additional novel predictions suggest a disulfide oxidoreductase regulatory mechanism for two subunits (OST3 and OST6) of the yeast oligosaccharyltransferase complex. Based on homology, this prediction can be extended to a potential tumor suppressor gene, N33, in humans, whose biochemical function was not previously known. Attempts to obtain a folded, active N33 construct to test the prediction were unsuccessful. The results show that structure prediction coupled with biochemically relevant structural motifs is a powerful method for the function annotation of genome sequences and can provide more detailed, robust predictions than function prediction methods that rely on sequence comparison alone.     

Comparison of X‐ray and NMR structures: Is there a systematic difference in residue contacts between X‐ray‐ and NMR‐resolved protein structures?

We have compared structures of 78 proteins determined by both NMR and X-ray methods. It is shown that X-ray and NMR structures of the same protein have more differences than various X-ray structures obtained for the protein, and even more than various NMR structures of the protein. X-ray and NMR structures of 18 of these 78 proteins have obvious large-scale structural differences that seem to reflect a difference of crystal and solution structures. The other 60 pairs of structures have only small-scale differences comparable with differences between various X-ray or various NMR structures of a protein; we have analyzed these structures more attentively. One of the main differences between NMR and X-ray structures concerns the number of contacts per residue: (1) NMR structures presented in PDB have more contacts than X-ray structures at distances below 3.0 A and 4.5-6.5 A, and fewer contacts at distances of 3.0-4.5 A and 6.5-8.0 A; (2) this difference in the number of contacts is greater for internal residues than for external ones, and it is larger for beta-containing proteins than for all-alpha proteins. Another significant difference is that the main-chain hydrogen bonds identified in X-ray and NMR structures often differ. Their correlation is 69% only. However, analogous difference is found for refined and rerefined NMR structures, allowing us to suggest that the observed difference in interresidue contacts of X-ray and NMR structures of the same proteins is due mainly to a difference in mathematical treatment of experimental results.     

Lymph node fine‐needle cytology in the era of personalised medicine. Is there a role?

The 2016 World Health Organisation revised classification of lymphoma has sub‐classified well‐defined entities and added a number of provisional entities on the basis of new knowledge on genetic, epigenetics and phenotypical data; prognostic and predictive features are also part of this classification. New knowledge on well‐defined entities further enlightens the mechanisms of lymphomagenesis, which are more complex and multifactorial than once believed. Therapies are also more complex because traditional clinical trials have been integrated with new drugs and compounds with unique mechanisms of actions against distinct molecular targets. As lymphoma acquires additional genetic and phenotypic features over the time, pathological assessment is also necessary. Histological evaluation and tissue collection by surgical biopsies are necessary for phenotypical and molecular purposes; however, these are demanding procedures for both the patient and the health care system. At the same time, the choice of the best treatment for a specific entity, in different phases and different patients requires information that may not be available when the biopsy is performed. Fine needle aspiration cytology (FNAC) is successfully used in lymph nodes (LNs) in combination with different ancillary techniques and might be used to assess the phenotypic and genetic profile of specific targets and to get key information for therapy, in different phases and stages of the disease, with the option to re‐check the same target over time, without surgical excision. This brief review describes LN‐FNAC diagnostic criteria, current therapies for lymphomas and the potential role of LN‐FNAC in selecting non‐Hodgkin lymphomas patients for specific targeted treatments.     

PARP‐mediated proteasome activation: A co‐ordination of DNA repair and protein degradation?

During the evolution of aerobic life, antioxidant defence systems developed that either directly prevent oxidative modifications of the cellular constituents or remove the modified components. An example of the latter is the proteasome, which removes cytosolic oxidised proteins. Recently, a novel mechanism of activation of the nuclear 20S proteasome was discovered: automodified poly-(ADP-ribose) polymerase-1 (PARP-1) activates the proteasome to facilitate selective degradation of oxidatively damaged histones. Since activation of the PARP-1 itself is induced by DNA damage and is supposed to play a role in DNA repair, these new results suggest a joint role of PARP-1 in the removal of oxidised nucleoproteins and in DNA repair. We hypothesise that PARP-1 could provide a co-ordinative link between two nuclear antioxidant defence systems, whose concerted activation would produce a fast and efficient restoration of the native chromatin structure following oxidative stress.     

Song and Sperm in Crickets: A Trade‐off between Pre‐ and Post‐copulatory Traits or Phenotype‐Linked Fertility?

When females mate multiply (polyandry) both pre‐ and post‐copulatory sexual selection can occur. Sperm competition theory predicts there should be a trade‐off between investment in attracting mates and investment in ejaculate quality. In contrast, the phenotype‐linked fertility hypothesis predicts a positive relationship should exist between investment in attracting mates and investment in ejaculate quality. Given the need to understand how pre‐ and post‐copulatory sexual selection interacts, we investigated the relationship between secondary sexual traits and ejaculate quality using the European house cricket, Acheta domesticus. Although we found no direct relationship between cricket secondary sexual signals and ejaculate quality, variation in ejaculate quality was dependent on male body weight and mating latency: the lightest males produced twice as many sperm as the heaviest males but took longer to mate with females. Our findings are consistent with current theoretical models of sperm competition. Given light males may have lower mating success than heavy males because females take longer to mate with them in no‐choice tests, light males may be exhibiting an alternative reproductive tactic by providing females with more living sperm. Together, our findings suggest that the fitness of heavy males may depend on pre‐copulatory sexual selection, while the fitness of light males may depend on post‐copulatory fertilization success.     

A gene for speed? The evolution and function of α‐actinin‐3

The alpha-actinins are an ancient family of actin-binding proteins that play structural and regulatory roles in cytoskeletal organisation and muscle contraction. alpha-actinin-3 is the most-highly specialised of the four mammalian alpha-actinins, with its expression restricted largely to fast glycolytic fibres in skeletal muscle. Intriguingly, a significant proportion ( approximately 18%) of the human population is totally deficient in alpha-actinin-3 due to homozygosity for a premature stop codon polymorphism (R577X) in the ACTN3 gene. Recent work in our laboratory has revealed a strong association between R577X genotype and performance in a variety of athletic endeavours. We are currently exploring the function and evolutionary history of the ACTN3 gene and other alpha-actinin family members. The alpha-actinin family provides a fascinating case study in molecular evolution, illustrating phenomena such as functional redundancy in duplicate genes, the evolution of protein function, and the action of natural selection during recent human evolution.     

Is a liquid‐based cytology more sensitive than a conventional Pap smear?

K. Sigurdsson
Is a liquid‐based cytology more sensitive than a conventional Pap smear? Background: The comparative sensitivity of liquid‐based cytology (LBC) test and conventional Papanicolaou (Pap) smears is controversial. Material and methods: This study analyses the distribution of cytology, histology, colposcopy and large loop excision of the transformation zone among women screened in Iceland with LBC at the Cancer Detection Clinic in Reykjavik and with a conventional Pap smear outside the Detection Clinic in 2007–2011. The study material included 42 654 LBC tests from 20 439 women and 103 909 Pap smears from 61 574 women. The period 2000–2004 is used to correct for potential bias as a result of unequal distribution of the studied parameters between the study sites before the introduction of LBC. Results: The observed results indicated that women screened with an LBC sample had significantly decreased detection rates of inadequate smears, increased detection of low‐grade squamous intraepithelial lesion (LSIL)/atypical cytology and referrals to colposcopy, and an increased detection rate of cervical intraepithelial neoplasia grade 2 or worse (CIN2+) irrespective of age. LBC increased significantly the detection rates of high‐grade squamous intraepithelial lesion or worse (HSIL+) cytology and CIN3+ histology only in women under 40 years of age. Taking into consideration the unequal prevalence of the studied parameters between the study sites in 2000–2004 indicated, however, that LBC only affected the rate of inadequate and low‐grade cytology tests under the age of 40 years. Positive predictive values for CIN2+ were not significantly different between the tests. Conclusions: The study results support the view that LBC is no more sensitive than Pap smears for the detection of HSIL+ and CIN2+ irrespective of age. LBC decreased the rate of inadequate smears, but increased the rate of low‐grade cytology under the age of 40 years and decreased the total rate of abnormal smears over the age of 40 years.     

Liquid‐based cytology: is this the way forward for cervical screening?

Liquid-based cytology (LBC) is currently being marketed as an alternative methodology to replace the conventional PAP smear in cervical cytology. A substantial body of literature exists in support of LBC, some of which is at least partially sponsored by product manufacturers. The majority of published literature in support of LBC employs Bethesda reporting terminology. In this study we have analysed published raw data and presented this in NHSCSP terminology. Claims relating to sensitivity, specificity and smear adequacy have then been considered with reference to this data. Our analysis of existing data does not support the nationwide implementation of LBC at present. Further studies are recommended in order to evaluate the place of this technology within the NHSCSP.     

Could co‐transplantation of iPS cells derived hepatocytes and MSCs cure end‐stage liver disease?

Orthotopic liver transplantation is, to date, the only proven effective treatment for end-stage liver disease. However, it suffers from lack of donors and immunorejection. Here, we speculate that co-transplantation of induced pluripotent stem (iPS) cells derived hepatocytes and mesenchymal stem cells (MSCs) may offer an alternative way to treat patients with end-stage liver disease. Recently, progress on iPS cells, homogeneous differentiation of hepatocyte-like cells from embryonic stem cells (ESCs), and paracrine effects by MSCs highlight the possibility. Safe, efficient and rapid generation of iPS cells has been reliably produced by several experimental laboratories. Methods for highly efficient and homogeneous differentiation of ESCs into functional hepatocytes have been established as well. Moreover, paracrine effects by MSCs have been proven to play an important role in liver regeneration and repair, and the effects can be used as an enhancer for engraftment. All of these remarkable developments lead to this hypothesis which may offer a novel therapeutic strategy for treatment of patients with end-stage liver disease, though some issues about safety and efficacy need to be further guaranteed.     

Left,right or both? Estimating and improving accuracy of one‐side‐only geometric morphometric analyses of cranial variation

Procrustes‐based geometric morphometric analyses of bilaterally symmetric structures are often performed using only one side. This is particularly common in studies of cranial variation in mammals and other vertebrates. When one is not interested in quantifying asymmetry, landmarking one side, instead of both, reduces the number of variables as well as the time and costs of data collection. It is assumed that the loss of information in the other half, on which landmarks are not digitized, is negligible, but this has seldom been tested. Using 10 samples of mammalian crania and a total of more than 500 specimens, and five different landmark configurations, I demonstrate that this assumption is indeed easily met for size. For shape, in contrast, one‐side landmarking has potentially more severe consequences on the estimates of similarity relationships in a sample. In this respect, microevolutionary analyses of small differences are particularly affected, whereas macroevolutionary studies are fairly robust. In almost all instances, however, a simple preliminary operation improves accuracy by making one‐side‐only shape data more similar to those obtained by landmarking both sides. The same operation also makes estimates of allometry more accurate and improves the visualization. This operation consists in estimating the missing side by a mirror reflection of bilateral landmarks. In the Supporting Information, I exemplify how this can be easily done using free user‐friendly software. I also provide an example data set for readers to repeat and learn the steps of this simple procedure.     

Macro and Microhabitat Associations of the Peter's Tent‐Roosting Bat (Uroderma bilobatum): Human‐Induced Selection and Colonization?

Understanding species‐specific habitat selection is essential to identify how natural systems are assembled and maintained, and how emerging natural and anthropogenic disturbances will affect ecosystem function. In the Neotropics, Peter's tent‐roosting bat (Uroderma bilobatum), known to roost in forests, has become abundant in human‐modified areas. To understand how habitat characteristics in both intact forest and human‐modified areas influence the presence and density of U. bilobatum, we characterized habitat use at two scales (macrohabitat and microhabitat) and used logistic and poisson regressions to determine which habitat characteristics best predicted the presence and density of U. bilobatum within each scale. Moreover, we performed a redundancy analysis to determine which habitat scale explained more variation. As these bats are obligate tent roosters, we used tent as a surrogate for bat presence and density. We found that both macrohabitat and microhabitat scales explained variation in presence and density. Characteristics of the microhabitat scale, however, had higher predictive power, revealing that U. bilobatum preferentially inhabits areas with high density of coconut palms. Coconut palms were introduced recently in the Neotropics and are found only in human‐modified areas. Therefore, we hypothesize that U. bilobatum is expanding its range into these areas following the expanded distribution of this exotic plant species.     

Trade‐offs in oviposition choice? Food‐dependent performance and defence against predators of a herbivorous sawfly

The sawfly Athalia rosae L. (Hymenoptera: Tenthredinidae) is a feeding specialist on plant species of the Brassicaceae, which are characterised by secondary metabolites, called glucosinolates. The larvae can take up the respective glucosinolates of their hosts and concentrate them in their haemolymph to protect themselves against predators. Oviposition preferences of naïve females were tested for three species, Sinapis alba L., Brassica nigra (L.) Koch, and Barbarea stricta Andrz., and were related to larval performance patterns. Larvae were reared on either one of these plants and it was investigated how host‐plant quality influences both the developmental times and growth of larvae (bottom‐up) and the defence efficiency against predators (top‐down). Innately, almost all adult females avoided B. stricta for oviposition and clearly preferred B. nigra over S. alba. On average, larvae developed best on B. nigra. Female larvae reached similar final body masses on all host‐plant species, but males reared on S. alba were slightly lighter. The developmental time of larvae reared on B. stricta was significantly longer than on the other two plants. However, larvae reared on B. stricta were best protected against the predatory wasp Polistes dominulus Christ (Hymenoptera: Vespidae). The wasps rejected these larvae most often, while they attacked larvae reared on S. alba most frequently. Thus, larvae feeding on B. stricta theoretically run a higher risk of predation due to a prolonged developmental time, but in practice they are better protected against predators. Overall, oviposition preferences of A. rosae seem to be more influenced by bottom‐up effects on larval performance than by top‐down effects.     

Obesity and triple‐negative‐breast‐cancer: Is apelin a new key target?

Epidemiological studies have shown that obese subjects have an increased risk of developing triple‐negative breast cancer (TNBC) and an overall reduced survival. However, the relation between obesity and TNBC remains difficult to understand. We hypothesize that apelin, an adipokine whose levels are increased in obesity, could be a major factor contributing to both tumour growth and metastatization in TNBC obese patients. We observed that development of obesity under high‐fat diet in TNBC tumour‐bearing mice significantly increased tumour growth. By showing no effect of high‐fat diet in obesity‐resistant mice, we demonstrated the necessity to develop obesity‐related disorders to increase tumour growth. Apelin mRNA expression was also increased in the subcutaneous adipose tissue and tumours of obese mice. We further highlighted that the reproduction of obesity‐related levels of apelin in lean mice led to an increased TNBC growth and brain metastases formation. Finally, injections of the apelinergic antagonist F13A to obese mice significantly reduced TNBC growth, suggesting that apelinergic system interference could be an interesting therapeutic strategy in the context of obesity and TNBC.     

Ecological filtering or random extinction? Beta‐diversity patterns and the importance of niche‐based and neutral processes following habitat loss

Although both niche‐based and neutral processes are involved in community assembly, most models on the effects of habitat loss are stochastic, assuming neutral communities mainly affected by ecological drift and random extinction. Given that habitat loss is considered the most important driver of the current biodiversity crisis, unraveling the processes underlying the effects of habitat loss is critical from both a theoretical and an applied perspective. Here we unveil the importance of niche‐based and neutral processes to species extinction and community assembly across a gradient of habitat loss, challenging the predictions of neutral models. We draw on a large dataset containing the distribution of 3653 individuals of 42 species, representing 35% of the small mammal species of the Atlantic Forest hotspot, obtained in 68 sites across three continuously‐forested landscapes and three adjacent 10 000‐ha fragmented landscapes differing in the amount of remaining forest (50%, 30% and 10%). By applying a null‐model approach, we investigated β‐diversity patterns by detecting deviations of observed community similarity from the similarity between randomly assembled communities. Species extinction following habitat loss was decidedly non‐random, in contrast to the notion that fragmented communities are mainly driven by ecological drift. Instead, habitat loss led to a strong biotic homogenization. Moreover, species composition changed abruptly at the same level of landscape‐scale habitat loss that has already been associated with a drastic decline in species richness. Habitat loss, as other anthropogenic disturbances, can thus be seen as a strong ecological filter that increases (rather than decreases) the importance of deterministic processes in community assembly. As such, critical advances for the development of conservation science lie on the incorporation of the relevant niche traits associated with extinction proneness into models of habitat loss. The results also underscore the fundamental importance of pro‐active measures to prevent human‐modified landscapes surpassing critical ecological thresholds.