Dear Editor,

A very important issue was assessed by Yang et al in their outstanding study recently published by this journal1. As pointed out by the authors, distant metastasis is the most preoccupant complication of differentiated thyroid carcinoma (DTC) and a very anguishing therapeutic challenge for the attending physician. A series of studies have been trying to establish a molecular pattern able to predict more aggressive follicular cell behavior. One of the most promissory markers integrating this molecular pattern is the expression of neural cell adhesion molecules (NCAM).

Yang et al studied a series of 365 surgical cases of thyroid disease - 214 DTC and 151 benign lesions. Immunohistochemistry showed that most benign lesions presented NCAM expression, whereas a significant proportion of DTC lost completely or showed a reduced NCAM expression, which confirms previous results suggesting that NCAM could be a diagnostic marker of DTC 2. We also studied NCAM expression in a series of 527 surgical cases of thyroid tissues - 395 DTC (343 papillary thyroid carcinomas and 52 follicular carcinomas) and 132 nonmalignant thyroid tissues (18 normal thyroids, 58 goiters and 56 adenomas). One hundred fifty-three of our patients presented metastasis at diagnostic and 58 developed distant metastasis during a follow-up of 12-298 months (43.50?33.29 months), Mo=21 months. NCAM expression was evaluated by immunohistochemistry and the same technique used by Yang et al, but with anti-NCAM monoclonal 123C3 clone antibody (DAKO- Carpenteria, CA, USA). We also considered NCAM positive those cases with NCAM expression in more than 30% of tumor cells. Fisher's exact test showed total loss or reduction of NCAM expression in 74.65% of DTC cases, while a significant portion (52.73%) of benign lesions were positive for NCAM (p< 0.0001). However, NCAM expression was not able to predict malignancy due to low sensibility (25.35%) and low specificity (47.27%), suggesting that NCAM alone is not a useful diagnostic marker.

In addition, Yang et al found that persistent NCAM expression in DTC is associated with a higher rate of metastasis. In our cohort, NCAM expression was not correlated with the presence of metastasis at diagnosis (p=0.4506), neither to tumor size (p=0.3814) nor to extrathyroid invasion (p=0.9855), multifocality (p=0.2747) or pTNM stage (p=0.6928). A log-rank test failed to show NCAM expression as a prognostic marker of relapse-free survival (p=0.8846). Nevertheless, NCAM positivity was more frequent in encapsulated tumors (37.78%) than in nonencapsulated tumors (20.62%; p=0.0399), suggesting that the peritumoral fibrotic reaction is associated with NCAM expression. In fact, 51.11% of our NCAM positive cases presented concurrent chronic lymphocytic thyroiditis (CLT), while only 25.89% of NCAM negative cases presented concurrent CLT (p=0.0045). We also evaluated the presence of tumor infiltrating lymphocytes (TIL) in DTC specimens by a routine HE staining. We found that NCAM expression was associated with the presence of TIL (p=0.0427). In order to distinguish TIL subsets, we performed immunohistochemical analysis using classical immune cell markers. We observed that NCAM expression was associated with the presence of CD4+ lymphocytes (p=0.0477), CD8+ lymphocytes (p=0.0015), CD20+ lymphocytes (p=0.0284) and FoxP3+ lymphocytes (p=0.0024). Interestingly, most NCAM negative cases (80.10%) were also negative for sodium/iodine symporter (NIS) protein immunohistochemical expression, whereas 71.43% of NCAM positive cases were positive for NIS as well (p< 0.0001), suggesting that NCAM could boost immunogenicity in DTC. These results suggest that NCAM expression is engaged in the antitumor immune response. However, the outcome of patients is not modified by NCAM expression, perhaps because an appropriate management of DTC patient is the most important and modifiable prognostic factor, impeding the natural course of malignancy.

The differences between Yang results and our data could be related to different population backgrounds, which are thought to affect antitumor immunity in DTC 3. Since the tumorigenic process is a complex biological system in which multiple molecular interactions may occur, minimum genetic differences in populations might affect dramatically the obtained results. An antitumor effect of NCAM may be expected in cases presenting genetic background that facilitates antitumor immune defense 3-4. We also cannot exclude that the different antibody used may lead to different results. In addition, it is worthy noting that NCAM may be engaged in pleiotropic functions in tumor progression, making the interpretation of NCAM expression a difficult task. More studies are warranted to understand the functional biologic role of NCAM expression in DTC tumors. Unfortunately, our data do not support the conclusion of Yang et al that NCAM expression in well differentiated thyroid carcinoma is an indicator for a higher risk of distant metastasis.

Sincerely,

Lucas Leite Cunha1, Elaine Cristina Morari2, Suely Nonogaki3, Fernando Augusto Soares4, Jose Vassallo5, and Laura Sterian Ward1.

1Laboratory of Cancer Molecular Genetics, Faculty of Medical Sciences - University of Campinas (Unicamp). 126 Tessalia Vieira de Camargo Street, Campinas, SP, Brazil.

2Department of biological sciences and health- State University of Roraima. 231, Sete de Setembro Street, Boa Vista, Roraima, Brazil.

3. Adolfo Lutz Institute. 355, Doutor Arnaldo Avenue, S?o Paulo, Brazil.

4Department of Pathology, A. C. Camargo Cancer Hospital. 211 Antonio Prudente Street, S?o Paulo, SP, Brazil.

5Laboratory of Investigative and Molecular Pathology (Ciped), Faculty of Medical Sciences - University of Campinas (Unicamp). 126, Tessalia Vieira de Camargo Street, Campinas, SP, Brazil.

REFERENCES

1. Yang AH, Chen JY, Lee CH. Expression of NCAM and OCIAD1 in well- differentiated thyroid carcinoma: correlation with the risk of distant metastasis. J Clin Pathol 2011;

2. El Demellawy D, Nasr AL, Babay S, Alowami S. Diagnostic utility of CD56 immunohistochemistry in papillary carcinoma of the thyroid. Pathol Res Pract 2009;205:303-9.

3. Cunha LL, Tincani AJ, Assumpcao LV, Soares FA, Vassallo J, Ward LS. Interleukin-10 but not interleukin-18 may be associated with the immune response against well-differentiated thyroid cancer. Clinics (Sao Paulo) 2011;66:1203-8.

4. Scarpino S, Di Napoli A, Melotti F, Talerico C, Cancrini A, Ruco L. Papillary carcinoma of the thyroid: low expression of NCAM (CD56) is associated with downregulation of VEGF-D production by tumour cells. J Pathol 2007;212:411-9.

Conflict of Interest:

None declared

CAUSES OF PULMONARY GRANULOMAS

In defining underlying causes of pulmonary granulomatous inflammation in their study population(s), Mukhopadhyay et al[1] correctly list immunodeficiency disorders as one possible association. They define a causal link between pathologist-observed granulomata and immune deficit where the latter has been already been identified clinically in individual patients. However, granulomatous disease can be a presenting feature of underlying, unsuspected immune deficiency, particularly in the context of some primary antibody deficiency disorders. The relationship of granulomatous inflammation in a biopsy and immunodeficiency needs to be considered beyond the simple circumstance of a patient with a previously defined immune deficit, in particular in the context of a) granulomatous disease with an alternative diagnostic label (e.g. sarcoidosis) and b) granulomatous disease of unknown aetiology. Awareness of the association of granulomata and immune deficiency is important, whether in the context of a geographically high incidence of sarcoidosis as a cause of pulmonary granulomata or of a high rate of no underlying aetiological factor being identified. Although relatively rare, primary immunodeficiency is an important issue for clinicians caring for patients with granulomatous disease to consider, identify, classify, risk assess and optimally manage.

Anecdotal local experience in the North of Scotland demonstrates that occasional 'sarcoid' patients (not included in the population studied by Mukhopadhyay et al) with pulmonary or extrapulmonary granulomatous inflammation are ultimately shown to have a primary immunodeficiency disorder (most frequently one of the common variable immune deficiency group of diseases, CVID[2]) but only after significant diagnostic delay. Such delay in these circumstances is relatively commonplace and is frequently associated with either overt or insidious secondary disease complications (usually pulmonary) which may be prevented or retarded by early immunoglobulin replacement and/or immunomodulatory treatment. Subtle histological differences have been described in the granulomata of sarcoid and CVID[3]. Granulomatous disease (particularly with splenic involvement), recurrent infections, cytopaenias and hypogammaglobulinaemia (rather than the hypergammaglobulinaemia of sarcoid) are, collectively, indicators of significant immune dysregulation and should prompt consideration of CVID as a potentially unifying diagnosis. Clinicians should consider routine measurement of serum immunoglobulins in granulomatous disease of unknown aetiology and as part of the diagnostic work-up in sarcoidosis. Similar recommendations, for similar reasons, have recently been made in the context of non-cystic fibrosis bronchiectasis[4]. Albeit relatively rarely, proactive detection of underlying immune deficiency as a cause of granulomatous inflammation will aid earlier diagnosis in conditions like CVID, allow more definitive and accurate aetiological classification of some cases at the clinician:pathologist interface and, not incidentally, will enhance opportunities for improvements in morbidity, mortality and quality of life for this group of complex patients.

REFERENCES

1. Mukhopadhyay S, Farver CF, Vaszar LT, et al. Causes of pulmonary granulomas: a retrospective study of 500 cases from seven countries. J Clin Pathol 2012; 65: 51-7

2. Morimoto Y, Routes JM. Granulomatous disease in common variable immunodeficiency. Curr Allergy Asthma Rep 2005; 5: 370-5

3. Bates CA, Ellison MC, Lynch DA et al. Granulomatous-lymphocytic lung disease shortens survival in common variable immunodeficiency. J Allergy Clin Immunol 2004; 114: 415-21

4. Pasteur MC, Bilton D, Hill AT. British Thoracic Society guideline for non-CF bronchiectasis. Thorax 2010; 65: i1-58

Conflict of Interest:

None declared

Dear Editor,

We read this article with great interest and would like to share our own similar experiences in support of this growing evidence base. Our department has the added complexity of being one of the UK ST1 training schools, with between ten and fifteen ST1 - ST5 trainees per year. We have trained and developed a senior Biomedical Scientist (BMS) in all specimen dissections who has gained the RCPath Diploma of Expert Practice in Histological Dissection and been appointed as an Advanced Practitioner (AP). This post covers all surgical specialties, and the AP currently dissects the majority of specimens, both simple and complex, as and when appropriate knowledge and experience has been gained. In terms of colorectal specimens, all types are dissected by the AP, including Extralevator Abdomino-Perineal Excision (ELAPE) specimens where the levator ani and/or coccyx are also resected.

Macroscopy

In terms of macroscopic assessment we follow a similar defined protocol to that of Sanders et al, with triage of each colorectal cancer specimen before dissection. Included in this discussion is most appropriate block selection. This process occurs for both the AP and the trainees, with the AP following the same pathway as that of the trainees. The AP and trainee take similar numbers of blocks, as appropriate to the case, in keeping with the departmental protocol (at least four blocks required). A macroscopic proforma is used to record data items during dissection. This is used by all staff, regardless of grade, and is seen as a method of ensuring that the minimum data items are recorded. Evidence has shown there to be more chance of identifying extramural vascular invasion (EVI) where additional tumour blocks are sampled [1]. In the study by Sanders et al it would be interesting to know how often EVI was identified where additional tumour blocks were taken by the BMS, as this may not be an appropriate criticism of practice.

Audit

We recently performed an audit to assess lymph node harvesting. This showed the average lymph node harvest of the AP was twenty-five percent higher that that of her histopathologist colleagues (consultant and trainee) (20 vs 15). All staff groups achieve the RCPath requirement of a harvest of twelve lymph nodes per case [2]. These findings were statistically significant (p=<0.001) and were presented at a national meeting [3]. The AP was less likely to perform resampling for additional lymph nodes (8.1% vs 16.6%). This was attributed to the level of training and experience of the AP in comparison to some trainee pathologists. In Sanders et al study the mean numbers of lymph nodes harvested by medical staff are similar to our own, but those harvested by the BMS are lower. This may be related to the experience of the BMS, as a similar decrease is seen with the least experienced trainees in our laboratory. Time spent on dissection may be a factor as our AP takes on average forty-five to sixty minutes per case.

Microscopy

As part of the Histopathology team our AP routinely reviews the slides generated from her colorectal cancer dissections. Reports are generated using a microscopic minimum dataset proforma and a paper copy of this is initially completed by the AP for joint review with the consultant before generation of the final report and authorisation by the consultant. This pathway is identical to that of the trainees within our department.

Development of scientific staff

Our AP has a specific interest in gastrointestinal pathology and is currently leading a research study around colorectal cancer diagnostics as part of a Professional Doctorate in Biomedical Science. This follows the Modernising Scientific Careers (MSC) programme which aims to provide the most appropriate developmental opportunities to high achieving scientists in their field [4]. Achieving the RCPath Specialist Diploma in lower gastrointestinal histopathology is also a current aim for our AP. To improve the knowledge base surrounding colorectal cancer management, she also regularly attends the local colorectal cancer multidisciplinary team meeting with one of the specialist consultants. This has the additional benefits in that it publicises the role of the AP within the team, and also provides an essential feedback mechanism on her performance. We feel that the performance of the AP is similar to that of an experienced registrar, and that she should be treated in the same way.

Benefits

The benefits to us are multiple. Consultant histopathologists benefit from more time to perform other important duties and the ability to delegate dissection training where appropriate. We would argue that in many cases reporting is quicker with the AP than with a trainee, as specimens are less likely to require resampling. Trainee histopathologists benefit from the additional dissection training, supervision and an additional source of advice. Due to the protocol driven nature of the AP's practice, she is able to provide the trainees with examples of agreed best practice within the department. APs benefit from career development which was previously unavailable, with the potential of eventual consultant healthcare scientist status when at doctoral level and with appropriate experience [4]. Patients benefit from the knowledge that dissection practice is standardised and that they are receiving the correct treatment based on appropriate staging.

In conclusion, we feel that there is an important clinical role for scientists within the histopathology team. Rather than simply working on the laboratory based aspects of the service, high achievers within this staff group should be utilised more appropriately. This may be in specimen dissection and microscopy, but could equally be within immunohistochemistry or molecular pathology. We agree that the evidence base needs to be improved in order to support further development within this area.

Conflict of Interest:

None declared

Re: EGFR gene copy number increase in vulvar carcinomas is linked with poor clinical outcome. Woelber et al. J Clin Pathol. 2012 Feb;65(2):133-9.

Dear editor,

A very novel issue was assessed by Woelber et al.[1] in their recently published study. This subject has major importance since the medical interest for vulvar carcinoma has increased in the last decade as the recognition of the increasing incidence of the disease, especially among young women. However, the fact that this carcinoma is an uncommon neoplastic disease makes its relative rarity an obstacle in designing studies to evaluate the effectiveness of the prognostic factors of molecular markers for this type of tumor. Thus, all experience on this type of tumor regarding the molecular aspects, becomes of paramount importance and we would like to share our experience in this field. Woelber et al.[1] studied a series of 183 formalin-fixed paraffin-embedded (FFPE) vulvar squamous cell carcinomas (VSCC) arranged in two TMAs, in which FISH analysis for EGFR, HER-2, CCND1 and MYC, IHQ for EGFR, HER-2 and CCND1, besides HPV detection by conventional PCR were performed. All data were associated to clinicopathological features. As the main results, EGFR copy number increase was found in 39.3% of the tumors and amplification of the EGFR gene in 9%. By IHQ, 53.3% of the samples showed 3+ staining for EGFR, being EGFR protein expression significantly correlated to EGFR copy number increase (p<0.05). Copy number gain of the EGFR locus was associated with high-invasion depth (p=0.045), non- basaloid phenotype (p=0.03), high-tumour stage (p<0.001), human papillomavirus negativity (p=0.04) and the number of lymph node metastases (p=0.02). Therefore, the authors showed that EGFR copy number gains were significantly related to unfavorable patient outcome and suggest the potential role of EGFR as a suitable therapeutic target in a subgroup of vulvar carcinomas. In a very similar fashion, our group studied 139 invasive VSCC arranged in two TMAs in which IHQ and FISH were performed for EGFR. In our approach, IHQ was held on automated Benchmark? Platform (Ventana Medical Systems) using the Zymed 31G7 mouse monoclonal antibody diluted 1:20, being this dilution previously standardized on whole slides of vulvar carcinomas. The absolute intensity of EGFR immunostaining was performed on a fourpoint scale as suggested by several authors [2,3,4] in vulvar carcinomas: (0=negative, 1=weakly positive, 2=positive, 3=strongly positive). In the same way of the study of Woelber and colleagues[1], we used FISH probes ZytoLight SPEC EGFR/CEN 7 Dual Color Probe (ZytoVision?) and the analysis were also performed as Woelber et al[1]. In order to analyze the relationship between the results obtained from IHC and FISH, associations between these data and histopathological features from the classification of the tumors (such as histological type, vascular and perineural invasion among others) and clinical data (such as recurrence, death by cancer and lymph node involvement), obtained from the clinical records of all the 139 patients were performed using the Chi-square test (X2) adopting p<0,05 as significant level. Regarding the amplification of EGFR gene, our results showed a lower rate of amplification (5 out of 78 cases - 6.4%) than those data raised by Woelber et al.[1] - 9%; and Growdon et al. (2008)[4] - 11,7%. Similarly, our IHQ analysis demonstrated only 3.9% of overexpression (3+) among our cases, which is contradictory to previous findings that reported an overexpression of 41-68% for this receptor [1,2,3]. Regarding the correlation between protein expression and amplification, among our cases, all tumors exhibiting amplification demonstrated an intense 3+ immunostaining by IHC, demonstrating a 100%correlation between FISH and IHC. These results corroborate the findings of Growdon et al. (2008) [4] to the observation that all tumors with intense 3+ expression of this receptor exhibited EGFR amplification and contradicts Woelber et al. [1] findings, regarding the occurrence of 20 vulvar carcinomas with EGFR copy number increase (including tumors with high polysomy or amplification) exhibiting low or intermediate levels of EGFR expression (scores 0, 1+ or 2+). Moreover, contrary to Woelber et al[1] study , our results showed no correlation between EGFR expression and the clinical and pathological features evaluated. The only association observed was statistically marginal and was related to FIGO staging, in which the most part of tumors stained negative for EGFR on the membrane (scores 0 and 1+) were classified as FIGO I or II, while the tumors EGFR positive (scores 2+ and 3+) were FIGO III or IV (p=0.08) which, as mentioned by Woelber et al.(2012)[1], may underline the influence of EGFR on tumor progression. Regarding the lack of association between EGFR expression and the clinical and pathological features evaluated, we attribute this result to the great heterogeneity of the intensity of immunostaining for EGFR observed in our cases, also described by Brustmann (2007) [2]. This heterogeneity leads us to believe that TMAs may not be useful for analyzing EGFR amplification in vulvar carcinomas. Anyway, it seems unanimous in the literature and among our results the fact that EGFR is, on greater or lesser extent, amplified in vulvar carcinomas and its overexpression could benefit a small group of patients with the therapy against this tyrosine kinase receptor and that more studies, regarding clinical trials, are required to elucidate this field.

References: [1] Woelber L, Hess S, Bohlken H et al. EGFR gene copy number increase in vulvar carcinomas is linked with poor clinical outcome. J Clin Pathol. 2012 Feb;65(2):133-9.

[2] Brustmann, H. Epidermal growth factor receptor is involved in the development of an invasive phenotype in vulvar squamous lesions, but is not related to MIB-1 immunoreactivity. Int J Gynecol Pathol. 2007;26(4):481-89.

[3] Oonk MH, de Bock GH, van der Veen DJ, Ten Hoor KA, de Hullu JA, Hollema H et al. EGFR expression is associated with groin node metastases in vulvar cancer, but does not improve their prediction. Gynecol Oncol 2007.104(1):109-13.

[4] Growdon WB, Boisvert SL, Akhavanfard S, Oliva E, Dias-Santagata DC, Kojiro S et al. Decreased survival in EGFR gene amplified vulvar carcinoma. Gynecol Oncol 2008.111(2):289-97.

Conflict of Interest:

None declared