Recent eLetters

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

To the Editor,

We noted with interest the study entitled "Breast cancer stem cell markers CD44, CD24 and ALDH1: expression distribution within intrinsic molecular subtype", published by Ricardo and colleagues [1]. Papers like this one have major importance since retrospective studies analyzing the proportion of cancer stem cells in breast tumor biopsies as prognosis factors are still required. Ricardo et al. also correlate the identification of breast cancer stem phenotypical markers with the molecular subtypes of breast cancer. Therefore, this report tries to address important and relevant questions in breast cancer cell biology. However, this study presents two major flaws. First, the authors performed single CD44 or CD24 staining to identify cells with the CD44+/CD24- cancer stem phenotype. Single immunohistochemistry is not the choice for analyzing the combined expression of two different markers on the same cell but, on the contrary, expression of both receptors needs to been analyzed simultaneously. Double-staining immunohistochemistry for the simultaneous detection of CD44 and CD24 in paraffin embedded sections from breast cancer patients has been developed and validated first by Abraham et al. [2] and subsequently by Mylona et al. [3]. In those reports, the authors quantified the intensity of staining and then the proportion of CD44+/CD24- tumor cells using software-based image analysis in order to avoid bias derived from pathologist inspection. Ricardo and colleagues quote both papers but they followed a different methodology. From our perspective, the double immunofluorescence for CD44 and CD24 performed by the authors in only 10% of the samples does not validate their methodology since it still considered the percentage of cells expressing the receptors rather than the intensity of labeling. On the other hand, they used flow cytometry for the simultaneous analysis of CD44 and CD24 expression in breast cancer cell lines. With this methodology, their results are consistent with those from previous publications [4]. The second imperfection of this study is directly related to the first. Ricardo and colleagues stratified their samples based on the percentage of cells expressing one of the receptors rather than in the number of cells with the CD44+/CD24- phenotype (as reported by Abraham et al. [2] and Mylona et al. [3]). They defined as "CD44 positive" the samples containing 10-100% of tumor cells immunoreactive for CD44 and as "CD24 negative/low" the samples with 0-25% of tumor cells expressing membranal CD24. With this methodology, 411/463 samples (88.6%) were classified as CD24-/low. Thus, nine out of ten samples fitted their description of CD24 "negativity", leaving CD44 "positivity" as the characteristic that is mainly in charge of their whole analysis. Accordingly, the correlation that they found between CD44 expression (not the cancer stem cell phenotype) and the basal subtype (where 94% of the samples were considered CD24-/low) has been previously described in breast cancer cell lines by Charafe-Jauffret and colleagues [5]. Given the clear methodological differences between the present studies from Ricardo and colleagues and those from Abraham et al. [2] and Mylona et al. [3], it is unclear how the authors classified their samples as "CD44+/CD24- <10%" and "CD44+/CD24- >10%" for the further analysis presented in tables 2-3, and figures 2-3. Therefore, we think that the results of this very interesting paper need to be carefully reinterpreted.

References: 1. Ricardo S, Vieira AF, Gerhard R, et al. Breast cancer stem cell markers CD44, CD24 and ALDH1: expression distribution within intrinsic molecular subtype. J Clin Pathol. 2011; doi:10.1136/jcp.2011.090456 2. Abraham BK, Fritz P, McClellan M, Hauptvogel P, Athelogou M, and Brauch H. Prevalence of CD44+/CD24-/low cells in breast cancer may not be associated with clinical outcome but may favor distant metastasis. Clin Cancer Res. 2005; 11(3):1154-9. 3. Mylona E, Giannopoulou I, Fasomytakis E, Nomikos A, Magkou C, Bakarakos P, and Nakopoulou L. The clinicopathologic and prognostic significance of CD44+/CD24(-/low) and CD44-/CD24+ tumor cells in invasive breast carcinomas. Hum Pathol. 2008; 39(7):1096-102. 4. Fillmore CM, and Kuperwasser C. Human breast cancer cell lines contain stem-like cells that self-renew, give rise to phenotypically diverse progeny and survive chemotherapy. Breast Cancer Res. 2008; 10(2):R25. 5. Charafe-Jauffret E, Ginestier C, Monville F, Finetti P, Adelaide J, Cervera N, et al. Gene expression profiling of breast cell lines identifies potential new basal markers. Oncogene. 2006; 25(15):2273-84.

Conflict of Interest:

None declared

To the Editor,

I read with interest the report of Chakupurakal and colleagues on a patient who developed peripheral neuropathy during imatinib treatment.(1) Their report highlights the importance of vigilance for late, unexpected adverse events in patients receiving potentially lifelong maintenance chemotherapy.

The authors assert that neuropathy has not previously been reported as a side effect of imatinib. I would like to draw the authors' attention to a case of neuropathy during imatinib treatment, which I reported some years ago.(2) In that case there was a temporal association between the initiation of a concomitant medication (amlodipine) that may increase imatinib exposure and the acute onset of neuropathic symptoms. A search of Pubmed (accessed 28 April 2011) using the search terms 'imatinib' and 'neuropathy' identifies this paper, the paper of Chakupurakal and two less relevant papers. Since the publication of my case report I have twice been contacted by colleagues who had each observed a single case of neuropathy during imatinib treatment with no other explanation identified. It is difficult to know whether the frequency of neuropathy on imatinib is greater than the frequency of idiopathic neuropathy in an age-matched population.

The same caution applies to the interpretation of cases of left ventricular dysfunction during imatinib treatment. The authors include heart failure and left ventricular dysfunction in a list of 'commonly reported side effects of imatinib'. However, the average age of patients at diagnosis of chronic myeloid leukaemia coincides with the age at which cardiac problems start to rise in incidence in the general population. The experiments of Kerkela and colleagues (3) might lead us to predict many more cases of left ventricular failure with dasatinib, which is 300 times as potent as imatinib as an inhibitor of the ABL1 enzyme,(4) yet this is not a major clinical problem in experience to date. Whilst the absence of significant cardiac impairment in a prospective evaluation of imatinib- treated patients (5) is somewhat reassuring, it remains possible that late effects might emerge after many years of treatment, and ongoing pharmacovigilance is required.

References

1. Chakupurakal, G., Etti, R.J. & Murray, J.A. Peripheral neuropathy as an adverse effect of imatinib therapy. J Clin Pathol 2011; 64: 456.

2. Ross, D.M. Peripheral neuropathy on imatinib treatment for chronic myeloid leukaemia: suspected adverse drug interaction with amlodipine. Intern Med J 2009; 39: 708.

3. Kerkela, R., Grazette, L., Yacobi, R., Iliescu, C., Patten, R., Beahm, C., et al. Cardiotoxicity of the cancer therapeutic agent imatinib mesylate. Nat Med 2006; 12: 908-916.

4. O'Hare, T., Walters, D.K., Stoffregen, E.P., Jia, T., Manley, P.W., Mestan, J., et al. In vitro activity of Bcr-Abl inhibitors AMN107 and BMS-354825 against clinically relevant imatinib-resistant Abl kinase domain mutants. Cancer Res 2005; 65: 4500-4505.

5. Estabragh, Z.R., Knight, K., Watmough, S.J., Lane, S., Vinjamuri, S., Hart, G., et al. A prospective evaluation of cardiac function in patients with chronic myeloid leukaemia treated with imatinib. Leuk Res 2011; 35: 49-51.

Conflict of Interest:

None declared

We thank Dr Naim for the interest in our paper. In our review of histiocytoid breast carcinoma (1), we have indicated that it is best categorized as a subtype of invasive lobular carcinoma, and this is stated in our title. The various terms mentioned in our review relate to historical descriptions that alluded to this unusual tumour, based on morphological evaluation and reports by authors who investigated this subject (2-6). The 'apocrine lobular' prefix is perhaps the most morphologically and immunophenotypically accurate among the terms, as histiocytoid breast carcinoma has been shown to express apocrine differentiation fairly consistently, and its underlying lobular origin is mostly well accepted. Eusebi et al used 'myoblastomatoid' to refer to its resemblance to granular cell tumour, which is a histologic differential diagnosis for histiocytoid breast carcinoma (6). The reference to 'pleomorphic' lobular breast carcinoma was because of the observation that histiocytoid carcinoma cells that mimicked 'foam' cells were noted in these aggressive pleomorphic tumours (2). While lipid-rich carcinoma was initially considered synonymous with histiocytoid breast cancer, lipid stains are generally negative in the latter (1, 3, 4). We are not suggesting that these myriad terms should be currently applied to histiocytoid lobular breast carcinoma. The purpose of their mention is to present a chronological sequence of how this entity was initially recognized and the subsequent work that led to our present understanding. We believe the term histiocytoid breast carcinoma is best applied to a tumour that consists of carcinoma cells that resemble benign histiocytes. Invariably, further workup usually establishes its lobular phenotype. 'Malignant histiocytoma', which implies a tumour of fibrohistiocytic origin, is not an appropriate term for histiocytoid lobular breast carcinoma which is epithelial and not fibrohistiocytic nature. When a breast tumour composed of histiocyte-like cells is established on immunohistochemistry to be epithelial in nature with lobular characteristics, the diagnosis of histiocytoid lobular breast carcinoma is firm. However, we acknowledge the potential challenges in making this diagnosis on needle core biopsy where limited sampling may pose diagnostic difficulty, and in such instances, further excision with more complete histological examination will allow a conclusive diagnosis. References 1.Tan PH, Harada O, Thike AA, Tse GM. Histiocytoid breast carcinoma: an enigmatic lobular entity. J Clin Pathol Mar 12.

2.Eusebi V, Magalhaes F, Azzopardi JG. Pleomorphic lobular carcinoma of the breast: an aggressive tumor showing apocrine differentiation. Hum Pathol 1992 Jun; 23(6): 655-62.

3.Hood CI, Font RL, Zimmerman LE. Metastatic mammary carcinoma in the eyelid with histiocytoid appearance. Cancer 1973 Apr; 31(4): 793-800.

4.Ramos CV, Taylor HB. Lipid-rich carcinoma of the breast. A clinicopathologic analysis of 13 examples. Cancer 1974 Mar; 33(3): 812-9.

5.Eusebi V, Betts C, Haagensen DE, Jr., et al. Apocrine differentiation in lobular carcinoma of the breast: a morphologic, immunologic, and ultrastructural study. Hum Pathol 1984 Feb; 15(2): 134- 40.

6.Eusebi V, Foschini MP, Bussolati G, Rosen PP. Myoblastomatoid (histiocytoid) carcinoma of the breast. A type of apocrine carcinoma. Am J Surg Pathol 1995 May; 19(5): 553-62.

Conflict of Interest:

None declared

The Hematologists(Pathologists trained in hematology) of kolkata, West Bengal( in private setup tertiary care hospitals or in diagnostic laboratories or in Govt. set up secondary or tertiary care teaching hospitals) are being mostly trained with performing, interpretation, evaluation and diagnosis of common hematological problems, requiring Bone Marrow studies, by Bone Marrow aspiration(for diagnostic and follow up after any therapy) than by Bone Marrow Trephine biopsy, unless there is1) failed aspirate due to no marrow fragments in conditions when there is Marrow fibrosis or Marrow aplasia or 2) when there is suspected Pathology in Bone or 3) Marrow is cellular but poor aspiration happens due to tightly packed marrow. In Kolkata the interpretation and reporting of Bone Marrow aspiration is usually done by consultant pathologists trained specially in hematology division in a laboratory or in a teaching hospital Pathology dept set up. A very few centers[ one or two] are there in kolkata in the medical colleges where there are post doctoral trainees in hematology who also perform and report Bone marrow aspiration mainly and occasionally by trephine biopsy.

We authors consider however that there are till many advantages of performing and reporting Bone Marrow aspiration then reporting Bone Marrow Trephine biopsy, some of them can be summarized as follows • The Marrow aspiration needles are less costly, supplied and easily available in the kolkata market, in each &every laboratories and can be easily sterilized then islam or Jamshedi needles for marrow trephine biopsy ** the procedure can be repeated if and when necessary *** Multiple numbers of slides can be drawn from a single aspirate and may be used thus for special stain, cyto-chemistry and immuno histochemistry when necessary **** Report can be handed over to patient by 24 hours in most cases unless special stains or immunostains are asked for.***** Cytogenetic studies including flow cytometry can be performed with aspirated materials******The technique and interpretation can be percolated even at secondary health care level where there is a trained pathologists(at district and sub divisional level hospitals or diagnostic laboratories) and thus necessary treatment can be given earlier by General physicians or primary care physicians. • However there remain recognized problems with Bone Marrow aspiration studies 1) That the aspirated material often becomes diluted with much aspirated blood and in that case it is wiser to suck off blood before making smears with a blotting paper edge 2) the smear drawn by the trainee Post doctorals may not be equally enough thin and spreaded and clumping of many cells at some patchy areas of the slide 3) Marrow material may be drawn inadequate for interpretation 4) while Interpretation of cases and diagnosis are given erythriod hyperplasia particularly in children-the underlying diseases is not well described and many reports realy are such 5) When there is suspected focal lesion –in the bone marrow itself marrow aspiration can miss diagnosis 6) suspected &focal bone marrow fibrosis 7) when there is need to study the bone marrow architecture or bone structure or bone marrow blood vessels, Bone marrow aspiration may not be adequate study • Besides there may be many indications for performing Bone marrow Trephine biopsy like in Aplastic anemia; Myelofibrosis; MDS; Hairy cell leukemia; smoldering Multiple Myeloma; Early Multiple Myeloma Granulomatous lesions in Marrow [ may be from bacterial, viral, rickettsial, fungi, parasitic and sarcoidosis]; Osteopatheis. hypocellular MDS and investigation of suspected MDS or MDS with fibrosis; investigation for suspected amylodosis in cases of Multiple Myeloma ; Hypoplastic acute leukemia; AML M7; After Bone marrow transplant assessments; in CML for sub-typing the disease or to detect early blast crisis and to assess marrow fibrosis; for staging of Hodgkin disease(Bone marrow involvement is(2-32%) diagnosis and staging of small cell tumors of childhood; investigations for unexplained luekoerythroblastic blood picture and trephine biopsy can diagnose occult or micro metastasis if any or necrosis of bone marrow when there is infraction of bone which are missed or become difficult to diagnose by Bone Marrow aspiration studies. • The problem of trephine biopsy is that needles( Islam or jamshidi or westermann-jensen) needles are not available in every laboratories even at tertiary care teaching hospitals Pathology department and disposable needles are very costly for patient* it is always necessary to carry out aspiration at same time** Requires tissue processing set up with fixation facility {microwave fixation is better then10% buffered formalin or zinker fixative as often requires immuno stain as style] and decalcification fluid and making paraffin or resin blocks *** Training for interpretation and evaluation of Trephine biopsy is not adequate; Adequacy of length of Marrow tissues often not obtained (25% shrinkage is natural for fixation and at least 5-6 trabecular space is required for interpretation as per authors] and there remains also word of cautions for patients with coagulation disorders, liver failure and in patients with thrombocytopenia{< 1.5 lack/cumm] • Finally Bone Marrow aspiration and Bone Marrow trephine biopsy are complementary to each other as per authors at least if aspirations are not done then bone marrow imprint should be seen before evaluating Trephine biopsy.

Conflict of Interest:

None declared