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Clinical Research & Trials
Dr. Paul Mellor, DECVIM

Update: We are recruiting formalin-fixed paraffin wax embedded tumour blocks NOW for the forthcoming studies:

1) "Mucosal-associated extramedullary plasmacytoma of the upper aero-digestive tract in cats"

Background: In one study of human patients, 82.2% of all EMP were located in the upper aero-digestive tract (Alexiou et al 1999). At present, there is virtually no published information on tumours in this location in the cat. We hope to describe a feline case series in a hypothesis-driven study, in order to enable general veterinarians, veterinary dental specialists, veterinary ENT specialists, veterinary oncologists and pathologists to make more informed decisions about these uncommon tumours. Collaboration is essential and currently the group involves the University of California-Davis, the Animal Medical Center (New York), Abbey Veterinary Services (UK), IDEXX laboratories (UK), Dacross Services (UK), the Royal Veterinary College (UK), the University of Cambridge and the University of Edinburgh.

Our request: Do you have formalin-fixed paraffin wax embedded samples from cat(s) with oral / pharangeal / nasal / laryngeal masses and histopathological confirmation or strong suspicion of any of the following: plasmacytomas / myeloma / plasma cell granulomas / plasma cell pseudo-tumour ? Contributions from pathology laboratories are very welcome and will be formally acknowledged. CLICK HERE TO EMAIL YOUR INFORMATION.

2) "Feline MRD: differentiating extramedullary MRD from intra- or combined intra- and extra- medullary MRD on the basis of immunophenotyping"

Background: We have previously shown that cats with MRD commonly present with marked extramedullary involvement (67% of cats versus 5% of humans with MRD, P <0.001) (Mellor et al 2006) and that morphological categorization of these tumours is consistent with an extramedullary origin in the majority of feline MRD (Mellor et al 2008). In this next study we will investigate further immunophenotypic markers that may help with sub-typing of these tumours. Improving the understanding of the possible tissue origins of these tumours and sub-typing may lead to better rationales for treatment and prognostication.

Our request: Do you have formalin-fixed paraffin wax embedded samples from cat(s) with histopathological confirmation / strong suspicion of any of the following: plasmacytomas / myeloma / plasma cell granulomas / plasma cell pseudo-tumour ? Cases that have undergone full post-mortem and have a well defined tissue distribution of myeloma cells are particularly welcome. CLICK HERE TO EMAIL YOUR INFORMATION.

Future Studies:
Comparative treatment trial - a design for a treatment trial is being outlined now. Please watch this space.

Acknowledgement & Co-authorship:
We will gratefully acknowledge your / your institution's contribution - both on the website and in any publications if so desired. Our suggestion is that persons contributing 10% or more of cases to a series along with intellectual input to the paper, would be welcomed as co-authors. Please feel free to discuss this further. In particular residents at academic institutions who would be willing to help gather cases and contribute to papers / new ideas and who are looking to build their authorship profile, should get in touch.

Previously Published Research:
"Myeloma-related disorders in cats commonly present as extramedullary neoplasms in contrast to myeloma in human patients: 24 cases with clinical follow-up".

Authors: Mellor PJ, Haugland S, Murphy S, Smith KC, Holloway A, Archer J, Powell RM, Polton GA, Tasker S, McCormick D, Tempest ME, McNeil PE, Scase TJ, Knott CD, Bonfanti U, Villiers EJ, Argyle DJ, Herrtage ME, Day MJ.

Publication: Journal of Veterinary Internal Medicine 2006 Nov-Dec; 20(6):1376-83.

Background: Myeloma-related disorders (MRD) are rare neoplasms of plasma cells. Published case reports describe a diversity of clinical presentations with confusing terminology and diagnostic criteria as a consequence of the assumption that MRD in cats are analogous to those in dogs or humans. There are no reports describing the clinical features of this disease in a large case series of histopathologically and immunohistochemically confirmed feline MRD.

Objectives: The aim of the study was to describe clinical, clinicopathologic and imaging findings, response to treatment, survival and possible associations with other diseases or vaccination in the largest cytological, histopathological and immunohistochemically confirmed case series thus far of this rare disease. A priori hypotheses were that cats with MRD commonly present with extramedullary involvement and uncommonly have radiographic bone lesions, in contrast to human patients.

Methods: Multicenter retrospective study. Cases were identified by database search or by direct contact with veterinarians, at all major public and private UK referral centers and laboratories (see acknowledgements). A spectrum of disciplines (oncology, pathology, clinical pathology, internal medicine, ophthalmology, soft tissue surgery, orthopedics, dermatology and neurology) was included in an attempt to avoid selection bias. Search terms were: plasma cell neoplasia, myeloma, plasmacytoma, macroglobulinemia, hyperglobulinemia, gammopathy and paraproteinemia. Cases were derived from the period 1990 to 2004. Permission for use of the clinical records, clinicopathologic data, radiographs, cytologic and histopathologic material was obtained from referral and primary veterinarians and their clients. The inclusion criteria were:

a.  a report of cytologic or histopathologic confirmation or both, of a tumor with plasma cell or plasma cell-like morphology or alternatively lymphoid morphology as long as the tumor was accompanied by hyperglobulinemia (> 5.2 g/dL) attributable to a paraproteinemia (as evidenced by serum protein electrophoresis),

b.  confirmation of the reported morphologic diagnosis by review of the original cytologic slides, histopathologic sections or both. All histopathologic specimens were evaluated using anti-immunoglobulin antibody immunohistochemistry against alpha, gamma and mu heavy chain as well as kappa and lamda light chain isotypes.

c.  complete clinical records, and

d.  complete follow-up information from either the clinical records or directly from the client.

Reports for 56 cases were obtained, of which 24 cases met the inclusion criteria. Serum (SPE) and urine (UPE) protein electrophoresis was conducted in 19 and 16 cases, respectively. Original SPE and UPE traces were retrieved in 14 and 6 cases, respectively and reviewed by a single clinical pathologist (JA). Serum paraprotein immunoglobulin type was assessed by agar gel immunodiffusion assay by a single pathologist (MJD). Thirteen cats were radiographed. All views were retrieved and reviewed by a single radiologist (AH). A complete past medical history was obtained for all 24 cats from the owner or the primary veterinarian(s) and referral veterinarian. Records were examined for evidence of (1) feline immunodeficiency virus (FIV), feline leukemia virus (FeLV) and feline infectious peritonitis virus (FIP) status, (2) presence of historical and concurrent diseases associated with chronic antigenic stimulation (e.g. severe gingivitis, cholangiohepatitis), (3) vaccination history and (4) non-plasma cell neoplasms.

Statistical Analysis: The differences in proportions of MRD cases between cats and that reported in humans with regard to (1) extramedullary involvement and (2) radiographically detectable bone lesions at initial presentation, were examined using the 2-sided z-test for differences in proportions. Significance was set at P<0.05.

Results: Two types of clinical presentation were observed. The first group (n = 17) had neoplasia involving abdominal organs, bone marrow, or both. All developed systemic clinical signs and paraproteinemia. Plasma cell neoplasia of an abdominal organ, primarily the liver or spleen accounted for 50% of cats with MRD at initial presentation and increased to 62.5% at necropsy. There was good correlation between an abdominal organ abnormality seen at imaging (e.g. radiography, ultrasonography) and demonstration of tissue infiltration by myeloma cells. The second group comprised 7 cats with skin masses, only 2 of which were paraproteinemic and developed rapidly worsening systemic signs. Lameness was suspected in 1 cat only, attributed to a subcutaneous mass on a limb. None of the cats presenting with lameness or pain had radiographic evidence of bony lesions. Of the first group, five of 7 cats that received chemotherapy improved clinically or had decreased serum globulin concentration (median survival, 12.3 months; range, 8.5-22 months). In the second group, in cats without systemic signs excision of the skin masses appeared to be associated with prolonged survival (up to 2.4 years). Cats with MRD commonly presented with extramedullary involvement (67%), versus humans with MRD (5%) (P < 0.001), and uncommonly presented with radiographic bone lesions (8%) versus humans with MRD (80%) (P < 0.001). In murine models, chronic antigenic stimulation has been shown to drive or enhance the chromosomal abnormalities that are necessary for the neoplastic transformation of plasma cells. In this series, examination of the complete past medical records for 24 cats did not identify any significant inflammatory or antigenic stimulatory factor (chronic diseases including FIV, FeLV or FIP, vaccination history (type or time of administration) or previous / intercurrent non-myeloma neoplasms).

Conclusions: Our hypotheses that cats with MRD at initial presentation (1) commonly display extramedullary involvement and (2) uncommonly show radiographically detectable bone lesions in comparison to human patients with MRD have been demonstrated. MRD in cats has a wide spectrum of dissemination and frequently displays aggressive neoplastic behavior. Additional careful clinical investigations are warranted. Although some skin masses truly may be CEMP and respond favorably to complete excision, there should be an awareness of the potential for underlying multi-organ involvement. In the presence of paraproteinemia, bone marrow aspirates and core biopsy samples should be examined. Abdominal organ involvement, especially of the spleen and liver, is common. In our series, there was good correlation between an abdominal organ abnormality seen at imaging (e.g. radiography, ultrasonography) and tissue infiltration. Cats with abdominal involvement or bone marrow infiltration commonly displayed systemic signs of illness, but the majority responded well to chemotherapy and the median survival time of responders was 12.3 months.

Limitations to the study & further notes: incomplete data sets due to retrospective nature of the study. Despite national recruitment resulting in this being the largest case series yet reported on feline MRD there are still relatively few cases. The methodology for the comparison of two different populations (cats versus humans) has limitations. We used the difference in proportions Z-test: If two samples are drawn from different populations, then the Z-test statistic is used in hypothesis tests to compare two proportions with independent random samples. The Z-statistic is the difference between the sample proportions divided by the standard error of the difference between the sample proportions. The denominator of this Z-statistic estimates the standard error of the difference between the sample proportions under the assumption that the two population proportions are equal, i.e., under the assumption that H0 is true. A one proportion Z-test can also be used against a hypothesized proportion where the population numbers are unknown.

Where P1 and P2 are proportions in the two populations and Q1 = 1 - P1, Q2 = 1 - P2 then


 

Under the hypothesis that the proportions in two populations are equal.
i.e. P1 = P2 = P and Q1 = Q2 = Q then


 

Following a systematic review of all published feline MRD cases thus far it seemed that extramedullary involvement was a very common presenting feature in cats and that radiographic bone lesions were uncommon. In humans, extramedullary involvement can be found in two-thirds of patients with multiple myeloma at autopsy (Kapadia 1980), but only rarely (< 5%) is extramedullary involvement the presenting manifestation of an MRD (Kapadia 1980, Soutar et al 2004). Radiographically detectable bone lesions are observed in 80% of all human patients with an MRD (International-Myeloma-Working-Group 2003, Kyle et al 2003). Uptil now, there has been an assumption that "myeloma" in cats is the same as "myeloma" in man (or dogs for that matter). The human diagnostic criteria for multiple myeloma have been applied to the cat, and there are confused descriptions / terminology in the feline literature. From the outset, our intention was to highlight that MRD seemed to be different in cats compared to MRD in man. The question was whether to do describe this in the form of a simple statement, or whether to do this and back this statement up with a simple statistical description. We decided that a hypothesis approach was preferable and possible. We were aware that there were a number of weaknesses in applying a statistical test to this data (cats v. humans, with differences in diagnostic standards, differences in geographical settings, differences in time period of case selection etc). However, the Z-test can be used to assess the differences in proportions between two independently sampled populations - so long as there is an awareness of the limitations of the test and of the populations sampled. We believe that there is value in attempting to describe the differences between cats and humans in this way (so long as the limitations are also noted) - as it seems desirable to counter the long held assumption that myeloma in cats is the same as myeloma in man.

New report: MARCH 2008:
"Histopathological, immunohistochemical and cytological analysis of feline myeloma-related disorders: further evidence for primary extramedullary development in the cat"

Authors: P.J. Mellor, S. Haugland, K.C. Smith, R.M. Powell, J. Archer, T.J. Scase, E.J. Villiers, P.E. McNeil, C. Nixon, C. Knott, D. Fournier, S. Murphy, G.A. Polton, C. Belford, A.W. Philbey, D.J. Argyle, M.E. Herrtage, M.J. Day

Publication: Veterinary Pathology 45 (2), 159-173.

Background: The multistep transformation model of myeloma in humans hypothesizes that plasma cells undergo neoplastic transformation primarily within the intramedullary compartment, and that over time they become poorly-differentiated and metastasize to extramedullary locations. Historically, diagnostic criteria used for human multiple myeloma have been applied to the cat, with the assumption that feline MRD commonly arises in the intramedullary compartment.

Objectives: To describe the cytological, histopathological, histochemical and immunohistochemical features of feline MRD and to categorize these tumours. A priori hypotheses were (1) tumour category predicts survival and (2) cats with well-differentiated tumours commonly have extramedullary involvement in contrast to human myeloma patients.

Methods: Multicenter retrospective study. For case recruitment details see Mellor et al JVIM 2006. Samples comprised ante mortem cytological and biopsy specimens as well as post mortem material. Confirmation of the original report of a MRD was achieved by review of:

1.  Cytology: Preparations were optimally stained with May-Grünwald-Giemsa, although some were received pre-stained with other Romanovsky stains (e.g. Rapi-Diff II®, Bios Europe Ltd, West Pimbo, UK).

2.  Histopathology: 4μm thick sections of formalin-fixed, paraffin wax-embedded tissues were stained with haematoxylin and eosin (HE).

3.  Ancillary histochemistry and immunohistochemical analysis: Where the diagnosis of a MRD was in question, we carried out further histochemistry and / or immunohistochemistry prior to inclusion or rejection: toluidine blue, astra blue, lysozyme, fascin, MHC II, Melan A, cytokeratin and BLA.36.

4.  Immunoglobulin heavy and light chain immunohistochemical labeling: 2μm thick sections of formalin-fixed, paraffin wax-embedded tissues were stained for α, γ and μ heavy chains, and λ and κ light chains in the presence of controls. In a subjective assessment of representative sections, case specimens were considered positive if the majority of the neoplastic population exhibited cytoplasmic labeling.

Reports for 56 cases were obtained, of which 26 cases met the inclusion criteria. The clinical features for 24 of these cases have been described elsewhere (Mellor et al JVIM 2006). All cytological specimens were reviewed by a joint-panel of cytologists (JA, EJV, RMP, PJM). A cytological diagnosis in bone marrow aspirates was achieved if representative slides contained >20% myeloma cells as a proportion of the total number of myeloid & lymphoid cells examined. Where there was discordance between cytological and histopathological findings in the same animal, the histopathological & immunohistochemical categorization was accepted on the basis that evaluation of repeat sections from a larger tissue sample was more likely to be representative than aspirated cells.

All histopathological and immunohistochemical specimens were reviewed by a joint-panel of histopathologists (SH, KCS, TJS, PEM, DF) who were blinded to the cytology results. A histopathological diagnosis from bone marrow core biopsies was achieved if representative bone core sections contained >20% myeloma cell infiltration and myeloma cell clustering. Sections displaying single Ig light chain immunolabeling were regarded as consistent with a diagnosis of MRD, whereas sections displaying mixed κ and λ light chain were diagnosed as reactive plasma cell lesions and were excluded from further analysis. Histopathological specimens that did not label with anti-Ig antibodies were still included in the case series on the basis of unequivocal cytological findings and/or histopathological, ancillary histochemical and immunohistochemical findings.

All confirmed MRD histopathological specimens were also examined for expression of CD79a and CD3 in the presence of controls according to standard techniques. Where HE sections revealed material consistent with amyloid deposition, the presence of AL amyloid (also known as Ig light chain associated amyloid) was confirmed by examination of Congo red stained, permanganate oxidised sections under polarized light. Mitotic rate (average of 10 fields at x400 total magnification) was assessed in histopathological sections only.

Cytological and histopathological sections were categorized according to an adaptation of the method described by Wutke et al. 1981. Myeloma cells were classified as mature myeloma cells, proplasmacytes, plasmablasts, giant myeloma cells or lymphoma cells. Immunoglobulin secreting lymphoma (IgSL) could be included as a MRD if accompanied by hyperglobulinaemia (total serum globulin >52g/L) attributable to a myeloma (M)-proteinaemia, as evidenced by serum protein electrophoresis (SPE). All MRD were then categorized according to the proportion of plasmablasts: (1) well-differentiated MRD, (2) intermediate-grade MRD and (3) poorly-differentiated MRD.

Statistical Analysis: In order to test the first hypothesis, the association between tumour morphological category and survival was examined by both (1) log rank analysis of Kaplan-Meier survival curves, followed by Holm-Bonferroni corrections for multiple comparisons (Ludbrook 1998) (significance was set at P<0.05) and (2) Cox proportional hazards analysis. Survival time was defined from the time of a cytological or histopathological diagnosis until death. Animals were censored if they were lost to follow-up, or they died or were euthanased due to causes unrelated to tumour development.

In the same model, Cox proportional hazards analysis was used to assess the association between other independent variables and survival (censored and all-cause mortality). In view of the small sample size, these were selected, dichotomized variables as follows: (1) tumour location (visceral and/or bone marrow versus cutaneous or subcutaneous mass), (2) CD79a expression (versus absence), (3) presence of mitoses (1-10 per field at x400 total magnification, versus absence), (4) treatment category (chemotherapy or surgical excision versus no treatment or glucocorticoids alone), and (5) gender (male and male-neutered versus female and female-neutered). Forward variable selection with P <0.10 was used, as appropriate for a study with small sample sizes (Katz 2001). Proportionality assumptions for each variable were assessed by Kaplan-Meier survival curves. Relative risks (RR) (along with confidence intervals) were reported for all independent variables where the 95% confidence interval (CI) of the RR did not include 1. In order to test the second hypothesis, the 2-sided z-test for the difference in proportions was used to examine the relative proportion of cats with well-differentiated tumours and extramedullary involvement versus the proportion of humans reported to have well-differentiated tumours and extramedullary involvement. Significance was set at P<0.05.

Results: There was good agreement between histopathological and cytological tumour categorization. Histochemistry and immunohistochemistry were shown to be valuable adjunctive tests in the diagnosis of MRD. The majority of cats in this series had well-differentiated tumours. Immunohistochemically confirmed giant myeloma cells (a rare but well described morphological feature in man) were observed and this is the first report documenting this pathological feature in cats. The gamma heavy chain and the lambda light chain were the most commonly expressed immunoglobulin components. Only 32% of feline MRD specimens expressed CD79a in this series. Cats with well-differentiated tumours had increased median survival relative to those with poorly-differentiated tumours (254 versus 14 days). There were also significant differences between those receiving no treatment / glucocorticoids alone and (1) chemotherapy treated animals and (2) surgical excision cases. There was no significant difference in survival between chemotherapy treated animals and surgical excision cases. Cats with well-differentiated tumours more commonly have extramedullary involvement than human myeloma patients with well-differentiated tumours (90% versus 20%, P<0.0002).

Discussion: The histopathological, histochemical and immunohistochemical joint-panel review of original specimens in multiple tissues, presence of Ig expression and absence of CD3 expression, provided a high degree of confidence in the final diagnosis of an MRD in our series. Similarly where cytological slides only were available, the joint-panel review of these specimens, together with the presence of a M-proteinaemia in those cases examined by SPE, provided a high degree of confidence in the final diagnosis of an MRD.

In man, the full morphological and functional transition of a B-lymphocyte to a normal, terminally-differentiated long lived plasma cell occurs after exiting lymph node and splenic germinal centres and upon maturation in bone marrow (Hallek et al 1998). Genetic and phenotypic studies have revealed that myeloma cells originate from these antigen-selected, post-germinal plasma cells, and that neoplastic transformation occurs in the intramedullary compartment (Takishita et al 1994, Hallek et al 1998). A multistep transformation model is hypothesized for the development of myeloma in man (Hallek et al 1998, Sirohi and Powles 2004). Myeloma cells develop karyotypic instability and it appears that translocations in IgH loci are an early and possibly universal event (Hallek et al 1998). The initial clinical manifestation of this transformation is monoclonal gammopathy of unknown significance (MGUS) (Zandecki et al 1995, Hallek et al 1998). Over time, myeloma cells accumulate further translocations, gene deletions and activating mutations, and these have been correlated with the onset of clinical disease development (typically MGUS to intramedullary myeloma then extramedullary metastases) and a worsening prognosis (Hallek et al 1998, Seidl et al 2003, Sirohi and Powles 2004). Furthermore, studies have clearly established correlations between accumulated cytogenetic aberrations and progressively poorly-differentiated myeloma cells (Weh et al 1995, Garand et al 2003).

Historically there has been an assumption that feline MRD is similar to human myeloma with intramedullary involvement being a key diagnostic feature (MacEwen and Hurvitz 1977, Drazner 1982, King et al 2002, Hanna 2005, Patel et al 2005). However, we have previously reported that marked extramedullary involvement at initial clinical presentation is significantly more common in the cat, in comparison to human MRD patients. In this study, we demonstrate that cats with well-differentiated tumours more commonly have extramedullary involvement than human myeloma patients with well-differentiated tumours (90% versus 20%, P<0.0002). These results contrast strongly with the human myeloma model of primary intramedullary neoplastic transformation. This is further evidence for a novel hypothesis: primary extramedullary neoplastic transformation may be more common in feline MRD.

Limitations to the study & further notes: incomplete data sets due to retrospective nature of the study. Despite national recruitment resulting in this being the largest case series yet reported on feline MRD there are still relatively few cases. The methodology for the comparison of two different populations (cats versus humans) has limitations - see notes above (Mellor et al 2006) for details on the difference in proportions Z-test. Further discussion of statistical limitations is discussed within the paper.

"Solitary plasmacytoma of bone in two successfully treated cats"

Authors: PJ Mellor, GA Polton, M Brearley, S Haugland, KC Smith, TJ Scase, PE McNeil, A Holloway, J Archer, RM Powell, EJ Villiers, ME Herrtage, DJ Argyle, MJ Day

Publication: Journal of Feline Medicine and Surgery 2007, 9, 72-77

Abstract: This is the first report of feline solitary plasmacytoma of bone. We describe the clinical, clinico-pathological, radiographic and pathological findings of two successfully treated cats with long term follow-up. The first case presented with spinal pain and neurological deficits. Radiographs demonstrated sclerosis of lumbar vertebra L6 and a myelogram confirmed interference to flow of contrast in the L4-7 region. A biopsy of L6 revealed neoplastic plasma cell infiltration. There was no evidence of paraproteinaemia on serum protein electrophoresis. The cat underwent hypofractionated megavoltage radiotherapy. Clinical signs resolved completely and four years after diagnosis the cat remains well and has no electrophoretically-detectable paraproteinaemia. The second case presented with neurological deficits of the tail and spinal radiographs revealed extensive osteolysis of the sacrum. A biopsy of sacral bone demonstrated neoplastic plasma cell infiltration. The animal was normoglobulinaemic. The cat improved clinically with induction chemotherapy (melphalan and methylprednisolone). The same chemotherapeutics were continued at maintenance doses for 4.3 years, at which time there was recurrence of neurological deficits and a palpable sacral mass. Cytological examination of a fine needle aspirate confirmed recurrence of plasma cell neoplasia. A low concentration monoclonal paraproteinaemia was detected. Vincristine was administered resulting in resolution of neurological deficits and a palpably smaller sacral mass. Eighteen months into vincristine therapy, there was recurrence of clinical signs and the cat was euthanased (> six years after the initial diagnosis).

Table of Contents
The Veterinary Myeloma Website
Dr. Paul Mellor, DECVIM
Introduction
What are myeloma related disorders
Clinical signs
Diagnosis
Imaging Findings
Clinico-pathological Findings
Anatomical Pathology
Cytology, Histopathology
Other Images
Immunohistochemistry
Mis-diagnosis of MRD
Treatment and Survival
Clinical Research & Trials
MRD References
History, Acknowledgements, Links
Bio

Date Published: September 25, 2007 Paul Mellor


Address (URL): https://www.vin.com/Projects/Myeloma/M08157.htm

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