Injection Site Sarcomas: New Data, New Understandings

Gary D. Norsworthy, DVM, DABVP (Feline) Alamo Feline Health Center San Antonio, Texas Injection site sarcomas (ISS) were first reported in the veterinary literature in 1991(Hendrick, 1991). My first personal experience occurred shortly thereafter. Our practice had been using a rabies vaccine for cats that was labeled for intramuscular (IM) use. I much preferred giving injections to cats using the subcutaneously (SC) route so I was off label when I gave it SC. When a rabies vaccine became available that was approved for SC use, we changed to it. Within six months we had three cats with fibrosarcomas at vaccine sites. Like most of our colleagues at the time we were stunned and shocked. We sought the advice of the experts as we tried to understand what was happening. During that era these very aggressive tumors were termed vaccine associated sarcomas or VAS. Largely based on a temporal relationship, they were assumed to be due to adjuvanted vaccines. This association continues even today as evidenced by reports that come to me from my externs. These fourth year students from over 10 American veterinary schools and from several foreign schools uniformly state that they are taught that sarcomas at injection sites are due to the adjuvant in vaccines. This is confusing to me because the papers of the past 10 years no longer make this connection. TEMPORAL RELATIONSHIPS A temporal relationship is one based on connecting two events, the first of which is supposed to be causative of the second. The report of 1991 (Hendrick, 1991) found aluminum in many of these tumors and assumed that it was causative, primarily based on a temporal relationship with the vaccines. Three events in the 1980s caused a spike in the use of adjuvanted vaccines. 1) In 1985, the USDA changed vaccine regulations so that it was no longer economically feasible to produce modified live rabies vaccine. Consequently, the only available ones were killed virus vaccines that contained adjuvant. 2) Norden Laboratories released the first feline leukemia virus (FeLV) vaccine. Because FeLV infections were nearly epidemic, this vaccine was widely embraced and recommended for all cats. It was a killed adjuvanted product. 3) During the time span of 1985-89, for the first time several states mandated that cats be vaccinated for rabies. By default, adjuvanted rabies vaccines were used. The second and third of these events resulted in a surge of cats being vaccinated, many for the first time in their lives. Authorities at the time stated that there was compelling evidence that adjuvant caused these tumors; therefore, the term VAS seemed very appropriate. In retrospect, it is surprising that they were not called adjuvanted vaccine associated sarcomas. Temporal relationships can be very meaningful, but they can also be very misleading. For example, immune-mediated hemolytic anemia (IMHA) of dogs was declared to be vaccine-associated (Duval, 1995). However, several others (Burgess 2000; Carr, 2002; Elliott, 1997; Jackson, 1985; Klag, 1993; Reimer, 1999) came to the conclusion that there was no relationship between vaccination and IMHA. This compels us to look for another interpretation between the three events stated above and the onset of sarcomas. It is possible that the surge of vaccinations in cats in the late 1980s, possibly as much as a 5-10 fold increase, meant that the number of sarcoma-susceptible cats had significantly more exposure to vaccines and other injections, and the presence of adjuvant may have been incidental. CHRONIC INFLAMMATION Chronic inflammation has been associated with certain types of tumors in humans and other species (cigarette smoking, chewing tobacco, asbestos, etc.) Regarding feline sarcomas, a recent publication stated: After an initial correlation with rabies and feline leukaemia virus vaccination, subsequent studies have demonstrated that an abnormal reaction of feline tissues to chronic inflammation was mainly

responsible for the disease (Martano, 2011). The paper went on to state that these tumors have been associated with long acting antibiotics or steroids, the benzoylurea pesticide lufenuron, non-absorbable suture material, and microchip implants. To that list could be added meloxicam (Munday, 2011). Based on these observations, another author concluded: Although only vaccines were initially implicated in sarcoma development, investigators have elucidated that any injected compound causing local inflammation may lead to oncogenesis (Romanelli, 2008). Interestingly, a peer-reviewed study has never been published establishing a link between inflammation and sarcomas. In fact, we have been warned that such an association is nothing more than a hypothesis. In 2005 the Vaccine Sarcoma Task Forces final report to the profession (Richards, 2005) stated inflammation secondary to injection is a necessary precedent to sarcoma formation is a hypothesis. It went on to state But, it is a big leap of faith to say that it is solely the inflammatory reaction that induces a sarcoma. That leap has not been filled in with any concrete structure. It takes the right kind of triggering event in the right individual cat to lead to neoplastic transformation. The 2006 vaccine recommendations of the American Association of Feline Practitioners (Richards, 2006) state: A direct association between the presence or severity of postvaccinal inflammation and tumor risk has not been specifically established, but after taking all currently available evidence into consideration, the Advisory Panel suggests that veterinarians use less inflammatory vaccines whenever possible; nonetheless, the subsequent impact of this practice on sarcoma risk is currently not known. (Italics by GDN) The most extensive published study on vaccines and inflammation (Day, 2007) stated: Despite extensive investigation of the pathogenesis of these lesions [sarcomas], there is no definitive information that explains their occurrence. The current working hypothesis suggests that a chronic inflammatory reaction at the site of injection may provide the trigger for subsequent malignant transformation. It has been suggested that the injection of adjuvanted vaccines may be particularly linked to the development of sarcoma due to the more intense local inflammation associated with such products. (Italics by GDN) Clearly the authors were aware that it is only a hypothesis that links inflammation and sarcoma formation. This study (Day, 2007) also showed that cats given a non-adjuvanted vaccine had similar post vaccination inflammation as compared to one adjuvanted vaccine and less inflammation than another adjuvanted vaccine. However, some inflammation is not surprising because that is how adjuvant works. In order for the body to respond and make antibodies to a killed virus or killed bacterium something (adjuvant) has to get the immune systems attention. The resulting acute inflammation is the vehicle for protective antibody formation. This study showed that two different adjuvants (alum-based and lipid-based) had significantly different local inflammatory responses. Vaccine adjuvants are proprietary products with secret formulas. Vaccine companies develop adjuvants to cause maximum antibody response with minimal inflammation. The alum-based adjuvant caused the greatest inflammation; however, there are no veterinary vaccines made in the U.S. with an alum-based adjuvant. One must also appreciate that there are at least 35 products that have been used as adjuvant in U.S. vaccines. They include: aluminum phosphate, aluminum hydroxide, a surfactant, a polyanion, a polycation, a detergent, a peptide, a metabolizable animal or vegetable oil, a mineral oil, a mineral oil emulsion, an immunomodulator, a polyoxyethylene-polyoxypropylene block copolymer, a copolymer of styrene with a mixture of acrylic acid and methacrylic acid, a Corynebacterium-derived adjuvant, a Propionibacterium-derived adjuvant, Mycobacterium bovis (Bacille Calmette-Guerin), an interleukin, an interferon, a liposome, an iscom adjuvant, a mycobacterial cell wall extract, a synthetic glycopeptide, pyridine, Lipid A, dextran sulfate, DEAE-Dextran, carboxypolymethylene, ethylene/maleic anhydride copolymer, an acrylic copolymer emulsion, an animal poxvirus protein, a subviral particle adjuvant, cholera toxin, dimethyldioctadecylammonium bromide. Thus, it is neither fair nor scientifically accurate to state that adjuvant produces any specific response. One must not use the blanket term adjuvant as if it is a specific entity.

The cited study (Day, 2007) concluded: The results of this investigation may also have implications for the possible development of sarcomas at the site of injection. The pathogenesis of such tumours is not clear. In this study, the late time point of 62 days was selected partially in order to detect any neoplastic or pre-neoplastic changes that might be induced post-vaccination. No such changes were observed (Italics by GDN) The two cited references (Richards, 2005; Day, 2007) used the term hypothesis to describe our level of understanding of these concepts. A hypothesis is an assumption used in an argument without its being endorsed; a supposition; an unproved theory; a conjecture (World, 2012) and the lowest level of proof in the scientific method (hypothesis theory fact). We must be very careful that we do not upgrade a hypothesis to a theory or a fact; based on my contact with veterinary students, some veterinary professors are guilty of doing this. The outcome of local, acute inflammation was examined by a study (Moore, 2007) that identified 428 cats returned following vaccination; each had a palpable injection-site swelling. These cats were followed for one to two years with the expectation that some or most would develop sarcomas. However, no sarcomas occurred in these cats in this time frame. There have been many comparisons of chronic inflammation caused by vaccine adjuvant to chronic inflammation that causes diseases such as lung cancer in humans. However, an injection of vaccine adjuvant every one to three years hardly qualifies as a chronic irritant. Therefore, we must not leave the subject of inflammation without noting that it is a hypothesis that the acute local inflammation that occurs at some vaccine sites progresses to chronic inflammation; no such progression has been documented. Our understanding of post-vaccinal local inflammation in relation to sarcoma formation is minimal. The study previously cited (Moore, 2007), seriously questions if there is any link between the two. GENETICS There is a wide variation in response when humans take any given drug. For example, one dose of diphenhydramine can cause several hours of drowsiness in some individuals. In others, there is no drowsiness and no antihistamine effect. This variation was documented in an interesting article (Medco, 2008) that placed individuals in one of four recognized categories: poor metabolizer (7-10%), intermediate metabolizer (10-15%), extensive metabolizer (73-82%), and ultrarapid metabolizer (1-2%). Those in the first category have exaggerated drug effects and many side-effects, so they need to be given lower than normal doses. Drugs given to those in the last category do not reach therapeutic effect and are ineffectual at standard doses. The paper states: Genetic variations are one of the primary factors accounting for the wide range in drug response. Many investigators have examined the role of genetics in ISS. The Vaccine Sarcoma Task Force alluded to genetics when it said that sarcomas occur in the right individual cat (Richards, 2005). It further validated the issue by noting sarcomas occurring in littermates (Dr. Dennis Macy: In my personal experience, I know of a situation in which cats from the same litter developed sarcomas at injection sites, even though they went to separate veterinarians and received various substances.) and differences in incidence in different communities (Dr. Glickman: A difference in the incidence of VAS in different communities may suggest a genetic predisposition.). Furthermore, papers have noted an allele in cats predisposed to developing VAS (Banerji, 2006) and sarcomas occurring months apart in different locations in the same cat (De Man 2007). (I have treated two cats in the latter category.) Another paper (Banerji, 2007) listed factors that suggest genetic predisposition as 1) occurrence at sites of injury, 2) occurrence at sites of other drugs or vitamins, 3) occurrence at suture sites and non-healing wounds, 4) occurrence in multiple related members, and 5) multiple tumors in the same cat.

The authors of the latter study (Banerji, 2007) were looking for a genetic marker for ISS-predisposed cats. They conclude: The results of this study indicate that analysis of the presence or absence of the identified genetic markers in apparently healthy disease-free cats may be helpful in predicting which individual animals are at a greater risk of developing the disease. THE ROLE OF ADJUVANT IN VACCINES Although demonized in the veterinary literature of the past 20 years, vaccine adjuvant is widely used in human and animal vaccines because it has very positive attributes. It has been shown to significantly increase efficacy of killed vaccines. In one study (Fischer, 2007), cats receiving one dose of a killed vaccine received a considerably higher level of immunity against the feline herpesvirus than those given one dose of a modified live virus vaccine. It is also used in human vaccines to stretch a limited antigen supply (Brownstein, 2009). This occurs when a new pathogen with epidemic or pandemic potential creates the need for rapid production of a needed vaccine. One must appreciate the diverse products that have been used as vaccine adjuvant. Some are more inflammatory than others (Day, 2007). Therefore, vaccine manufacturers investigate multiple adjuvants before selecting the one to be used in one of their vaccines. INCIDENCE OF ISS In the mid-1990s Dr. Dennis Macy estimated that 22,000 cats in the U.S. would develop an injection site sarcoma (Smith, 1995). In light of my experience (three in six months), that shocking number seemed believable. In 2002, a study (Gobar, 2002) involving over 30,000 cats concluded that the incidence was 0.63 per 10,000 vaccinated cats. It was estimated that this would equal about 2000 sarcomas per year in the U.S. (Kirpensteijn, 2006). To put it in perspective, this incidence is about the same as the occurrence of mammary neoplasia in male dogs (Saba, 2007). Using Kirpensteijns formula, 22,000 cases per year would equal about 11 per 10,000 cats. If these two estimates are accurate, it appears that there was a dramatic (nearly twenty-fold) decrease in the incidence of ISS between 1995 and 2002. The 1995 estimate fanned the flames of panic that contributed heavily to changes in vaccine protocols. Unfortunately, it also resulted in cat owners becoming fearful of vaccines and refusing to have their cats vaccinated, including rabies vaccine. Those same owner attitudes persist today. It is not uncommon for one of my clients to refuse rabies vaccine for his or her cat. THE TIE BREAKER The etiology of ISS is a very personal issue for me and other practitioners who treat and vaccinate cats. Recommendations to clients and clients responses to those recommendations often hinge on this issue. It is confusing to hear what veterinary students are taught (adjuvanted vaccines) and to read the literature of the past decade (genetics). Simply put, the two do not agree. When there is a disagreement of this nature, it is best to get data from an unbiased third party. I have done so with my patients. I researched the number of each type of vaccine that was used and compared it to the occurrence of ISS in my practice over an 11 year period. Here are the findings:

Alamo Feline Health Center San Antonio, Texas March 6, 2000 July 12, 2011 Doses of non-adjuvanted vaccines administered Rabies (recombinant) FVRCP (MLV) Herpes-Calici (MLV) Total 20,739* 10,376** 1,464** 32,579*

(53%)

Doses of adjuvanted vaccines administered FVRCP FeLV FIV FeLV/FIV VS-Calici Total Combined Total *Injection Site Sarcomas: 1 13,905** 6,588** 4,410** 1,731** 2,139** 28,773**

(47%)

61,352 Doses **Injection Site Sarcomas: 0

These data create some interesting observations. 1) The incidence of ISS is MUCH lower than that experienced by me and others in the early 1990s. 2) The ISS that occurred in cats vaccinated in my practice was at the site of a non-adjuvanted rabies vaccine. 3) Of the 61,000+ doses of vaccine given in my practice, nearly half were adjuvanted. WHY OLDER CATS? Injection site sarcomas typically occur in mature to older cats instead of young cats. Since this is a disease of genetic predisposition, why is it not just the opposite? A recent article (Kong, 2012) in the world renowned scientific journal Nature gives us an insight into this phenomenon. It points out that genetic mutation is an ongoing process, and increasing age increases the number of mutations within an individual. In light of this, it is logical that mature and older cats are more likely to have mutations that lead to predisposition to ISS occurrence. NOMENCLATURE There is a move in veterinary pathology to abandon inappropriate terms. The term vaccine associated sarcoma is one that should be abandoned. The term injection site sarcoma is an appropriate term. Another approach that I find appealing is to use the terms induced sarcoma and spontaneous sarcoma. SUMMARY The literature of the past 10 years and my experience over the past 11 years lead me to these conclusions: 1. The incidence of ISS has changed over the past 20 years. It is now a rare disease and should not be used to scare cat owners regarding vaccination. We have done that to the point many cats are no longer vaccinated, even against rabies. We also have provided a springboard for emotion-driven owners of ISS

cats to become aggressive in seeking retribution for a perceived viscous act on the part of the veterinarian or the vaccine manufacturer. We need to reverse these trends using a re-education process. 2. The underlying cause of ISS is a genetic predisposition in a few rare cats. Obviously, the injection of some material is required, but without genetic predisposition, it is virtually impossible for an ISS to form. 3. The incidence of ISS is no different in cats receiving adjuvanted vaccines and non-adjuvanted vaccines. Therefore, one should not base vaccine selection on the presence or absence of adjuvant. Choose your vaccines based on the desired antigens and the desired vaccine characteristics.

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