Lactate dehydrogenase is a tetrameric enzyme that converts lactate to pyruvate. It is composed of 2 immunologically distinct subunits, the combination of which results in 5 isoenzymes.1 Lactate dehydrogenase is synthesized in several cell systems, including skeletal muscle, heart, kidney, small intestine, liver, lung, pancreas, bone, and hematopoietic cells,1,2 and its activity may increase in many pathological conditions, such as myocardial or pulmonary infarction, hepatic disorders, hemolytic disease, myopathy, and malignancy.1,2 In humans, serum LDH activity can increase with a variety of cancers, including non-Hodgkin lymphoma,3,4 acute lymphoblastic leukemia,5 chronic granulocytic leukemia,6 multiple myeloma,7 and nonseminoma germ cell tumors.8 Increased LDH activity can indicate a shift toward increased glycolysis in malignant cells that is necessary to sustain accelerated tumor growth.9
Of interest, LDH activity represents a strong and validated factor that can be used as a predictor of survival in humans with non-Hodgkin lymphoma, and it is one of the factors listed in the International Prognostic Index.10 High LDH activity at the time of lymphoma diagnosis reflects elevated tumor bulk and correlates with a poor prognosis, irrespective of the histologic subtype.3,11 Although LDH comprises multiple isoenzymes, measurement of total LDH activity is preferred for use in making clinical decisions.12 Indeed, although total LDH activity generally reflects tissue damage, measurement of individual isoenzymes may provide clues as to the specific organ or tissue that has been damaged. Because lymphoma is principally a systemic disease, diffuse organ involvement is to be expected, thereby reducing accuracy of the predictive ability when measuring individual isoenzyme actvities.
Total serum LDH activity ≥ 2 times the upper limit of the reference range at the time of admission consistently is associated with CNS involvement for patients with non-Hodgkin lymphoma, which requires that drugs able to penetrate the blood-brain barrier be incorporated in the treatment plan.13 Additionally, elevated serum LDH activities in patients without clinical signs may precede the onset of the clinical stage for occult lymphoma.14 Consequently, evaluation of LDH activity is considered important in the diagnosis, staging, and management for human patients with non-Hodgkin lymphoma.13–15
Measurement of LDH activity in small animals with lymphoma has gained attention16–19; however, little information is available on the clinical importance of LDH activity in the canine species regarding prognosis or follow-up monitoring.16,18,19 In 1 study,16 high LDH activities at the time of lymphoma diagnosis were associated with a shorter duration of survival, regardless of clinical stage or grade of the malignancy.
Improving the ability to identify subsets of dogs with lymphoma during the initial stages of disease that have a better (or worse) prognosis would greatly contribute to appropriate treatment planning and determination of risk-benefit ratios. Furthermore, early recognition of disease progression in dogs with lymphoma may be helpful in the optimal treatment of these tumors by targeting the newly expanded cluster of neoplastic cells before the disease is clinically apparent. Increased serum concentrations of the inflammatory protein α-1-acid glycoprotein and activities of the cytoplasmic enzyme thymidine kinase can be used to predict clinical recurrence of lymphoma in dogs.20,21 Unfortunately, these compounds are not routinely measured in dogs because the assays are not widely available.
The study reported here was designed to prospectively investigate pretreatment and posttreatment serum activities of LDH to evaluate its prognostic value and its role in predicting early recurrence during follow-up monitoring in dogs with lymphoma. We hypothesized that high LDH activities at the time of initial lymphoma diagnosis would be associated with shorter disease-free and survival intervals and that increased LDH activities after treatment would be associated with early recurrence of lymphoma.
Materials and Methods
Animals—Client-owned dogs with de novo cytologically diagnosed lymphoma admitted to the Clinica Veterinaria L'Arca were prospectively enrolled in the study. None of the dogs had received any treatment (including corticosteroids) prior to admission. Written informed consent was obtained from owners for enrollment of dogs in the study.
Lymphoma in all dogs was staged in accordance with the World Health Organization clinical staging system22 on the basis of results of physical examination, a CBC, and serum biochemical analysis (including total LDH activity); cytologic evaluation of a lymph node; abdominal ultrasonography; cytologic evaluation of the spleen, liver, and bone marrow; thoracic radiography; and immunophenotypic analysis of an aspirate obtained from a lymph node and samples of blood obtained from a peripheral vein and from the bone marrow. Additionally, a biopsy specimen was surgically obtained from the lesion of dogs with cutaneous involvement and used to differentiate between nonepitheliotrophic and epitheliotrophic lymphoma.
For inclusion in the study, each dog had to have a high-grade lymphoma (as determined by use of the updated Kiel classification scheme23), receive the same chemotherapy treatment until first tumor recurrence,24,25 and receive rescue treatments thereafter. For the first-line chemotherapy, dogs were treated in accordance with a 9-week modified VCAA protocol24,25 unless they had bone marrow infiltration, in which case cytosine arabinoside was incorporated, as described elsewhere.25
Response was classified as CR, partial remission, stable disease, or progressive disease. Complete remission was defined as 100% reduction in size of all measurable tumors for at least 21 days. Partial remission was defined as reduction in size of > 50% but < 100% of all measurable tumors for at least 21 days. Stable disease was defined as reduction in size of < 50% or no change in size of all measurable tumors and lack of appearance of new neoplastic lesions for at least 21 days. Progressive disease was defined as an increase in size of > 25% of all measurable tumors or the appearance of new neoplastic lesions. To ascertain tumor response, lymphoma was again staged at the end of the 9-week chemotherapy period. Follow-up monitoring at 1, 3, and 6 months after completion of chemotherapy was conducted. Overall survival interval, disease-free interval, response rate, and frequency of recurrence were recorded.
Measurement of LDH activity—Food was with-held from each dog enrolled in the study for 12 hours overnight. The following morning, a blood sample was collected from a jugular vein. Serum was obtained by centrifugation and immediately processed.
For all dogs, total LDH activity was determined at the time of initial lymphoma diagnosis (ie, prior to treatment), at the completion of chemotherapy administered in accordance with the modified 9-week VCAA protocol,24,25 and at 1, 3, and 6 months after completion of chemotherapy. Total LDH activity was measured in accordance with the manufacturer's instructions by use of a commercial assay.a The reference range for total serum LDH activity at our laboratory was 10 to 280 U/L.
Statistical analysis—Several factors were investigated to determine whether they were associated with overall survival rate and the disease-free interval. These factors included age, sex (male or female), body weight, clinical substage (a or b), immunophenotype (T cell or B cell), bone marrow involvement, hypercalcemia (ionized calcium concentration), hypoalbuminemia, hypercreatininemia, and anemia. The association of these factors was evaluated by use of the Kaplan-Meier product-limit followed by use of a log-rank test. Factors with a value of P < 0.2 for the univariate analysis were included in the Cox proportional hazard model to investigate the effect of LDH activity on overall survival and disease-free intervals. Overall survival interval was defined as the interval between diagnosis and death. Dogs were censored when they were lost to follow-up monitoring, death was not caused by lymphoma, or they were alive at the end of the study. Disease-free interval was defined as the interval between completion of chemotherapy and tumor recurrence. Dogs were censored when CR was not achieved, lymphoma had not recurred before the end of the study, or dogs were lost to follow-up monitoring. For overall survival and disease-free intervals, LDH activity was considered as a continuous variable. Hazard ratios and 95% CIs were calculated.
To assess the value of LDH activity for use in predicting recurrence of lymphoma in dogs without clinically evident disease, the proportion of dogs in CR that had a high LDH activity (> 280 U/L) at the end of chemotherapy was compared between dogs with tumor recurrence or no tumor recurrence during the successive 45 or 90 days. These time points were arbitrarily selected on the basis of post hoc data analysis because recurrence was frequently observed between 45 and 90 days after completion of chemotherapy. Comparison of proportions was performed with a Fisher exact test. Sensitivity and specificity were calculated. The analysis was also performed for dogs in CR at 1, 3, or 6 months after completion of chemotherapy. Values of P < 0.05 were considered significant. All statistical analyses were conducted with a commercially available program.b
Results
Patient and tumor characteristics—Fifty dogs with high-grade lymphoma were enrolled; of these, 26 (52.0%) were male (24 sexually intact and 2 neutered) and 24 (48.0%) were female (13 sexually intact and 11 spayed). There were 22 purebred dogs. The German Shepherd Dog (n = 5), Rottweiler (4), American Staffordshire Terrier (3), and Yorkshire Terrier (3) were the breeds most commonly represented. Age ranged from 2 to 18 years (median, 8 years). Body weight ranged from 2.6 to 52.4 kg (5.72 to 115.28 lb), with a median of 28.6 kg (62.92 lb). Four (8.0%) dogs had stage III lymphoma (3 with substage a and 1 with substage b), 28 (56.0%) had stage IV lymphoma (18 with substage a and 10 with substage b), and 18 (36.0%) had stage V lymphoma (6 with substage a and 12 with substage b), as determined by use of the World Health Organization classification scheme. Of the 18 dogs with stage V lymphoma, 9 had bone marrow involvement. Of the 23 dogs classified in the substage b category, 5 (21.7%) had hypercalcemia (high ionized calcium concentration). Immunophenotype distribution for B cells and T cells was 34 and 16, respectively.
All dogs were treated in accordance with a VCAA-based chemotherapeutic protocol, as described elsewhere.24,25 Of the 50 dogs, 41 (82.0%) achieved CR at the end of the 9-week treatment period. Of those, 29 (70.7%) dogs had recurrence of lymphoma; the interval until recurrence ranged from 8 to 420 days (median, 87 days). In contrast, 12 (29.3%) dogs were still in CR at the end of the study.
Thirty-five of the 50 (70.0%) dogs had died at the end of the study as a result of lymphoma (n = 30 dogs) or causes not obviously attributable to lymphoma or its treatment (5). There were 15 (30.0%) dogs still alive at the end of the study. Overall survival time ranged from 4 to 1,945 days (median, 320 days).
LDH activity at diagnosis and overall survival and disease-free intervals—At the time of initial diagnosis of lymphoma, the LDH activity in the 50 dogs ranged from 86 to 3,325 U/L (median, 416 U/L). Ten (20.0%) dogs had an LDH activity within the reference range, whereas 40 (80.0%) dogs had a high LDH activity. On the basis of results of univariate analysis, confounding factors included to assess the effect of LDH activity on survival were clinical substage, immunophenotype, bone marrow involvement, hypercalcemia, hypoalbuminemia, hypercreatininemia, and anemia. The Cox proportional hazard model revealed that LDH activity at the time of initial diagnosis of lymphoma was not significantly (P = 0.368) associated with overall survival (hazard ratio, 1.000; 95% CI, 0.999 to 1.001).
Univariate analysis revealed that only hypercreatininemia was retained as a confounding factor to assess the effect of LDH activity on disease-free interval. The Cox proportional hazard model revealed that LDH activity at the time of initial diagnosis of lymphoma was not significantly (P = 0.892) associated with disease-free interval (hazard ratio, 1.000; 95% CI, 0.998 to 1.002).
LDH activity at follow-up monitoring and anticipation of recurrence—Proportions of dogs that had increased LDH activities during follow-up monitoring and then had recurrence of lymphoma during the successive 45 or 90 days were summarized (Tables 1 and 2). Sensitivity and specificity were also determined. In brief, 41 dogs had CR at the end of the 9-week treatment period. Nine dogs had recurrence of lymphoma within 45 days after completion of chemotherapy, and 3 of these dogs had a high LDH activity (> 280 U/L) at completion of chemotherapy. None of the remaining 32 dogs without recurrence of lymphoma within 45 days after completion of chemotherapy had an increased LDH activity at the end of chemotherapy. The proportion of dogs with CR that had an increased LDH activity and recurrence of lymphoma within 45 days after completion of chemotherapy was significantly (P = 0.007) higher than the proportion of dogs without recurrence of lymphoma. Sensitivity of LDH activity for detecting the recurrence of lymphoma in dogs was 33.3%, whereas specificity was 100%. The proportion of dogs with an increased LDH activity that had recurrence or no recurrence of lymphoma within 90 days after completion of chemotherapy did not change significantly (3/15 vs 0/26, respectively). However, sensitivity decreased to 20.0%, but specificity remained at 100%.
Proportion of dogs with CR that had increased LDH activity and that had or did not have recurrence of lymphoma within the successive 45 days.
Time of LDH measurement | Recurrence of lymphoma* | No recurrence of lymphoma* | Sensitivity (%)† | Specificity (%)† | PPV (%)† | NPV (%)† |
---|---|---|---|---|---|---|
End of chemotherapy | 3/9 (33.3) | 0/32 (0) | 33.3 (7.5–70.1) | 100 (89.1–100) | 100 (29.2–100) | 84.2 (68.8–94.0) |
1 month after chemotherapy | 7/9 (77.8) | 1/26 (3.8) | 77.8 (40.0–97.2) | 96.2 (80.4–99.9) | 87.5 (47.4–99.7) | 92.6 (75.7–99.1) |
3 months after chemotherapy | 2/6 (33.3) | 1/16 (6.3) | 33.3 (4.3–77.7) | 93.8 (69.8–99.8) | 66.7 (9.4–99.2) | 79.0 (54.4–94.0) |
6 months after chemotherapy | NA | 0/7 (0) | ND | ND | ND | ND |
Value reported is number of dogs with high LDH activity/number of dogs with CR at time of LDH measurement (percentage).
Value reported is mean (95% CI).
NA = Not applicable because none of the dogs had recurrence of lymphoma. ND = Not determined. NPV = Negative predictive value. PPV = Positive predictive value.
Proportion of dogs with CR that had increased LDH activity and that had or did not have recurrence of lymphoma within the successive 90 days.
Time of LDH measurement | Recurrence of lymphoma* | No recurrence of lymphoma* | Sensitivity (%)† | Specificity (%)† | PPV (%)† | NPV (%)† |
---|---|---|---|---|---|---|
End of chemotherapy | 3/15 (20.0) | 0/26 (0) | 20.0 (4.3–48.1) | 100 (86.8–100) | 100 (29.2–100) | 68.4 (51.4–82.5) |
1 month after chemotherapy | 7/15 (46.7) | 1/20 (5.0) | 46.7 (21.3–73.4) | 95.0 (75.1–99.9) | 87.5 (47.4–99.7) | 70.4 (49.8–86.3) |
3 months after chemotherapy | 2/7 (28.6) | 1/15 (6.7) | 28.6 (3.7–71.0) | 93.3 (68.1–99.8) | 66.7 (9.4–99.2) | 73.4 (48.8–90.9) |
6 months after chemotherapy | 1/2 (50.0) | 0/5 (0) | 50.0 (1.3–98.7) | 100 (47.8–100) | 100 (25.0–100) | 83.3 (35.9–99.6) |
See Table 1 for key.
At 1 month after completion of chemotherapy, 35 dogs were in CR. In 9 of these dogs, recurrence of lymphoma was detected within the successive 45 days, and 7 of the 9 had an increased LDH activity at 1 month after chemotherapy. Of the remaining 26 dogs that did not have recurrence of lymphoma within the successive 45 days, only 1 (3.8%) had an increased LDH activity at 1 month after chemotherapy. The proportion of dogs in CR with an increased LDH activity at 1 month after chemotherapy that had recurrence of lymphoma within the successive 45 days was significantly (P < 0.001) higher than the proportion of dogs that had an increased LDH activity at 1 month after chemotherapy but that did not have recurrence of lymphoma. Sensitivity of LDH activity at 1 month after chemotherapy for the detection of dogs with recurrence of lymphoma was 77.8%, and specificity was 96.2%. The proportion of dogs with an increased LDH activity at 1 month after chemotherapy that had recurrence or no recurrence of lymphoma within the successive 90 days did not change significantly (7/15 vs 1/20, respectively). However, sensitivity decreased to 46.7% and specificity decreased to 95%.
At 3 months after chemotherapy, 22 dogs were in CR. The proportion of dogs with increased LDH activity at 3 months after chemotherapy was not significantly different between dogs with or without recurrence of lymphoma within the successive 45 (2/6 vs 1/16, respectively) or 90 (2/7 vs 1/15, respectively) days. Mean LDH activities at 3 months after chemotherapy in dogs that had recurrence of lymphoma within the successive 45 days was slightly higher but did not differ significantly (P = 0.052) from the mean LDH activities for dogs without recurrence of lymphoma within that successive 45 days (227 ± 57 U/L vs 122 ± 23 U/L, respectively). For recurrence within either the successive 45 or 90 days, sensitivity was < 35% and specificity was > 90%.
At 6 months after chemotherapy, 7 dogs were in CR. None of them had recurrence of lymphoma in the successive 45 days, and only 2 had recurrence of lymphoma within the successive 90 days. Of these 2 dogs with recurrence, 1 had an increased LDH activity at 6 months after chemotherapy, whereas the other dog had an LDH activity within the reference range. In the remaining 5 dogs without recurrence within the successive 90 days, LDH activity was within the reference range. Comparison between proportions was not possible because of the small sample size.
Discussion
Lymphoma represents a heterogeneous group of neoplasms, characterized by clonal expansion of lymphoid cells with distinctive morphological, immunophenotypic, and biological features.22 Traditionally, dogs with lymphoma have been grouped into 5 clinical stages on the basis of anatomic tumor extension,22 with a more advanced clinical stage reflecting a more aggressive clinical course. However, in each stage, the clinical syndrome and prognosis can vary, with some dogs not responding to chemotherapy and dying within a short time, whereas others survive for extended periods without tumor recurrence.22 It has become clear that use of only anatomic staging provides inadequate prognostic information, thereby making it difficult for clinical investigators to identify useful factors associated with the disease-free interval and overall survival. During the past decades, several variables have emerged as negative prognostic factors for dogs with lymphoma, including substage b, T-cell immunophenotype, prolonged corticosteroid treatment that leads to multidrug resistance, hypercalcemia, bone marrow involvement, and selected anatomic locations (including diffuse cutaneous, alimentary, and hepatosplenic).22,25–31
In humans with non-Hodgkin lymphoma, the correlation between LDH activity at time of diagnosis and disease aggressiveness has been clearly established; increased serum LDH activities are closely associated with marked disease activity and diminished response to treatment.3,4,10,12 This finding has important clinical implications because high pretreatment LDH activities allow clinicians to rationally select an initial treatment for patients likely to die as a result of treatment failure.12,13
In the study reported here, serum LDH activity at the time of initial lymphoma diagnosis was not associated with disease-free interval or overall survival interval, which is in contrast with another study16 in dogs in which the LDH activity at diagnosis was associated with a shorter survival interval. The reason for this difference may reside in the relatively low number of dogs or in distinct characteristics of the dog groups evaluated.
Nevertheless, by serially determining LDH activities after treatment, we recognized LDH activity as a dynamic variable during follow-up monitoring. Intriguingly, we were able to determine that an increase in LDH activity (> 280 U/L) over time preceded the clinical stages of lymphoma. More specifically, dogs with an increase in LDH activity at the completion of chemotherapy or 1 month after completion of chemotherapy were more likely to have recurrence of lymphoma within the successive 45 days. As a consequence, evaluating LDH activity during follow-up monitoring may be helpful in predicting early disease progression. However, it is worth mentioning that increased LDH activity had limited sensitivity, especially when measured at the end of chemotherapy (ie, sensitivity of 33.3%); the sensitivity increased (77.8%) when LDH activity was measured at 1 month after chemotherapy. On the other hand, measurement of LDH activity at the end of chemotherapy or 30 days after the end of chemotherapy yielded a specificity of approximately 100%. Therefore, if increased LDH activity were detected at the completion of chemotherapy or 1 month after completion of chemotherapy, there was a high likelihood that the dog would have recurrence of lymphoma during the successive 45 days, even if clinically in CR at those times of LDH measurement.
The results did not change significantly when recurrence was evaluated for the successive 90 days, rather than the successive 45 days. This finding has important clinical implications because rescue chemotherapy may be initiated before recurrence becomes clinically evident, thereby improving treatment response and outcome in selected dogs.
However, until the implications of abnormal LDH activities are better assessed, it may be unsuitable to plan further treatments (eg, rescue chemotherapy) solely on the basis of an increased LDH activity. Thus, we propose that clinicians determine LDH activity at the end of chemotherapy and at 1 month after completion of chemotherapy. If LDH activity is increased, clinicians should suggest to the owner that they carefully observe the clinical status of their dog because an early recurrence may be expected. In addition, an increase in LDH activity should encourage a thorough reevaluation of the disease via complete restaging (possibly including an evaluation of minimal residual disease) to differentiate between apparent and actual CR and definitely prove the need for further treatment.
With regard to LDH activities measured at 3 and 6 months after completion of chemotherapy, the chance to identify lymphoma recurrence within the successive 45 or 90 days on the basis of increased LDH activity appeared to decrease. However, the number of these dogs with recurrence was too limited to make an assumption about the usefulness of the measurement of LDH activity at these specific time points.
Mechanisms underlying the increase in LDH activity in dogs with progressing lymphoma in the study reported here are unknown. It is known that after chemotherapy, highly responsive proliferating cells have been killed, whereas the dormant neoplastic cells (which account for the minimal residual disease) can resume the cell cycle.32 We assume that the accelerated turnover of 1 or more novel neoplastic clusters accounted for the increased LDH activities.
Therefore, LDH activity is an appealing variable for use in follow-up monitoring in dogs with lymphoma. Measurement of LDH activity may assist clinicians in early recognition of disease progression and thus help in risk stratification and case selection for interventional treatments. In dogs with lymphoma, all prognostic factors proposed so far are primarily used as pretreatment, static variables.22,25–31 We believe that anticipating the dynamics of the disease would be of great value and will provide new opportunities for treating and possibly improving survival in affected dogs. However, despite the specificity for the use of LDH activity, its low sensitivity limits its usefulness as a good screening tool for use in predicting recurrence of lymphoma in dogs.
ABBREVIATIONS
CI | Confidence interval |
CR | Complete remission |
LDH | Lactate dehydrogenase |
VCAA | Vincristine, cyclophosphamide, adriamycin, and l-asparaginase |
Kinetic UV method pyruvate-lactate, Biogamma SRL, Rome, Italy.
SPSS, version 11.0, SPSS Inc, Chicago, Ill.
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