Mediterranean Journal of Hematology and Infectious Diseases Cutaneous Hyperpigmentation in Megaloblastic Anemia: a Five Year Retrospective Review

Background: Cutaneous hyperpigmentation is an often overlooked clinical sign in megaloblastic anemia (MA) which has been sporadically reported in the literature. Methods: We describe the bone marrow (BM) changes and clinicolaboratory characteristics of 25 of 198 adult cases (>16 years) with cutaneous hyperpigmentation who underwent BM evaluation for cytopenia (s). Results: Twenty-one of 25 cases (84%) had MA, while MA without hyperpigmentation occurred only in 12 of remainder 173 cases (P<0.001). Knuckle pad hyperpigmentation (KP) was noted in 16 (64%) cases; whereas 9 (36%) had diffuse brownish black discoloration (DP) of the palms and /or soles. Eighteen of 25 (72%) cases had pancytopenia (13 with KP) and 7 of 25 (28%) had bicytopenia (3 with KP). In addition, five cases (20%) presented with pyrexia. Of the 17 cases where data available, eleven were B 12 deficient [<190 pg/ml; eight had severe deficiency (<100 pg/ml); ref.; 190-800pg/ml], while 4 had pure folate deficiency (< 4.0 ng/ml; ref.; 4-20ng/ml); and remainder 2 had combined B 12 and folate deficiency. Compared to those with diffuse) pg/ml] (P>0.05). In six cases where follow-up data were available, there was a significant reversal of hyperpigmentation at 12 weeks following parenteral cobalamin therapy. In all five cases with pyrexia, fever subsided after 24 to 72 hours following administration of parenteral cobalamin therapy. Conclusion: Cutaneous hyperpigmentation and cytopenia (s) are strongly associated with megaloblastic anemia. Knuckle pad hyperpigmentation is much more frequent than diffuse pigmentation of the palms and/or soles in such patents. A nonsignificant trend towards a greater degree of MA was found in cases with pigmentation of the knuckles. hyperpigmentation in megaloblastic anemia: a five-year retrospective review. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Introduction. Megaloblastic anemia (MA) is a heterogeneous group of reversible bone marrow failure syndromes characterized by a variable degree of peripheral blood cytopenia (s) in the

Introduction. Megaloblastic anemia (MA) is a heterogeneous group of reversible bone marrow failure syndromes characterized by a variable degree of peripheral blood cytopenia (s) in the presence of a normo or hypercellular bone marrow.
The hallmark pathophysiologic mechanism of MA is an impairment of DNA synthesis in all nucleated cells secondary to vitamin B 12 (B 12 ) and/or folate deficiency, resulting in nuclear-cytoplasmic asynchrony; distinctive megaloblastic changes, increased apoptosis, and ineffective hematopoiesis in the bone marrow. 1 The manifestations of MA are diverse and may range from nonspecific signs and symptoms of anemia to gastrointestinal disturbances and potentially fatal neuropsychiatric and cardiovascular disorders. 2 Megaloblastic anemia is not uncommon in the Indian subcontinent as well as other parts of Asia with females and vegetarians being more susceptible to B 12 deficiency. Various studies in the past have shown that occult B 12 deficiency may be rather prevalent among Indian urban and rural population. [3][4][5] Cutaneous manifestations associated with B 12 deficiency include characteristic mucocutaneous hyperpigmentation (most common), vitiligo, angular cheilitis, and hair-nail changes, which are often missed or overlooked in early, asymptomatic phases of the disease. 6 In this manuscript, we describe the association of cutaneous hyperpigmentation (CP) with bone marrow changes in a series of 25 cases from a tertiary center in South India with a correlation of hematological and biochemical parameters, and also present a concise review of relevant literature. Furthermore, the association of pyrexia in MA is also briefly highlighted.

Materials and Methods.
In this retrospective study the bone marrow records of all adult cases (> 16 years), who underwent bone marrow aspiration (BMA) and trephine biopsy (Bx) over the last five years (October 2010 to December 2015) in the Department of Pathology of our Institute, were reviewed for the presence of CP. Informed written consent had been obtained from each case prior to the BM procedure; and the study was approved by the Institutional Ethics Committee.
As a part of the protocol for BM procedure, CP was prospectively documented in the bone marrow record by one of the authors (SP), prior to performing the procedure. Two types of pigmentation were documented: 1) dominant brownish-black pigmentation over dorsal aspect of hands and/or feet with accentuation over interphalangeal joints and periungual areas [knuckle pad (KP) group], and 2) diffuse and/or patchy, macular, dusky brownish-black discoloration/pigmentation over palms and/or soles (diffuse pigmentation (DP) group). Nature of BMA and gross appearance of Bx core were also recorded in each case. Data pertaining to routine hematological parameters such as hemoglobin (Hb), mean corpuscular volume (MCV), total leukocyte count (TLC), total platelet count (Plt), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and peripheral blood smear (PBS) findings were collected from laboratory records in each case at the time of BM procedure.
BMA smears and Bx sections of all the above cases were retrieved from the departmental archives. Two pathologists (AR, RGV) who were blinded to the presence and nature of the pigmentation, reviewed the slides for the presence of definite megaloblasts. In each case, two MGG (May Grunewald Giemsa) and one Prussian blue (Perl stain) stained BMA smears were available for morphological study and assessment of the iron stores, respectively. A definitive diagnosis of MA was rendered with a constellation of PBS and BM findings such as erythroid hyperplasia, altered myeloid to erythroid ratio (M:E), presence of definite megaloblasts with sieve-like chromatin, nuclearcytoplasmic asynchrony, giant abnormal shaped metamyelocytes and/or band forms, and/or abnormal (multinucleated) megakaryocytes with adequate (1-3+) or increased (≥ 4+) iron stores. A diagnosis of dimorphic marrow picture was rendered when there was admixture of both megaloblast and micronormoblast (with reduced hemoglobinization) and reduced (1+) or absent (0) iron store on Perl stain. 7 When definite megaloblasts were not seen or very sparse, but dyspoietic changes were present, a possibility of macronormoblastic erythroid maturation with dyspoiesis was reported. Furthermore, those with dyspoietic changes and increased iron stores (≥ 4+) were also screened for the presence of ringed sideroblasts for a possible diagnosis of myelodysplastic syndrome (MDS) in the appropriate clinical setting.
Bone marrow changes were then correlated with the presence or absence and nature of hyperpigmentation, and serum biochemical parameters such as serum B 12 , folate, ferritin, and iron levels (wherever available). Serum B 12 [2]. Similarly, cases with folate levels < 4ng/ml were considered as folate deficient. Cases with serum ferritin < 30 ng/ml were considered as depleted iron stores; and those with < 15 ng/ml were considered as a severe iron deficiency.
Demographic profile, place of origin, dietary habits, history of prior illness and medications (if any), provisional clinical diagnoses, relevant clinical, microbiological and/or serological data were collected from the case records. All cases where a diagnosis of MA was made received parenteral (intramuscular) cyanocobalamin (1000 μg/day) for 7 days, followed by a weekly dose of the same for a minimum of 10 to 12 weeks. Those with dimorphic anemia were prescribed both parenteral and/or oral cobalamin in addition to hematinics and multivitamins. Statistical analysis: Continuous variables were described by mean (± SD) and Box plot were used to highlight the distribution of variables between two groups. Comparison between two groups was made using Fisher's exact method for categorical variables, student t-test for normally distributed continuous variables, and Mann-Whitney test for not normally distributed continuous variables. All the tests were 2 sided; and a P value less than 0.05 was considered as statistically significant. SPSS software (version 20.0) was used for analyzing the data.
Results. During the study period, a total of 304 patients underwent a BM examination, out of which 198 were personally performed by one of the authors (SP). All of these cases had been assessed for the presence or absence of CP at the time of the BM examination and hence were included in the analysis. CP was documented in 25 of these 198 patients (12.6%).
The demographic and clinico-laboratory characteristics of all 25 cases are presented in Tables 1  and 2. There were 16 males (64%) and 9 females (36%) with a mean age of 41.2 ± 16.7 years. Among the cases, 21 (84%) were dark skinned South Indians (in and around Puducherry). Seventeen of 25 (68%) were taking a mixed diet while only 8 (32%) were vegetarians. Seven of 16 (43.7%) males and none of the females had a history of alcohol abuse. Fatigue was the most common clinical presentation noted in 15/25 (60%) cases; 3/25 (12.0%) were diagnosed cases of autoimmune hepatitis on periodic follow-up with maintenance drugs such as azathioprine and prednisolone, 3/25 (12%) had evidence of gastric atrophy (anti-intrinsic factor antibody positive in one), 5 (20%) presented with fever, 2 (8%) had history of diarrhoea, 1 (4%) was a case of non-alcoholic steatohepatitis (NASH) secondary to uncontrolled type 2 diabetes mellitus, and 1 (4%) was a case of paranoid schizophrenia on olanzapine.
The association of MA with CP was highly significant (P<0.001) with 21 (out of 25) patients having MA [20 had pure MA and 1 had mixed MA and iron deficiency anemia (IDA)] as compared to only 12 patients with MA (5 had pure MA and 7 had mixed MA and IDA) among 173 patients without pigmentation ( Table 3). Prominent knuckle pad hyperpigmentation (KP) was documented in 16 of 25 (64%) cases whereas 9/25 (36%) cases had patchy or diffuse, dusky, brownish-black pigmentation over palms and/or soles with accentuation over palmar creases (DP) (Figures: 1A, 1B, 1C, 1D). Eighteen of Table 1. Base line characteristics of cases with cutaneous hyperpigmentation which was documented during bone marrow procedure (October 2010-December 2015).
Six of 25 cases where follow-up data were available, showed dramatic improvement (> 85%) in their hyperpigmentation following 12 weeks B 12 therapy (Figures 4A and 4B), whereas the rest were lost to follow-up. In all the five cases who presented with pyrexia, their fever subsided following day 3 to 4 of starting parenteral cyanocobalamin therapy.

Discussion.
Megaloblastic anemia is a multisystemic disorder where hematological and neuropsychiatric manifestations usually dominate the clinical picture. In 1944, Dr Bramwell Cook first observed that hyperpigmentation of the skin was associated with a macrocytic anemia and that both it and the anemia responded to crude liver extract. 8 Since then, there have been sporadic case reports and small case series with descriptions of the peculiar skin-hair-nail changes in patients with megaloblastic anemia. [9][10][11][12][13] In our series, we observed a strong association of cutaneous hyperpigmentation with megaloblastic anemia, and knuckle pad pigmentation was much more frequent than the diffuse type.
We also observed a good correlation between the presence of CP and BM changes with serum biochemical parameters. Barring one case of dimorphic anemia, none of the cases with hyperpigmentation had depleted marrow iron stores which in turn correlated well with the normal or increased serum ferritin levels in 24/25 cases. A greater proportion of our cases were B 12 deficient (<190 pg/ml); and eight had significant B 12 deficiency (<100 pg/ml). Cases with KP were associated with a greater degree of B 12 deficiency, macrocytosis, and pancytopenia; though this lacked statistical significance. Our observation was in accordance with that published in the literature. 8,9,11 Though majority (68%) of our subjects were on a mixed diet, a higher proportion of MA diagnosis suggests that factors other than poor intake like impaired absorption might be responsible for B 12 and/or folate deficiency; and alcohol abuse was a contributing factor among our male subjects. However, the presence of hyperpigmentation in a case of suspected MDS as well as in other 3 cases with reactive marrow changes contradicts the above hypothesis. Furthermore, an interesting aspect of our cohort was the fact that 5 of 21 cases (23.8%) with MA presented with pyrexia requiring extensive work up. However, in all cases, fever subsided within 24 to 72 hours of initiation of parenteral cyanocobalamin therapy. This reinforces our previous observation that florid ineffective hematopoiesis in MA, in conjunction with other but yet unidentified mechanisms, might be the underlying cause for such phenomenon. 14,15 A brief comparative review of literature regarding mucocutaneous hyperpigmentation in MA and/or B 12 deficiency is presented in Table 5. [8][9][10][11][12][13]15 In 1963, Baker and colleagues described characteristic reversible brownish-black pigmentation over dorsal aspect of interphalangeal joints of hands and feet (KP) in a large series of 21 South Indian subjects with MA (15 adults, 6 infants/children). Malabsorption was the commonest cause of B 12 deficiency in that series; and the mean serum B 12 level among cases was very low (49 pg/ml) by using microbiological assay method. 8 Similarly, reversible Addisonian type of mucocutaneous hyperpigmentation and nail changes were also reported in a dermatologic setting among nine South Indian  . Note that the median and interquartile range of Hb, Platelets, and serum B 12 were lower in the KP group than in the DP group; whereas the median and interquartile range of MCV were higher in KP group than the DP group. Also note that the group with DP has a wider B 12 value compared to the KP group (D). The median value of serum folate was similar among two groups.  patients with biochemical evidence of B 12 deficiency. 9 Aaron et al 10 described mucocutaneous changes as a significant physical finding in 26 of 63 patients (41%) with neurological manifestations secondary to B 12 deficiency. Cutaneous hyperpigmentation was found to be the most common (19% of cases) whereas hair changes and vitiligo were described in 9% and 3% of cases, respectively. Hyperpigmentation did not show any correlation with duration of symptoms, severity of megaloblastosis, and MCV; and follow-up data of skin changes were not reported in that series. 10 A recent prospective study from Turkey 11 recruited 57 pediatric subjects (mean age; 12.75 ± 4.75 months) of which 49 (86%) were exclusively breastfed. A higher proportion (63%) of cases had a severe B 12 deficiency (<100 pg/ml); and 44 of 57 mothers were also B 12 deficient (<200 pg/ml). Forty-nine of 57 (86%) babies had CP and 40 (70%) had atrophic glossitis. On serial followup at the end of 1 week, 4weeks, and 12 weeks, there was a dramatic improvement in mucocutaneous changes at 12 weeks following parenteral cobalamin therapy. 11 Similarly most of the other published reports have also noted a dramatic improvement or near complete reversal of the pigmentary changes following 8 to 12 weeks of parenteral cobalamin therapy. 12,13,15 The pathophysiologic mechanism associated with hyperpigmentation in B 12 and/or folate deficiency seems to be complex and is poorly understood. 6,16 However, the most accepted hypotheses are i) increased melanin synthesis, and ii) defective melanin transfer from melanocytes to adjacent megaloblastic keratinocytes ( Figure 5).

Figure 5:
The postulated biochemical mechanism of hyperpigmentation in megaloblastic anemia. 6,16 The 4 most accepted mechanisms involved are: 1) low methylcobalamin level in melanocytes leads to reduced level of reduced glutathione (GSSH); which in turn activates Tyrosinase enzyme in melanin synthesis pathway, 2) defective DNA synthesis activates Microphthalmia-associated transcription factor (MITF); which causes activation of both Tyrosinase and Tyrosinase related protein 1 and 2 (TRP 1and 2), 16 3) hyperhomocysteinemia leads to accumulation of cysteine leading to increased melanin synthesis, 4) defective melanin transfer from the melanocytes to adjacent megaloblastic keratinocytes. Increased angiogenesis secondary to upregulation of dermal vascular endothelial growth factor (VEGF) may also lead to increased pigmentation. 18 Both histopathologic and ultrastructural studies have postulated that hyperpigmentation is due to increased number of basal melanocytes as well as increased melanosomes. 19 Reduced methylcobalamin causes a reduction in intracellular reduced glutathione (GSSH) which in turn, activates Tyrosinase enzyme in the L-phenylalanine -L-tyrosine -melanin pathway. Also, defective DNA synthesis leads to activation of micro-ophthalmia associated transcription factor (MITF), which upregulates both Tyrosinase and Tyrosinase related  proteins  (TRP  1  and  2). 16 Furthermore, hyperhomocysteinemia in B 12 deficiency leads to increased cysteine level augmenting melanin synthesis. Both histopathologic and ultrastructural studies in skin biopsies have suggested that hyperpigmentation is not due to a defect in melanin transport but is secondary to an increase in melanin synthesis. 17,18 Moreover, increased angiogenesis secondary to upregulation of vascular endothelial growth factor (VEGF) has also been postulated to be responsible for the reddish brown discoloration seen in some cases. 19 However, the reason for the localized regional hyperpigmentation over the knuckle regions and greater prevalence among dark skinned individuals remains an enigma. It is open to speculation whether genetic and racial differences are responsible for this peculiar phenomenon.
The present study has certain limitations. 1) The retrospective nature of the survey led to the fact that CP was not assessed in all patients undergoing a bone marrow evaluation, resulting in a limited sample size. Not all patients with MA diagnosed on the basis of hematological parameters patients would have had a bone marrow examination performed. We also do not have data as to how many patients who presented to the hospital during this period had the typical cutaneous hyperpigmentation. 2) A lack of correlation of MA with biochemical parameters was found in up to 40% of cases. This can be explained by the poor sensitivity and wider reference range of the assay technique (ECLIA) used in our laboratory, which generally gives higher values as compared to the more accurate microbial assay technique. 2 3) Lastly, lack of follow-up in the 15 of 21 cases with MA was a major limitation in our study.
Conclusions. This is the first study that has systematically evaluated cutaneous hyperpigmentation among patients undergoing a bone marrow examination in which a definite association with megaloblastic anemia was observed. The present study reinforces the fact that cutaneous hyperpigmentation and pyrexia are helpful clinical signs in megaloblastic anemia. These in the presence of cytopenia (s) are reliable markers in the megaloblastic anemia and clinicians should be aware of these valuable clinical signs.