The bone marrow aspirate and biopsy in the diagnosis of unsuspected nonhematologic malignancy: A clinical study of 19 cases

Background Although bone marrow metastases can be found commonly in some malignant tumors, diagnosing a nonhematologic malignancy from marrow is not a usual event. Methods To underscore the value of bone marrow aspiration and biopsy as a short cut in establishing a diagnosis for disseminated tumors, we reviewed 19 patients with nonhematologic malignancies who initially had diagnosis from bone marrow. Results The main indications for bone marrow examination were microangiopathic hemolytic anemia (MAHA), leukoerythroblastosis (LEB) and unexplained cytopenias. Bone marrow aspiration was not diagnostic due to dry tap or inadequate material in 6 cases. Biopsy results were parallel to the cytological ones in all cases except one; however a meticulous second examination of the biopsy confirmed the cytologic diagnosis in this patient too. The most common histologic subtype was adenocarcinoma, and after all the clinical and laboratory evaluations, the primary focus was disclosed definitively in ten patients (5 stomach, 3 prostate, 1 lung, 1 muscle) and probably in four patients (3 gastrointestinal tract, 1 lung). All work up failed in five patients and these cases were classified as tumor of unknown origin (TUO). Conclusion Our series showed that anemia, thrombocytopenia, elevated red cell distribution width (RDW) and hypoproteinemia formed a uniform tetrad in patients with disseminated tumors that were diagnosed via bone marrow examination. The prognosis of patients was very poor and survivals were only a few days or weeks (except for 4 patients whose survivals were longer). We concluded that MAHA, LEB and unexplained cytopenias are strong indicators of the necessity of bone marrow examination. Because of the very short survival of many patients, all investigational procedures should be judged in view of their rationality, and should be focused on treatable primary tumors.


Background
Diagnosis and management of many hematologic diseases depends on examination of the bone marrow, which usually involves two separate specimens: a cytologic and a histologic preparation. While cytologic preparation of bone marrow, obtained by aspiration, allows excellent visualization of cell morphology, the second one, usually obtained with a Jamshidi needle, allows optimal evaluation of cellularity, fibrosis or infiltrative disease. In addition to hematologic malignancies, bone marrow examination has been increasingly useful in documenting metastatic involvement of tumors. During the past four decades, prospective evaluation of bone marrow aspirates and biopsy specimens has come into widespread use for accurate staging of many malignant diseases. Recognition of metastasis in random biopsies presents challenges to hematologists and pathologists when diagnosing the primary focus [1,2] Although marrow metastases can be found commonly in some tumors, especially when newer sensitive methods are applied for the detection of tumor cells, diagnosing a nonhematologic malignancy from marrow is a rare event. We reviewed 19 patients with nonhematologic malignancies who were diagnosed initially from bone marrow, to underscore the value of bone marrow aspiration and biopsy as a short cut in establishing a diagnosis for disseminated tumors. Additionally we reported the details of the management and survival of the cases to offer a practical suggestion about work up of these patients to find a primary focus.

Methods
This study is based on our retrospective analysis of 19 patients with solid tumors whose diagnoses were made from bone marrow seen at the Department of Hematology, Uludag University Medical Faculty, over a period of 9 years. Patients with non-Hodgkin's lymphomas and Hodgkin's diseases were not included in this study and patients with known neoplastic disease were also excluded.
The standard technique was employed in obtaining the samples from posterior iliac crest using a Jamshidi needle (Regular/Adult, 11-gauge). All of the trephine biopsies were performed unilaterally, because clinically none of the diseases that focally involve the bone marrow were included in the differential diagnosis prior to the biopsy procedure. Length of the biopsy cores ranged between 1.2 cm and 2.2 cm (mean 1.7 cm). Trephine biopsies were fixed in 10% neutral buffered formaline for at least 24 hours, and then decalcified overnight in a decalcifying solution which is a mixture of 8% HCl and 10% formic acid at equal amounts of volume. Following the automated tissue processing, biopsies were embedded in parafin blocks, and 0.3 micrometer sections were cut. In some cases the sternum was sampled using a Rosenthal needle for aspiration. Touch preparations were done if the aspiration resulted in a "dry tap" or if aspiration material was considered to be technically inadequate for evaluation, or if it was hemodilute. Bone marrow aspiration, touch preparations and peripheral blood smears which were obtained at the same time by biopsy were stained by May Grunwald-Giemsa. Hematoxylene-eosin, Giemsa and reticulin stains were routinely performed in biopsy sections. If the nonhematologic properties of the tumor could be identified with routine stains, and if the primary of the metastatic tumor could easily be diagnosed morphologically with routine hematoxylene-eosin stains, as in the cases of signet ring cell carcinoma; no immunohistochemical study was held. The pathologist's approach to definitive diagnosis of the patient with metastasis of unknown primary effectively followed a few sequential steps: First of all we tried to determine the cell line of differentiation e.g. carcinoma, lymphoma, melanoma, sarcoma, or germ cell, with the help of morphological findings and if needed, immunohistochemical stains. Our panel of antibodies contained pancytokeratin, HMB45, Leukocyte Common Antigen (LCA), Vimentin and Placental Alkalen Phosphatase (PLAP). If it was an epithelial tumor we tried to determine the cytokeratin (CK) type or types of distribution in the tumor cells, since some subsets of cytokeratins are uniqe to certain tumor types. Our panel contained AE1/AE3, CAM5.2, CK7, CK20, and 34 beta E12. In our further studies we tried to determine if there was expression of supplemental antigens of epithelial or germ cell derivation, that was Carcinoembryonic Antigen (CEA), Epithelial membrane Antigen (EMA) or PLAP. Last step was to determine if there was expression of cell-specific structures or receptors that are unique identifiers of cell types, for example neuroendocrine granules, peptide hormones, thyroglobulin, Prostate Specific Antigen (PSA), Gross Cystic Disease Fluid Protein (GCDFP) or Thyroid Transcription Factor-1 (TTF-1). Our panel of antibodies contained Synaptophysin, Chromogranin, Neuron Specific Enolase (NSE), Thyroglobuline, PSA, GCDFP, and TTF-1. Cases with sarcomatous properties were immunostained with Desmin, Smooth Muscle Actin, S100, Vimentin and Myoglobulin. After the confirmation of original peripheral blood and bone marrow cytological findings and histopathological diagnoses by a senior hematologist and an expert pathologist the patient charts were reviewed. Cases with a history of malignancy at the time of presentation were excluded.
Patient characteristics were recorded in each case, including: presenting symptoms, onset of symptoms, physical examination findings, peripheral blood counts, peripheral blood morphology, diagnostic evaluation, management, and survival.

Results
Between 1995 and 2004, bone marrow metastasis was diagnosed in 19 samples among 5420 bone marrow aspirations and 856 bone marrow trephine biopsies. The ages of the patients were between 25 and 83 years (median age: 56), eight of them were female.
The main indications for bone marrow examination were microangiopathic hemolytic anemia (MAHA), leukoeryhtroblastosis (LEB) and peripheral cytopenias. Constitutional symptoms and pain were the most prominent presenting symptoms of the patients. Clinical findings were highly variable according to the underlying disease.
In the laboratory, the most common findings were anemia and thrombocytopenia, which were found in all patients. White blood cell counts (WBC) were between 3.3 × 10 9 /l and 17.1 × 10 9 /l (median: 8.1 × 10 9 /l), red cell distribution width (RDW) was increased in all cases, whereas mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), reticulocyte percentage (RET) and erythrocyte sedimentation rate (ESR) were highly variable. Coagulation tests were carried out in 17 patients and in 8 of them at least one anomaly was detected at presentation; additionally, in two patients who had normal test results initially subsequent tests were found abnormal. All patients had hypoproteinemia; the second most common anomaly was elevated serum lactate dehydrogenase (LDH) level, which was found normal in only one patient (patient # 13) in biochemical analyses; some hepatic and renal parameters were abnormal in some patients but none of them was a constant finding.
In 6 cases bone marrow aspiration was not diagnostic due to dry tap or inadequate material. Histopathological examinations confirmed the nonlymphohematopoietic cell infiltration in first evaluation except one (patient # 15). However, a meticulous second evaluation of biopsy confirmed the cytologic diagnosis in this patient too. The most common histologic subtype was adenocarcinoma.
After the all clinical and laboratory evaluations, the primary focus was disclosed in ten patients definitively (5 stomach, 3 prostate, 1 lung, 1 muscle) and in four patients probably (3 gastrointestinal tract, 1 lung). In five patients all work up failed and these cases were classified as tumor of unknown origin (TUO).   The prognosis of patients was very poor and survivals were only a few days or weeks except for 4 patients whose survivals were longer (patients # 9, 10, 17, 18). To avoid missing the individual features, detailed tables were prepared: Clinical and cytopathologic characteristics are shown in Table 1, clinical courses and survivals are shown in Table 2, and some hematological and biochemical features are shown in Table 3.

Discussion
Although bone marrow metastases are frequently encountered in patients with disseminated solid tumors, it is not a common event as a presenting sign. It stems from two possible reasons: i) in general, bone marrow is seldom the sole site of systemic involvement by malignant disease and ii) bone marrow examination in evaluating patients with suspected malignancy has a limited value unless supported by some other clinical finding, such as a leukoerythroblastic reaction [1,3,4]. Although there are a number of correlates that may be useful, there is no single specific finding that is an indicator of marrow infiltration. Furthermore, in most patients with neoplastic infiltration of the marrow, the peripheral blood findings do not differ significantly from those without marrow involvement. Our series showed that MAHA, LEB and unexplained cytopenias are strong indicators of the necessity of bone marrow examination. When we reviewed routine hematologic and biochemical parameters we found that only four were present in all patients: anemia, thrombocytopenia, elevated RDW and hypoproteinemia. However, our results  should be interpreted with caution, as the current study was not designed to determine the predictive parameters of marrow metastases.
LEB is the term used to describe the combination of nucleated red cells (erythroblasts) and immature myeloid precursors (e.g. myelocytes and myeloblasts) in the peripheral blood film. The mechanism of leukoerythroblastic reactions is not defined. The invasion of metastatic cancer cells may cause the early release of some cytokines, leading to the development of a myelophthisic blood picture even before the marrow is completely replaced. This is a possible explanation for some instances of leukoerythroblastic changes in the blood of the patients with tumors in whom marrow metastases are not documented by histologic examination. Although metastatic foci can be found in a high percentage of some carcinomas, the development of frank LEB occurs much less frequently, so its absence should not exclude marrow involvement. If there is LEB in a case of suspected malignancy, bone marrow examination should be considered. Our series confirmed this judgment, because fifteen of the patients were presented with LEB. On the other hand despite clear bone marrow involvement in 4 patients there were no erythroid and myeloid precursors in their peripheral blood films (patients # 10, 11, 14, 15). LEB was reported at different rates in different series consisting of cancer patients with bone marrow metastases: 10/27 [5]; 19/25 [6]; 5/63 [7]; 26/73 [2]. Our relatively high LEB ratio (15/19) is not comparable with other series because they consisted of cancer patients who were diagnosed before bone marrow examination. Our high LEB ratio, when considered along with the bad outcomes, may reflect the late and advanced cases. In recent years there is a scarcity of publications on the association of myelophthisis with cancer. As mentioned by the authors in a recent report [8], early diagnosis and more effective therapies are possible explanations for this decrease.
MAHA or thrombotic microangiopathy (TM) describes the association of hemolytic anemia with red cell fragmentation caused by microangiopathy mechanically. Cancer related thrombotic microangiopathy (CR-TM) is a rare and severe complication; it usually occurs in the late or terminal stage of cancer with a short-term life-threatening prognosis [9,10]. CR-TM shares certain clinical similarities with thrombotic thrombocytopenic purpura such as neurological and renal impairment; also both are characterized by circulating platelet aggregates containing ultra large multimers of Von Willebrand factor (VWF). A recent report showed no VWF cleaving protease deficiency in a patient with metastatic adenocarcinoma of the colon and microangiopathic hemolysis, who is refractory to plasma exchange [11]. We do not have any data about protease activity but, because of an almost identical clini-cal picture with TTP as presentation, we started plasma exchange immediately in patient # 1 and # 2 but therapy failed as expected. Hemolysis, in our CR-TM patients, was so severe that several units of transfusion per day were required to maintain a safe hemoglobin level. Reticulocytosis is expected but this finding could not be a reliable indicator of hemolysis, as seen in our patients, because of marrow infiltration or chronic disease. In our series, seven out of eight MAHA patients showed LEB and six showed abnormality in some coagulation tests suggesting disseminated intravascular coagulation. When we reviewed our eight MAHA cases there were two TUO and one prostate carcinoma. All definitive stomach carcinomas and a probable gastrointestinal carcinoma were in the MAHA group. The survivals of our MAHA patients were between 11 and 51 days except for one (patient # 18; prostate carcinoma). This suggests that once microangiopathic hemolysis is seen in a patient with disseminated carcinoma, the overall prognosis is poor, especially in stomach adenocarcinoma presented with MAHA.
"Dry tap" is a term used to describe failure to obtain bone marrow on attempted marrow aspirations. Extensive marrow fibrosis and hypercellularity have been proposed as mechanisms to account for the inability to withdraw marrow by aspiration [12,13]. Because it can be attributed to faulty technique it should only be used retrospectively after review of the biopsy. We have two dry taps (patients # : 4,6). If the definition of dry tap includes cases in which material is obtained but no, or inadequate marrow cells found in films we have four additional cases (patients # 1, 2, 3, 13). We concluded that bone marrow biopsy is certainly indicated whenever aspiration results in an insufficient material especially in the presence of LEB, MAHA, cytopenias and an elevated serum LDH.
Classically, marrow biopsy is unequivocally the best method of detecting lymphomatous involvement because in approximately one-third of cases, the aspirate is unremarkable and the biopsy shows tumor. In solid tumors other than those of lymphomatous origin the data in the literature comparing the relative value of bone marrow aspiration and biopsy in detecting marrow involvement are not so conclusive. [2,14]. Naturally trephine biopsies have a definitive advantage over aspirates in cases of dry tap. Additionally histologic sections may allow classification of the type of tumor cells, and this is of particular value in the investigation of a patient with a malignancy of unknown primary site. Aspirations were diagnostic in 12 patients in our series. The other attempts yielded no, or inadequate material. Cytological diagnoses could not be confirmed in one patient because of the patient's objection to biopsy procedure. Only in one instance was cytology superior to biopsy in first examination (a retrospective examination of biopsy confirmed the cyto-  logical finding in this patient). Although biopsies have some advantages, tumor cells occasionally can be seen in aspirate preparations when biopsy sections are normal as mentioned in literature, these two procedures should therefore be regarded as complementary.
The management of a patient, whose solid malignancy is disclosed from bone marrow, depends on his/her primary tumor. The pathologist can assist the clinician by thoroughly examining the histologic specimen. In some cases an immunoperoxidase staining for organ-specific antigen examination might be sufficient to bring out of the primary focus. As an example, an immunoperoxidase stain for prostatic acid phosphatase or prostate-specific antigen may be helpful in establishing a diagnosis of metastatic prostate cancer. On the opposite side of the spectrum in some cases the pathologist could not comment on the primary site because of a poorly differentiated tumor. Maximum effort should be made to minimize the target. In these tumors as a first step leukocyte common antigen may be used to differentiate lymphohematopoietic neoplasm from other cancers. Patient # 7 and 10, who were reported recently elsewhere separately [15,16], are good examples for bringing out the primary focus by extra stainings. The examination of patients with bone marrow metastases of unknown origin should focus on detecting treatable primary tumors [17]. This work up may result in certain improvements in survivals of patients with prostate carcinoma. Bone marrow metastases commonly arise from lung, breast and prostate cancers; therefore, in case of no clinical sign a reasonable work up should include a chest roentgenogram, a prostate examination with serum prostate specific acid phosphotase (PAP) in men; a breast examination and mammography in women; computed tomography scans also should be performed in suspected cases. Serum tumor markers are not so useful in many cases except PAP; considering their less specificity it is no surprising. Because the expected survival of many patients is quite short, laboratory and imaging studies should be considered in view of their rationality.

Conclusion
In conclusion, MAHA, LEB and unexplained cytopenias are strong indicators of the necessity of bone marrow examination. Anemia, thrombocytopenia, elevated RDW and hypoproteinemia form a uniform tetrad in patients with disseminated tumors that are diagnosed via bone marrow aspiration and biopsy. Because of the very short survival of many patients, all investigational procedures should be focused on treatable primary tumors.