2000 Update of Recommendations for the Use of Hematopoietic Colony-Stimulating Factors: Evidence-Based, Clinical Practice Guidelines*

Stem cell transplantation represents a critical approach for the treatment of many malignant and non-malignant diseases. The foundation for these approaches is the ability to cryopreserve marrow cells for future use. This technique is routinely employed in all autologous settings and is critical for cord blood transplantation. A variety of cryopreservatives have been used with multiple freezing and thawing techniques as outlined in the later chapters. Freezing efficiency has been proven repeatedly and the ability of long-term stored marrow to repopulate has been established. Standard approaches outlined here are used in many labs as the field continues to evolve.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/ajh.20707

Cryopreservation of hematopoietic stem cells.

Heat shock protein 90 (Hsp90) is an emerging therapeutic target of interest for the treatment of cancer. Its role in protein homeostasis and the selective chaperoning of key signaling proteins in cancer survival and proliferation pathways has made it an attractive target of small molecule therapeutic intervention. 17-Allylamino-17-demethoxygeldanamycin (17-AAG), the most studied agent directed against Hsp90, suffers from poor physical-chemical properties that limit its clinical potential. Therefore, there exists a need for novel, patient-friendly Hsp90-directed agents for clinical investigation. IPI-504, the highly soluble hydroquinone hydrochloride derivative of 17-AAG, was synthesized as an Hsp90 inhibitor with favorable pharmaceutical properties. Its biochemical and biological activity was profiled in an Hsp90-binding assay, as well as in cancer-cell assays. Furthermore, the metabolic profile of IPI-504 was compared with that of 17-AAG, a geldanamycin analog currently in clinical trials. The anti-tumor activity of IPI-504 was tested as both a single agent as well as in combination with bortezomib in myeloma cell lines and in vivo xenograft models, and the retention of IPI-504 in tumor tissue was determined. In conclusion, IPI-504, a potent inhibitor of Hsp90, is efficacious in cellular and animal models of myeloma. It is synergistically efficacious with the proteasome inhibitor bortezomib and is preferentially retained in tumor tissues relative to plasma. Importantly, it was observed that IPI-504 interconverts with the known agent 17-AAG in vitro and in vivo via an oxidation-reduction equilibrium, and we demonstrate that IPI-504 is the slightly more potent inhibitor of Hsp90.

https://www.pnas.org/content/pnas/103/46/17408.full.pdf

Development of 17-allylamino-17-demethoxygeldanamycin hydroquinone hydrochloride (IPI-504), an anti-cancer agent directed against Hsp90.

BACKGROUND: Preservation of the male germ line in prepubertal boys undergoing gonadotoxic treatment is a crucial consideration in terms of their future quality of life. As these patients do not yet produce spermatozoa for freezing, only immature tissue is available for storage. We studied the survival, proliferation and differentiation capacity of spermatogonia after cryopreservation and long-term transplantation of immature testicular tissue pieces. METHODS: Single pieces of testicular tissue (2-8 mm(3)) from prepubertal boys (7-14 years) were cryopreserved, thawed and transplanted into the scrotum of mice for 6 months. Upon removal, histological, immunohistochemical and ultrastructural analyses and testicular sperm extraction (TESE) were used to evaluate the tissue. RESULTS: Histology showed 55 +/- 42% of tubules to be intact. MAGE-A4 immunostaining showed mean spermatogonial recovery to be 3.7 +/- 5.5%, with 35% of these cells expressing Ki67, evidencing proliferation in tissue from boys <14 years of age. No apoptosis was found, as demonstrated by the absence of active caspase-3 and TUNEL staining. Numerous premeiotic spermatocytes, a few spermatocytes at the pachytene stage and spermatid-like cells were observed. No immunostaining was observed for lactate dehydrogenase-C, ACE or proacrosin, indicating that the spermatid-like structures observed by histology did not express the meiotic and post-meiotic markers characteristic of normal spermatids. No ultrastructural alterations of the tissue were encountered. CONCLUSIONS: The present study demonstrates that spermatogonia are able to survive and proliferate after cryopreservation and long-term transplantation. Complete regeneration of normal spermatogenesis was not observed since, beyond the pachytene stage, no adequate characterization of germ cells was obtained. Further studies are thus required to investigate the differentiation potential of cryopreserved germ cells.

Long-term spermatogonial survival in cryopreserved and xenografted immature human testicular tissue

The cryoprotectant dimethyl sulfoxide (DMSO) is known to have toxic side effects, yet guidelines for its use in stem cell transplantation do not exist. To assess current practice in the use of DMSO and the incidence of DMSO-related complications, a single page questionnaire was mailed to 444 EBMT centres involved in autologous transplantation. The responses from 97 centres showed a wide variation in practice between transplant units regarding the concentration of DMSO used, daily DMSO dose restriction and the use of cell washing. The overall incidence of DMSO toxicity was approximately one in 70 transplants and most cases were cardiovascular and respiratory in nature. There was a trend to reduced complication rates in centres using lower concentrations of DMSO or washing cells prior to return. A large-scale prospective study of the strategies for reduction in exposure to DMSO and reduction in toxic effects is required before guidelines in the use of DMSO in stem cell cryopreservation can be promulgated.

Variation in dimethyl sulfoxide use in stem cell transplantation: a survey of EBMT centres

Background: We evaluated the infusion-related toxicity of cryopreserved autologous circulating progenitor cells transplanted in 22 patients receiving high dose chemotherapy and stem cells transplantation for malignancy. Materials and Methods: Progenitor cells were collected following mobilization with chemotherapy plus filgrastim and stored in liquid nitrogen in the presence of 10% dimethylsulfoxide (DMSO). Before infusion of the graft, patients were medicated with mannitol, hydrocortisone and clorphenamine. The amount of DMSO infused as well as the number of dead and damaged cells were evaluated as possible cause of toxicity. Results: Eleven patients (50%) experienced symptoms related to graft infusion, nausea and vomiting being the most common adverse events. Hypotension was documented in 3 patients (one of them developing transient bradycardia resolved with atropin administration) and one had hypertension with tachycardia. Other observed side effects were: chest tightness (2 pts), fever and chills (3 pts), associated with abdominal cramps (2 pts). 7 out of 8 (88%) patients infused with greater than 30 mL volume of DMSO experienced side-effects, the grade of toxicity being significantly less in those receiving lower amount ( 10×10 9 ) developed toxicity. Conclusions: In our exeperience the infusion of cryopreserved peripheral blood progenitors caused minor to moderate toxicity in most cases and, when present, side effects were observed only during infusion. The amount of DMSO present in the graft is related to the grade of toxicity.

Clinical toxicity of cryopreserved circulating progenitor cells infusion

One of the possible drawbacks to autologous stem cell transplantation in breast cancer (BC) patients is the potential for tumor contamination in the transplanted product. We present a patient with advanced disease who received high-dose chemotherapy (HDC) and PBPC support as consolidation therapy after achieving complete remission with standard-dose first-line treatment, and suffered recurrence of the disease 6 months after transplantation. Retrospective analysis revealed the presence of contaminating cells in the leukapheretic product, and clinical evidence suggested a role for these cells in the tumor relapse.

Role of tumor cells contaminating the graft in breast cancer recurrence after high-dose chemotherapy

Hematopoietic stem cell (HSC) enumeration is crucial to predict the engraftment potential of a given HSC collection, and currently involves the surrogate count of nucleated cells, CFU or CD34+ cells. However, there is raising evidence that CFU are HSC involved in short-term but not in long-term reconstitution, and that only a small fraction of all CD34+ cells have long term multilineage engraftment potential. In this regard, there is evidence that cord blood (CB), bone marrow (BM) and peripheral blood (PB) derived HSC are highly heterogeneous for a number of antigens useful for HSC enumeration by flow cytometry. Moreover, there is a raising evidence that a CD34 human HSC might exist. The CD34 HSC has been already described in animals and in human Hoechst 33342 negative HSC. This notwithstanding, clinical data have clearly demonstrated that purified allogeneic CD34+ cells can reconstitute the myeloid and the lymphoid lineages in myeloablated recipients. In the lack of a suitable marker for CD34 HSC enumeration, it is hard to predict the role of CD34 HSC in hematopoietic reconstitution after transplantation. On the other hand, these cells might be a better target for HSC expansion and gene transfer.

Hematopoietic stem cells from different sources: biological and technical aspects.

We have evaluated the combination of ifosfamide, carboplatin and etoposide (ICE regimen) along with mesna in 26 previously untreated patients with non small cell lung cancer (NSCLC). Thirteen stage III B and 13 stage IV patients received intermediate doses of ifosfamide (1000 mg/m2), carboplatin (120 mg/m2) and etoposide (120 mg/m2) given intravenously on day 1 to 3 every 4 weeks. Except for one patient who experienced grade 3 transient thrombocytopenia no major events of hematological or systemic toxicity were observed. Response rate (27%, 95% C.I., 10 to 44%), median duration of response (9 months, range 6-15), and survival (9.5 months, range 2-44+) were comparable to those achieved with conventional cisplatin-containing regimens. Our ICE combination, as compared to standard or high dose schedules appears effective, safe, well tolerated, and devoid of severe hematological toxicity.

Phase II Study with Ifosfamide, Carboplatin, Etoposide (ICE Regimen) at Intermediate Dosage for Advanced Non Small Cell Lung Cancer (NSCLC)

Fifty consecutive patients with stage IIIB-IV non-small cell lung cancer (NSCLC) received the ICE regimen at intermediate doses (ifosfamide 1 g/m2, carboplatin 120 mg/m2, etoposide 80 mg/m2, day 1 to 3, q.4 weeks, for a maximum of 6 cycles). Overall 2 complete response (CR) and 10 partial response (PR) (overall response, OR: 24%, 95% C.I. 14-37%) were observed. An additional 7 patients had stable disease (SD) lasting more than 6 months, therefore a clinical benefit (CR+PR+SD >6 mos) was achieved in 19 patients (38%). Median time-to-progression (TTP) was 7 months and median overall survival (OS) was 11 months; 1- and 2-year survival rates were 36% and 10%. The ICE regimen was well tolerated and devoid of any cardiac, hepatic or neurologic toxicity. Moderate nausea and vomiting were easily manageable, grade 2 alopecia was universal, while hematological toxicity was mild (grade 2 leuko-and thrombocytopenia). Due to its efficacy and safety profile, this 3-drug regimen can be considered for routine com...

A. Cuomo

Papers

Clinical toxicity of cryopreserved circulating progenitor cells infusion

Role of tumor cells contaminating the graft in breast cancer recurrence after high-dose chemotherapy

Hematopoietic stem cells from different sources: biological and technical aspects.

Phase II Study with Ifosfamide, Carboplatin, Etoposide (ICE Regimen) at Intermediate Dosage for Advanced Non Small Cell Lung Cancer (NSCLC)

Three conventional-drug combination (ifosfamide, carboplatin, etoposide--ICE regimen) in advanced non-small cell lung cancer (NSCLC).